The aim of this study to purify GPCR from a local strain of S. cerevisiae using gel filtration
chromatography techniques , by packing materials for columns which will be chosen of low cost comparing to
the already used in published researches, which depend on the costly affinity chromatography and other
expensive methods of purification. Local strain of S. cerevisiae chosen for extraction and purification of Gprotein
coupled receptor (GPCR) .The strains were obtained from biology department in Al- Mosul University,
Iraq. The isolated colony was activated on Yeast Extract Pepton Dextrose Broth (YEPDB) and incubated at 30
C˚ for 24 h .Loop fully of the yeast culture was transferred to (10ml) of yeast extract peptone glucose agar
(YEPGA) slant , then incubated at 30C˚for 24h , after that it was stored at 4C˚ ,the yeast cultures were
reactivated and persevered after each two weeks period. S.cerevisiae was identified by morphological,
microscopic characterization and biochemical test . The GPCR that extract from membrane of S.cerevisiae was
purified by gel filtration chromatography in two steps using Sepharose 6B. The optical density for each fraction
was measured at 280 nm by UV-VS spectrophotometer then the GPCR concentration was determined by using
ELISA Kit . The fractions which gave the highest absorbance and concentration of GPCR were collected .The
molecular weight of GPCR was determined by gel filtration chromatography using blue dextrin solution.
Standard curve was plotted between log of molecular weight for standard protein and the ratio of Ve/Vo of
GPCR . The purity of the GPCR that extracted and purified from whole cell of S, cerevisiae were carried out by
using SDS-PAGE electrophoresis In the first step 5ml of crude extract was applied on sepharose 6B column
(1.6x 96 cm) which previously equilibrated with 50 mM phosphate buffer saline pH= 7.4 . Multiple proteins
peaks appeared after elution with elution buffer (PBS PH= 7.4 containing 0. 5 % DDM). One peak only give
positive result with GPCR assay, fractions representing GPCR were collected , pooled and concentrated by
sucrose. In the second step five active fractions from the previous step were collected and applied once again on
the same column and same conditions. This step gave a single peak that was identical with the peak of GPCR
concentration ,maximum concentration of GPCR that observed in the fractions (34-38) was 18.541 (ng/ml) . The
specific activity for these fractions was 261.14 (ng/mg) protein with yield of 47.717%. The present study a chive
a relatively high purification of GPCR from membrane fraction of a local strain S. cerevisiae with fold
purification 5.094 and a yield of 47.717%. and molecular weight about~55KD.
Efficient transformation of lactococcus lactis il1403 and generation of knock...CAS0609
This document describes an optimized protocol for efficiently transforming Lactococcus lactis IL1403 by electroporation. Key aspects of the protocol include growing cells in media supplemented with glycine and sucrose, harvesting them at mid-late log phase, washing them in buffers containing sucrose and EDTA, and electroporating them with a resistor in series. The utility of the protocol was demonstrated by generating single and double gene knock-out mutants using non-replicating vectors. Transformation efficiencies as high as 106 cfu/μg of DNA were achieved, allowing for genetic manipulation of L. lactis IL1403.
Optimization of experimental protocols for cellular lysisExpedeon
In this project, we have compared existing sample preparation methods for proteomics studies against newly developed FASP method and our in-house developed SDS-TCA protocol. For our
preliminary studies, we have chosen a very well characterized soil microbe Pseudomonas putida.
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Linemdmitc
The document describes experiments characterizing a HepaRG cell line with MRP2 (an efflux transporter) knocked out using zinc finger nuclease technology. Western blot and immunostaining showed loss of MRP2 protein expression in knockout cells. Assays found control cells accumulated the fluorescent MRP2 substrate CDCF in bile canaliculi, while knockout cells did not, demonstrating functional loss of MRP2. The MRP2 knockout cell line allows investigation of MRP2-associated drug toxicity without its protective efflux.
Culture of Renal Proximal Tubule Epithelial Cell Line SA7K Using Extracellula...mdmitc
MilliporeSigma's Toni Steiner recently presented a poster at the 2016 AAPS/ITC Transporter Workshop demonstrating how culture conditions can influence drug transporter expression and activity in renal proximal tubule epithelial cells.
Rice is the principal food crop for more than half of the
world's population. Rice, as a staple food, supports more
than three billion people and comprises 50%–80% of their
daily calorie intake [1]. Adverse environmental factors
such as excessive cold, heat, drought, and salinity stresses
result in a considerable yield loss of crop plants all over
the world. Plant adaptations to environmental stresses
depend on the activation of cascades of molecularnetworks involved in signal transduction, stress perception,
and expressions of stress‐related genes. These
abiotic stresses elicit complex cellular responses in the
plant system, resulting in the production of excessive
reactive oxygen species (ROS) such as hydrogen peroxide
(H2O2), hydroxyperoxyl (HO2·), superoxide (O2
−), and
singlet oxygen (1O2) radicals. To protect themselves from
adverse conditions, plants have evolved a number of
cellular defense mechanisms including antioxidants such
as ascorbate, glutathione, and tocopherols as well as
ROS‐detoxifying enzymes such as superoxide dismutases
(SODs), peroxidases, and catalases (CATs) [2,3].
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
This study aims to clone and analyze GAPDH genes from various plant species to compare amino acid sequences related to catalytic function. Previous work successfully cloned GAPDH from Oxalis corniculata and Plectranthus amboinicus but not Myrtaceae psidium. The current study will continue work on the cloned samples through midipreps, restriction enzyme digestion and sequencing. Sequence data will undergo bioinformatics analysis to compare conserved amino acids of the GAPDH protein between plant species. Results could provide new genetic information published in GenBank and further the understanding of energy production pathways in plants.
Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles
Efficient transformation of lactococcus lactis il1403 and generation of knock...CAS0609
This document describes an optimized protocol for efficiently transforming Lactococcus lactis IL1403 by electroporation. Key aspects of the protocol include growing cells in media supplemented with glycine and sucrose, harvesting them at mid-late log phase, washing them in buffers containing sucrose and EDTA, and electroporating them with a resistor in series. The utility of the protocol was demonstrated by generating single and double gene knock-out mutants using non-replicating vectors. Transformation efficiencies as high as 106 cfu/μg of DNA were achieved, allowing for genetic manipulation of L. lactis IL1403.
Optimization of experimental protocols for cellular lysisExpedeon
In this project, we have compared existing sample preparation methods for proteomics studies against newly developed FASP method and our in-house developed SDS-TCA protocol. For our
preliminary studies, we have chosen a very well characterized soil microbe Pseudomonas putida.
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Linemdmitc
The document describes experiments characterizing a HepaRG cell line with MRP2 (an efflux transporter) knocked out using zinc finger nuclease technology. Western blot and immunostaining showed loss of MRP2 protein expression in knockout cells. Assays found control cells accumulated the fluorescent MRP2 substrate CDCF in bile canaliculi, while knockout cells did not, demonstrating functional loss of MRP2. The MRP2 knockout cell line allows investigation of MRP2-associated drug toxicity without its protective efflux.
Culture of Renal Proximal Tubule Epithelial Cell Line SA7K Using Extracellula...mdmitc
MilliporeSigma's Toni Steiner recently presented a poster at the 2016 AAPS/ITC Transporter Workshop demonstrating how culture conditions can influence drug transporter expression and activity in renal proximal tubule epithelial cells.
Rice is the principal food crop for more than half of the
world's population. Rice, as a staple food, supports more
than three billion people and comprises 50%–80% of their
daily calorie intake [1]. Adverse environmental factors
such as excessive cold, heat, drought, and salinity stresses
result in a considerable yield loss of crop plants all over
the world. Plant adaptations to environmental stresses
depend on the activation of cascades of molecularnetworks involved in signal transduction, stress perception,
and expressions of stress‐related genes. These
abiotic stresses elicit complex cellular responses in the
plant system, resulting in the production of excessive
reactive oxygen species (ROS) such as hydrogen peroxide
(H2O2), hydroxyperoxyl (HO2·), superoxide (O2
−), and
singlet oxygen (1O2) radicals. To protect themselves from
adverse conditions, plants have evolved a number of
cellular defense mechanisms including antioxidants such
as ascorbate, glutathione, and tocopherols as well as
ROS‐detoxifying enzymes such as superoxide dismutases
(SODs), peroxidases, and catalases (CATs) [2,3].
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
This study aims to clone and analyze GAPDH genes from various plant species to compare amino acid sequences related to catalytic function. Previous work successfully cloned GAPDH from Oxalis corniculata and Plectranthus amboinicus but not Myrtaceae psidium. The current study will continue work on the cloned samples through midipreps, restriction enzyme digestion and sequencing. Sequence data will undergo bioinformatics analysis to compare conserved amino acids of the GAPDH protein between plant species. Results could provide new genetic information published in GenBank and further the understanding of energy production pathways in plants.
Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles
Directed enzyme evolution is a technique that mimics natural selection to engineer proteins. It involves introducing random mutations into genes and screening proteins for modified activity. The key steps are selecting a gene, creating a library of mutant genes through error-prone PCR or other mutagenesis methods, expressing the proteins, and selecting variants with improved properties. Examples where directed evolution has been applied include improving the activity of enzymes used in producing the antibiotic cephalosporin and in the cholesterol-lowering drug atorvastatin. The goal is to leverage natural selection to develop enzymes with desired industrial applications like increased stability, activity, or substrate specificity.
This document provides an outline for a presentation on directed evolution. It discusses the process of directed evolution, which involves randomly introducing mutations at the genetic level followed by selection of variants with desired protein characteristics. The document also covers types of mutations, naturally evolutionary processes like random mutagenesis and gene recombination that directed evolution mimics, library size, selection and screening strategies, applications, and advantages of directed evolution over rational design.
Structure of a carotenoid gene cluster from Pantoea sp. strain C1B1YArunkumar K.R.
Structure of a carotenoid gene
cluster from Pantoea sp. strain C1B1Y and characterization of β-carotene hydroxylase (crtZ) gene
by functional complementation in Escherichia coli.
The document describes research into the function of the putative helix-turn-helix protein gp73 in mycobacteriophage HelDan. The researchers created an epitope-tagged version of gp73 and found that it may bind to HelDan genomic DNA based on crosslinking experiments. Future work aims to purify gp73 under native conditions in HelDan-infected M. smegmatis to identify interacting protein partners via mass spectrometry and examine gp73 expression during infection by RT-PCR.
- The document describes an experiment to produce and purify green fluorescent protein (GFP) from E. coli bacteria and then crystallize the purified GFP.
- GFP was expressed in E. coli bacteria containing a plasmid with the GFP gene. The bacteria were induced to produce GFP, which was then purified using nickel affinity and hydrophobic interaction chromatography.
- Bradford assays and SDS-PAGE were used to analyze the purified GFP samples and determine concentration and purity. Finally, purified GFP was crystallized using vapor diffusion.
This study analyzed the salicylic acid methyltransferase (SAMT) protein in Asclepias curassavica milkweed. The researcher extracted RNA from A. curassavica leaf tissue, amplified the SAMT gene, cloned it into a vector plasmid, and performed assays. Analysis of the SAMT amino acid sequence showed motifs predicting preference for salicylic acid over benzoic acid. Enzyme assays using GC-MS confirmed SAMT preferentially methylated salicylic acid. Statistical analysis supported the hypothesis that SAMT amino acid sequence correlates with substrate preference.
This document summarizes research on the protein Prl2a1 which is highly upregulated in B lymphocytes with impaired receptor editing. Experiments showed that purified mouse Prl (mPrl) and mPrl2a1 proteins have similar secondary structures. While mPrl stimulated cell proliferation in assays, mPrl2a1 did not demonstrate any mitogenic effects and its effects are independent of the prolactin and growth hormone receptors. Fusion proteins with mPrl and mPrl2a1 both bound to mouse liver tissue, but the specific cellular target of mPrl2a1 remains unknown. Future work aims to determine mPrl2a1's role and mechanism of action.
This document discusses protein engineering through directed evolution. It explains that directed evolution involves randomly recombining genes from protein libraries and screening mutant proteins for improved functions. This leaves beneficial changes up to chance but generates diversity. Rational design is also used to map important protein interactions to guide evolution. The document provides an example of using directed evolution to modify a cytochrome P450 enzyme to more efficiently convert alkanes to alcohols over multiple generations. However, it notes that expressing evolved proteins in vivo and requiring expensive cofactors limit the commercial potential of this approach.
IRJET- Subcellular Localization of Transmembrane E-cadherin-GFP Fusion Pr...IRJET Journal
1. The study aimed to express an E-cadherin-GFP fusion protein in HeLa cells and examine its subcellular localization. E-cadherin tagged with GFP at its carboxy terminus was cloned into a mammalian expression vector and transfected into HeLa cells.
2. Confocal microscopy of transfected HeLa cells did not show significant GFP signals from the transmembrane region, suggesting the fusion protein was not properly localized to the cell membrane.
3. Quantitative PCR found similar expression levels of the GFP-E-cadherin fusion regardless of whether GFP was on the carboxy or amino terminus, implying tag position did not affect gene expression.
The document discusses techniques for gene manipulation and introduction used in plant biotechnology. It describes how genes can be identified and modified, such as isolating a gene that controls a desired trait or creating a new allele. The modified gene is then introduced into an organism using transformation methods, creating transgenic plants. Specific examples discussed include creating Roundup-resistant crops by introducing a bacterial gene and developing Golden Rice by adding genes to produce vitamin A in rice.
This document discusses directed evolution, which mimics natural selection to evolve proteins or nucleic acids towards a defined goal. It does not create new organisms and focuses on specific molecular properties. The process involves randomly mutating a gene of interest, generating a library of variants, screening or selecting for desired properties, and repeating rounds of mutation and selection until the goal is achieved. Directed evolution was first used in the 1970s and has since advanced protein engineering techniques. It provides a way to customize protein reactions and improve properties like yield, substrate specificity, and stability.
Protein engineering and its techniques himanshuhimanshu kamboj
b pharma 6th sem
pharmaceutical biotechnology
Protein engineering
Objectives of protein engineering
Rationale of protein engineering
Protein engineering methods
Rational design -site-directed mutagenesis methods
Advantages and disadvantages of rational design
Directed evolution -random mutagenesis
Advantages and disadvantages of directed evolution
Peptidomimetics
Classification of peptidomimetics
Advantages and disadvantages of peptidomimetics
Flow cytometry
Instrumentation
Principle
components
This document discusses protein engineering techniques for modifying proteins, including rational protein design using site-directed mutagenesis and directed evolution using random mutagenesis. Site-directed mutagenesis involves introducing point mutations in a particular known area to modify a specific protein function, while directed evolution generates genetic diversity through random mutagenesis and screens variants to identify successful mutations without requiring structural information. Common random mutagenesis methods discussed are error-prone PCR and DNA shuffling, which can be used to engineer properties like protein folding, stability, binding, and catalysis.
LanglaisC_2007_Systematic approach to protein experssion in cell free systemsClaudia Langlais
This document describes a study that tested the expression of 960 human full-length proteins using both in vivo and in vitro expression systems. The researchers found that E. coli cell-free expression systems and wheat germ cell-free expression were better able to express proteins that did not express in E. coli in vivo. They optimized expression in the E. coli cell-free system through sequence modifications and achieved a 93% success rate for cell-free expression overall. Wheat germ expression showed high solubility and protein yield for the proteins tested.
Classical RIPA buffer is comprised of low concentration of sodium dodecyl sulfate (SDS, a denaturing detergent), deoxycholate for disruption of protein-protein interactions and other components. To get More information, come to Invent Biotechnologies
This paper describes a novel method for generating large libraries for directed evolution of proteins using error-prone PCR and a Kunkel-like mutagenesis reaction. The method uses error-prone PCR to generate a mutated DNA fragment, with one primer containing phosphorothioate modifications. Treatment with exonuclease preferentially removes the non-modified strand, generating a single-stranded "megaprimer". This megaprimer is then used in a Kunkel-like reaction with a uracilated template to introduce mutations efficiently without subcloning. This approach enables generation of libraries with over 108 clones from a single E. coli transformation, which is more efficient than conventional error-prone PCR
This document provides an overview of biotechnology principles and applications. It defines biotechnology as the application of technology to modify biological organisms by adding genes from other organisms. The document discusses how genes are identified, isolated, and manipulated to introduce desired traits. It describes techniques such as homology cloning, complementary genetics, and map-based cloning used to isolate genes. The document explains how genes are introduced into plants using transformation methods like Agrobacterium and biolistics. It provides examples of transgenic crops and their applications in agriculture.
A number of developments have been made in the molecular biology of oat (Avena spp.) in recent years. Many of these were recently described at the Fourth International Oat Conference, held on 18 to 23 October, in Adelaide, South Australia. These advances include a report of oat transformation and regeneration, the characterisation of J3-glucanase genes in oat, the further development of a molecular genetic map in oats, and the characterisation of genes encoding novel oat grain proteins. A technique for assessing pedigrees in the oat and other cereal crops has been reported using a modified electrophoretic technique.
- The study characterized the kinetic properties of wild-type human Δ1-pyrroline-5-carboxylate reductase 2 (HsPYCR2) and its R251C mutant found in patients.
- Kinetic analysis showed the R251C mutant had slightly lower catalytic efficiency (4-5 fold) for NADH and NADPH substrates compared to wild-type, suggesting the mutation impacts proline biosynthesis.
- Additional studies on protein structure may help understand how the R251C mutation contributes to neurological disease phenotypes seen in patients.
This document summarizes research on engineering bivalent affinity ligands. Key points include:
- A combinatorial bivalent ligand library was generated that identified ligands binding non-overlapping epitopes and maintained thermostability. This library performed better than a monovalent library.
- The best ligands from the bivalent library were sequenced, with 9 found to be bivalent and 1 trivalent. Mutations outside the original library positions were found in some ligands.
- Ligand M2 was identified that bound with low nanomolar affinity and could engage in multi-epitope binding to intrinsically disordered domains of beta-catenin. M2 showed promise as a new binding scaffold.
- Dimer
This document summarizes an experiment optimizing expression of G-protein coupled receptors (GPCRs) in HEK293 cells using a bioreactor. The researchers found that using a bioreactor allowed for higher cell densities and expression levels of over 1 mg of recombinant GPCR per liter of culture, compared to 0.1-0.2 mg/L using spinner flasks. A large-scale run in the bioreactor produced 10 mg of purified, bioactive recombinant GPCR. The bioreactor setup improved expression levels for structural characterization of this important class of membrane proteins.
This document describes a high-content screen to identify small molecules that selectively affect internalization of individual G protein-coupled receptors (GPCRs). The screen used a panel of 7 cell lines expressing fluorescent GPCR fusion proteins and tested 88 kinase and phosphatase inhibitors. Compounds were identified that selectively induced internalization of a single GPCR without affecting other receptors or general endocytosis pathways, allowing selection of potentially targeted molecules from a broad chemical library. One such selective compound was identified that affected only the muscarinic acetylcholine receptor 2.
Directed enzyme evolution is a technique that mimics natural selection to engineer proteins. It involves introducing random mutations into genes and screening proteins for modified activity. The key steps are selecting a gene, creating a library of mutant genes through error-prone PCR or other mutagenesis methods, expressing the proteins, and selecting variants with improved properties. Examples where directed evolution has been applied include improving the activity of enzymes used in producing the antibiotic cephalosporin and in the cholesterol-lowering drug atorvastatin. The goal is to leverage natural selection to develop enzymes with desired industrial applications like increased stability, activity, or substrate specificity.
This document provides an outline for a presentation on directed evolution. It discusses the process of directed evolution, which involves randomly introducing mutations at the genetic level followed by selection of variants with desired protein characteristics. The document also covers types of mutations, naturally evolutionary processes like random mutagenesis and gene recombination that directed evolution mimics, library size, selection and screening strategies, applications, and advantages of directed evolution over rational design.
Structure of a carotenoid gene cluster from Pantoea sp. strain C1B1YArunkumar K.R.
Structure of a carotenoid gene
cluster from Pantoea sp. strain C1B1Y and characterization of β-carotene hydroxylase (crtZ) gene
by functional complementation in Escherichia coli.
The document describes research into the function of the putative helix-turn-helix protein gp73 in mycobacteriophage HelDan. The researchers created an epitope-tagged version of gp73 and found that it may bind to HelDan genomic DNA based on crosslinking experiments. Future work aims to purify gp73 under native conditions in HelDan-infected M. smegmatis to identify interacting protein partners via mass spectrometry and examine gp73 expression during infection by RT-PCR.
- The document describes an experiment to produce and purify green fluorescent protein (GFP) from E. coli bacteria and then crystallize the purified GFP.
- GFP was expressed in E. coli bacteria containing a plasmid with the GFP gene. The bacteria were induced to produce GFP, which was then purified using nickel affinity and hydrophobic interaction chromatography.
- Bradford assays and SDS-PAGE were used to analyze the purified GFP samples and determine concentration and purity. Finally, purified GFP was crystallized using vapor diffusion.
This study analyzed the salicylic acid methyltransferase (SAMT) protein in Asclepias curassavica milkweed. The researcher extracted RNA from A. curassavica leaf tissue, amplified the SAMT gene, cloned it into a vector plasmid, and performed assays. Analysis of the SAMT amino acid sequence showed motifs predicting preference for salicylic acid over benzoic acid. Enzyme assays using GC-MS confirmed SAMT preferentially methylated salicylic acid. Statistical analysis supported the hypothesis that SAMT amino acid sequence correlates with substrate preference.
This document summarizes research on the protein Prl2a1 which is highly upregulated in B lymphocytes with impaired receptor editing. Experiments showed that purified mouse Prl (mPrl) and mPrl2a1 proteins have similar secondary structures. While mPrl stimulated cell proliferation in assays, mPrl2a1 did not demonstrate any mitogenic effects and its effects are independent of the prolactin and growth hormone receptors. Fusion proteins with mPrl and mPrl2a1 both bound to mouse liver tissue, but the specific cellular target of mPrl2a1 remains unknown. Future work aims to determine mPrl2a1's role and mechanism of action.
This document discusses protein engineering through directed evolution. It explains that directed evolution involves randomly recombining genes from protein libraries and screening mutant proteins for improved functions. This leaves beneficial changes up to chance but generates diversity. Rational design is also used to map important protein interactions to guide evolution. The document provides an example of using directed evolution to modify a cytochrome P450 enzyme to more efficiently convert alkanes to alcohols over multiple generations. However, it notes that expressing evolved proteins in vivo and requiring expensive cofactors limit the commercial potential of this approach.
IRJET- Subcellular Localization of Transmembrane E-cadherin-GFP Fusion Pr...IRJET Journal
1. The study aimed to express an E-cadherin-GFP fusion protein in HeLa cells and examine its subcellular localization. E-cadherin tagged with GFP at its carboxy terminus was cloned into a mammalian expression vector and transfected into HeLa cells.
2. Confocal microscopy of transfected HeLa cells did not show significant GFP signals from the transmembrane region, suggesting the fusion protein was not properly localized to the cell membrane.
3. Quantitative PCR found similar expression levels of the GFP-E-cadherin fusion regardless of whether GFP was on the carboxy or amino terminus, implying tag position did not affect gene expression.
The document discusses techniques for gene manipulation and introduction used in plant biotechnology. It describes how genes can be identified and modified, such as isolating a gene that controls a desired trait or creating a new allele. The modified gene is then introduced into an organism using transformation methods, creating transgenic plants. Specific examples discussed include creating Roundup-resistant crops by introducing a bacterial gene and developing Golden Rice by adding genes to produce vitamin A in rice.
This document discusses directed evolution, which mimics natural selection to evolve proteins or nucleic acids towards a defined goal. It does not create new organisms and focuses on specific molecular properties. The process involves randomly mutating a gene of interest, generating a library of variants, screening or selecting for desired properties, and repeating rounds of mutation and selection until the goal is achieved. Directed evolution was first used in the 1970s and has since advanced protein engineering techniques. It provides a way to customize protein reactions and improve properties like yield, substrate specificity, and stability.
Protein engineering and its techniques himanshuhimanshu kamboj
b pharma 6th sem
pharmaceutical biotechnology
Protein engineering
Objectives of protein engineering
Rationale of protein engineering
Protein engineering methods
Rational design -site-directed mutagenesis methods
Advantages and disadvantages of rational design
Directed evolution -random mutagenesis
Advantages and disadvantages of directed evolution
Peptidomimetics
Classification of peptidomimetics
Advantages and disadvantages of peptidomimetics
Flow cytometry
Instrumentation
Principle
components
This document discusses protein engineering techniques for modifying proteins, including rational protein design using site-directed mutagenesis and directed evolution using random mutagenesis. Site-directed mutagenesis involves introducing point mutations in a particular known area to modify a specific protein function, while directed evolution generates genetic diversity through random mutagenesis and screens variants to identify successful mutations without requiring structural information. Common random mutagenesis methods discussed are error-prone PCR and DNA shuffling, which can be used to engineer properties like protein folding, stability, binding, and catalysis.
LanglaisC_2007_Systematic approach to protein experssion in cell free systemsClaudia Langlais
This document describes a study that tested the expression of 960 human full-length proteins using both in vivo and in vitro expression systems. The researchers found that E. coli cell-free expression systems and wheat germ cell-free expression were better able to express proteins that did not express in E. coli in vivo. They optimized expression in the E. coli cell-free system through sequence modifications and achieved a 93% success rate for cell-free expression overall. Wheat germ expression showed high solubility and protein yield for the proteins tested.
Classical RIPA buffer is comprised of low concentration of sodium dodecyl sulfate (SDS, a denaturing detergent), deoxycholate for disruption of protein-protein interactions and other components. To get More information, come to Invent Biotechnologies
This paper describes a novel method for generating large libraries for directed evolution of proteins using error-prone PCR and a Kunkel-like mutagenesis reaction. The method uses error-prone PCR to generate a mutated DNA fragment, with one primer containing phosphorothioate modifications. Treatment with exonuclease preferentially removes the non-modified strand, generating a single-stranded "megaprimer". This megaprimer is then used in a Kunkel-like reaction with a uracilated template to introduce mutations efficiently without subcloning. This approach enables generation of libraries with over 108 clones from a single E. coli transformation, which is more efficient than conventional error-prone PCR
This document provides an overview of biotechnology principles and applications. It defines biotechnology as the application of technology to modify biological organisms by adding genes from other organisms. The document discusses how genes are identified, isolated, and manipulated to introduce desired traits. It describes techniques such as homology cloning, complementary genetics, and map-based cloning used to isolate genes. The document explains how genes are introduced into plants using transformation methods like Agrobacterium and biolistics. It provides examples of transgenic crops and their applications in agriculture.
A number of developments have been made in the molecular biology of oat (Avena spp.) in recent years. Many of these were recently described at the Fourth International Oat Conference, held on 18 to 23 October, in Adelaide, South Australia. These advances include a report of oat transformation and regeneration, the characterisation of J3-glucanase genes in oat, the further development of a molecular genetic map in oats, and the characterisation of genes encoding novel oat grain proteins. A technique for assessing pedigrees in the oat and other cereal crops has been reported using a modified electrophoretic technique.
- The study characterized the kinetic properties of wild-type human Δ1-pyrroline-5-carboxylate reductase 2 (HsPYCR2) and its R251C mutant found in patients.
- Kinetic analysis showed the R251C mutant had slightly lower catalytic efficiency (4-5 fold) for NADH and NADPH substrates compared to wild-type, suggesting the mutation impacts proline biosynthesis.
- Additional studies on protein structure may help understand how the R251C mutation contributes to neurological disease phenotypes seen in patients.
This document summarizes research on engineering bivalent affinity ligands. Key points include:
- A combinatorial bivalent ligand library was generated that identified ligands binding non-overlapping epitopes and maintained thermostability. This library performed better than a monovalent library.
- The best ligands from the bivalent library were sequenced, with 9 found to be bivalent and 1 trivalent. Mutations outside the original library positions were found in some ligands.
- Ligand M2 was identified that bound with low nanomolar affinity and could engage in multi-epitope binding to intrinsically disordered domains of beta-catenin. M2 showed promise as a new binding scaffold.
- Dimer
This document summarizes an experiment optimizing expression of G-protein coupled receptors (GPCRs) in HEK293 cells using a bioreactor. The researchers found that using a bioreactor allowed for higher cell densities and expression levels of over 1 mg of recombinant GPCR per liter of culture, compared to 0.1-0.2 mg/L using spinner flasks. A large-scale run in the bioreactor produced 10 mg of purified, bioactive recombinant GPCR. The bioreactor setup improved expression levels for structural characterization of this important class of membrane proteins.
This document describes a high-content screen to identify small molecules that selectively affect internalization of individual G protein-coupled receptors (GPCRs). The screen used a panel of 7 cell lines expressing fluorescent GPCR fusion proteins and tested 88 kinase and phosphatase inhibitors. Compounds were identified that selectively induced internalization of a single GPCR without affecting other receptors or general endocytosis pathways, allowing selection of potentially targeted molecules from a broad chemical library. One such selective compound was identified that affected only the muscarinic acetylcholine receptor 2.
The document discusses research into producing recombinant forms of the Cynara cardunculus Cyprosins, which are aspartic proteinases with milk clotting activity found in cardoon flowers. Researchers constructed expression cassettes containing genes for the Cyprosins with different signal sequences and expressed them in Saccharomyces cerevisiae. Cyprosin A was produced intracellularly but was inactive. Cyprosin B was actively secreted when expressed with the MFα1 presequence and native prosequence. A site-directed mutagenesis was performed to change the active site sequence of Cyprosin B from Asp-Ser-Gly to Asp-Thr-Gly as in other aspartic proteinases, but
Evaluation of the changes in the gene CYP3A4 expression in HepG2 cells under ...Angela Farngren
This study evaluated the effects of rifampicin and alpha-ketoglutarate on expression of the CYP3A4 gene in HepG2 liver cells. HepG2 cells were treated with either 10mM rifampicin or 4mM alpha-ketoglutarate for 7 days. RNA was extracted on days 0, 3, and 7 and analyzed via qPCR. Rifampicin treatment significantly increased CYP3A4 expression, peaking on day 3 and decreasing slightly by day 7. Alpha-ketoglutarate initially decreased CYP3A4 expression for the first 3 days but then increased expression levels. The study demonstrates that rifampicin and alpha-ket
Biotechnology and gene expression profiling for mechanistic understanding of ...Hana Fayed
1) The study characterized aggregation of baby hamster kidney (BHK) cells cultivated in perfusion bioreactors by analyzing expression of 84 genes related to extracellular matrix and adhesion. 2) They found that genes THBS2 and NCAM1 were significantly up-regulated and down-regulated, respectively, in late-stage versus early-stage cultures of an aggregating cell line. 3) Comparing an aggregating versus non-aggregating cell line, they found that genes COL1A1, COL4A1, THBS2, and VCAN were up-regulated while NCAM1 and THBS1 were down-regulated in the aggregating line, indicating differences in extracellular matrix and adhesion pathways contribute to aggregation
This document describes a study that evaluated the use of anion exchange chromatography to purify polyclonal immunoglobulin G (IgG) antibodies from rabbit serum. Two anion exchangers - DEAE and Q XL - were tested for their ability to remove albumin from rabbit serum while retaining IgG. The optimal conditions for albumin removal using DEAE were a pH of 8.0 and initial protein concentration of 0.5 mg/ml. Under these conditions, DEAE removed over 90% of albumin with less than 20% IgG loss, yielding 80% of IgG at 83% purity. Q XL also removed 90% of albumin but yielded only 70% of IgG at 62% purity. Therefore, DEAE
This document summarizes a student research project that aims to sequence and analyze the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes from two plant species endemic to Puerto Rico: Cyperus alternifolius and Schefflera actinophylla. The student will transform and clone the GAPDH genes from these plants into E. coli, purify and sequence the cloned genes, and perform bioinformatics analysis to compare the sequences to other known GAPDH genes. The GAPDH gene codes for an important enzyme involved in cellular processes like glycolysis and apoptosis. While well-studied in model organisms, the GAPDH genes from many native Puerto Rican plants have yet to be sequenced.
1) The document evaluates sample disruption techniques for extracting live E. coli and recombinant DNA from food using a bead mill and rotor stator homogenizer. Both techniques recovered similar amounts of viable cells as controls and allowed detection of E. coli down to 1x10-6 CFU/ml by PCR.
2) Spinach and beef samples inoculated with known levels of fluorescent E. coli were processed with each homogenizer. Live cell recovery was evaluated by plating and PCR detection limits were analyzed. A linear relationship was observed between CFU levels and DNA amounts amplified by PCR.
3) Both homogenizers fully disrupted samples without compromising bacterial viability, recovering up to 82% of cells. This vigorous processing could
1) There are several methods for selecting and screening recombinant transformants, including blue-white screening using X-gal, insertional inactivation of antibiotic resistance genes, and using reporter genes like luciferase or GFP.
2) Gene transfer in animals can be done using viral vectors like retroviruses which incorporate the transgene into their genome, or using non-viral methods like microinjection, gene guns, electroporation, and ultrasound to deliver naked DNA into cells.
3) Stable transfection integrates transferred DNA into the host chromosome for permanent expression, while transient transfection expresses DNA temporarily without integration.
DNA construct instability in bacteria used for Agrobacterium mediated plant t...iosrjce
This document summarizes a study on the instability of DNA constructs in bacteria used for Agrobacterium-mediated plant transformation. The researchers evaluated the stability of a plasmid (p8114) carrying genes for a transcription factor and antibiotic resistance in E. coli and different Agrobacterium strains. They found 16-100% instability in E. coli colonies stored at -80°C, with rearrangements observed. When transformed into Agrobacterium strains, p8114 showed 50-100% instability. Specifically, LBA4404 had 25-30% instability, EHA105 was 100% unstable, and AGL1 was 50% unstable. The results demonstrate plasmid and DNA construct instability in bacteria, which could compromise
This document describes the development of an overexpression system for the freshwater sponge Ephydatia muelleri. The author identifies putative promoter regions in the E. muelleri genome and amplifies the promoters for EF1α, Piwi and DAD genes. Expression vectors are constructed containing these promoters driving GFP expression. Sponges are collected and cultured to test the overexpression constructs. The goal is to generate a tool to induce targeted gene expression in E. muelleri and gain insights into early animal development and gene regulation.
Fluorescence activated cell sorted assay for Gaucher's diseaseMayank Sagar
Gaucher disease results from mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GC) (D-glucosyl-acylsphingosine glucohydrolase, and can be divided into three clinical types on the basis of the presence and severity of neurological involvement. Gaucher disease results from mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GC) (D-glucosyl-acylsphingosine glucohydrolase, and can be divided into three clinical types on the basis of the presence and severity of neurological involvement.
In the case of type 1 Gaucher disease, deficient GC activity leads to an accumulation of the catabolic intermediate glucocerebroside, primarily in macrophages of the reticuloendothelial.
- β-glucuronidase (GUS) is a commonly used reporter gene in plant molecular biology and genetic engineering to indicate successful introduction of foreign DNA into cells.
- GUS expression can be detected through fluorometric or histochemical assays, allowing visualization of promoter activity, protein localization, and transgenic events.
- The GUS gene is fused to genes of interest, and GUS activity is used to study processes like tissue-specific expression, response to stresses, and transformation efficiency.
- While destructive, GUS is a stable and non-toxic reporter enabling versatile applications in fundamental and applied plant research.
This study investigated the role of glycerol biosynthesis in lifespan extension from hyperosmotic conditions in C. elegans. The authors found that both 10x peptone and 5% sorbitol conditions increased wild type C. elegans lifespan by around 35%, but this lifespan extension was absent in gpdh-1;gpdh-2 mutant worms, which are unable to accumulate glycerol. Future experiments will determine if overexpressing glycerol biosynthesis is sufficient to extend lifespan without osmotic stress. This supports the role of glycerol accumulation, mediated by glycerol 3-phosphate dehydrogenase, in the longevity response to hyperosmotic stress in C. elegans.
This study used zymography to detect proteolytic enzymes in wheat leaves. One-dimensional (1D) zymography revealed 7 bands of proteolytic activity, which were assigned to proteinase families using specific inhibitors. Two-dimensional (2D) zymography provided additional separation of proteases according to isoelectric point, revealing multiple isoforms within families. Metalloproteinases were detected as a 150 kDa band inhibited by EDTA. Aspartic proteinases appeared as a 115-118 kDa double band inhibited by pepstatin. Serine and cysteine proteinases of various sizes were also observed. 2D zymography enabled visualization of isoforms within proteinase families.
Lab: Differential Expression Differential gene expression provides the ability for a cell or
organism to respond to a constantly changing external environment. The specific constellation of
proteins required for optimal function and growth varies with cellular age and environmental
context. Thus, protein production is carefully regulated by multiple mechanisms that modulate
both transcriptional and translational pathways. Control of transcription initiation by RNA
polymerase is a predominant mechanism for regulating expression of specific proteins,
presumably because it provides maximal conservation of energy for the cell. We can often
observe the consequence of differential transcription due to the presence or absence of particular
proteins or the growth in particular environments. Control can also occur at translation; the
mRNA is synthesized, but only in certain circumstances is it translated. Control can also occur at
the level of protein function; the protein is inactive, or activity is not observed due to the lack of
the substrate. In this lab we will observe differential expression of two different genes encoded
on plasmids. We will analyze transcriptional activity, translational activity, and protein function.
Plasmids are extra-chromosomal DNA. Bacteria often have plasmids and will replicate the
plasmid and pass it to daughter cells (vertical transmission) and to neighboring cells (horizontal).
Plasmids are a mechanism of gene diversity. In order to stably retain the plasmid, there needs to
be some type of metabolic reason for the bacteria to maintain the plasmid. In other words, the
plasmid confers an advantage. Plasmids contain: 1. Ori: the plasmid may present is low or high
copy number. 2. Lab generated plasmids typically also contain a selectable marker (antibiotic
resistance), 3. Additional gene for ease of visual screening 4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant plasmids. The transformed cells containing the plasmid with the gene of interest ca.
Lab: Differential Expression Differential gene expression provides the ability for a cell or
organism to respond to a constantly changing external environment. The specific constellation of
proteins required for optimal function and growth varies with cellular age and environmental
context. Thus, protein production is carefully regulated by multiple mechanisms that modulate
both transcriptional and translational pathways. Control of transcription initiation by RNA
polymerase is a predominant mechanism for regulating expression of specific proteins,
presumably because it provides maximal conservation of energy for the cell. We can often
observe the consequence of differential transcription due to the presence or absence of particular
proteins or the growth in particular environments. Control can also occur at translation; the
mRNA is synthesized, but only in certain circumstances is it translated. Control can also occur at
the level of protein function; the protein is inactive, or activity is not observed due to the lack of
the substrate. In this lab we will observe differential expression of two different genes encoded
on plasmids. We will analyze transcriptional activity, translational activity, and protein function.
Plasmids are extra-chromosomal DNA. Bacteria often have plasmids and will replicate the
plasmid and pass it to daughter cells (vertical transmission) and to neighboring cells (horizontal).
Plasmids are a mechanism of gene diversity. In order to stably retain the plasmid, there needs to
be some type of metabolic reason for the bacteria to maintain the plasmid. In other words, the
plasmid confers an advantage. Plasmids contain: 1. Ori: the plasmid may present is low or high
copy number. 2. Lab generated plasmids typically also contain a selectable marker (antibiotic
resistance), 3. Additional gene for ease of visual screening 4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant plasmids. The transformed cells containing the plasmid with the gene of interest ca.
This document describes an improved method for quantitative transcript profiling using cDNA-AFLP (cDNA amplified fragment length polymorphism). The key improvements allow it to be used as an efficient tool for genome-wide expression analysis as an alternative to microarrays. Unique transcript tags are generated from mRNA and screened through selective PCR amplifications. Based on in silico analysis, the enzyme combination BstYI and MseI was chosen to represent at least 60% of transcripts. The method was able to accurately detect differentially expressed genes and subtle expression differences. It was demonstrated to be useful by screening for cell cycle-modulated genes in tobacco.
A panel of recombinant monoclonal antibodies against zebrafishShahnaz Yusaf
This document describes the development of 10 recombinant monoclonal antibodies against neural receptors and secreted proteins in zebrafish. The antibodies were generated by expressing the extracellular domains of the target proteins in mammalian cells and using them as antigens. The antibodies were characterized, cloned into expression plasmids, and shown to specifically stain their antigens in fixed zebrafish embryo tissues. The staining patterns matched the known expression patterns of the target genes, demonstrating these antibodies will be useful tools for studying neural development in zebrafish.
Similar to Purification of G-Protein Coupled Receptor from Membrane Cell of Local Strain of Saccharomyces cerevisiae (20)
An Examination of Effectuation Dimension as Financing Practice of Small and M...iosrjce
IOSR Journal of Business and Management (IOSR-JBM) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of business and managemant and its applications. The journal welcomes publications of high quality papers on theoretical developments and practical applications inbusiness and management. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Does Goods and Services Tax (GST) Leads to Indian Economic Development?iosrjce
IOSR Journal of Business and Management (IOSR-JBM) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of business and managemant and its applications. The journal welcomes publications of high quality papers on theoretical developments and practical applications inbusiness and management. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Childhood Factors that influence success in later lifeiosrjce
IOSR Journal of Business and Management (IOSR-JBM) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of business and managemant and its applications. The journal welcomes publications of high quality papers on theoretical developments and practical applications inbusiness and management. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Emotional Intelligence and Work Performance Relationship: A Study on Sales Pe...iosrjce
IOSR Journal of Business and Management (IOSR-JBM) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of business and managemant and its applications. The journal welcomes publications of high quality papers on theoretical developments and practical applications inbusiness and management. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Customer’s Acceptance of Internet Banking in Dubaiiosrjce
IOSR Journal of Business and Management (IOSR-JBM) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of business and managemant and its applications. The journal welcomes publications of high quality papers on theoretical developments and practical applications inbusiness and management. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
A Study of Employee Satisfaction relating to Job Security & Working Hours amo...iosrjce
IOSR Journal of Business and Management (IOSR-JBM) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of business and managemant and its applications. The journal welcomes publications of high quality papers on theoretical developments and practical applications inbusiness and management. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Consumer Perspectives on Brand Preference: A Choice Based Model Approachiosrjce
IOSR Journal of Business and Management (IOSR-JBM) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of business and managemant and its applications. The journal welcomes publications of high quality papers on theoretical developments and practical applications inbusiness and management. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Student`S Approach towards Social Network Sitesiosrjce
IOSR Journal of Business and Management (IOSR-JBM) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of business and managemant and its applications. The journal welcomes publications of high quality papers on theoretical developments and practical applications inbusiness and management. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Broadcast Management in Nigeria: The systems approach as an imperativeiosrjce
IOSR Journal of Business and Management (IOSR-JBM) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of business and managemant and its applications. The journal welcomes publications of high quality papers on theoretical developments and practical applications inbusiness and management. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
A Study on Retailer’s Perception on Soya Products with Special Reference to T...iosrjce
IOSR Journal of Business and Management (IOSR-JBM) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of business and managemant and its applications. The journal welcomes publications of high quality papers on theoretical developments and practical applications inbusiness and management. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
A Study Factors Influence on Organisation Citizenship Behaviour in Corporate ...iosrjce
IOSR Journal of Business and Management (IOSR-JBM) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of business and managemant and its applications. The journal welcomes publications of high quality papers on theoretical developments and practical applications inbusiness and management. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Consumers’ Behaviour on Sony Xperia: A Case Study on Bangladeshiosrjce
IOSR Journal of Business and Management (IOSR-JBM) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of business and managemant and its applications. The journal welcomes publications of high quality papers on theoretical developments and practical applications inbusiness and management. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Design of a Balanced Scorecard on Nonprofit Organizations (Study on Yayasan P...iosrjce
1. The document describes a study that designed a balanced scorecard for a nonprofit organization called Yayasan Pembinaan dan Kesembuhan Batin (YPKB) in Malang, Indonesia.
2. The balanced scorecard translated YPKB's vision and mission into strategic objectives across four perspectives: financial, customer, internal processes, and learning and growth.
3. Key strategic objectives included donation growth, budget effectiveness, customer satisfaction, reputation, service quality, innovation, and employee development. Customers perspective had the highest weighting, suggesting a focus on public service over financial growth.
Public Sector Reforms and Outsourcing Services in Nigeria: An Empirical Evalu...iosrjce
IOSR Journal of Business and Management (IOSR-JBM) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of business and managemant and its applications. The journal welcomes publications of high quality papers on theoretical developments and practical applications inbusiness and management. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Media Innovations and its Impact on Brand awareness & Considerationiosrjce
IOSR Journal of Business and Management (IOSR-JBM) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of business and managemant and its applications. The journal welcomes publications of high quality papers on theoretical developments and practical applications inbusiness and management. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Customer experience in supermarkets and hypermarkets – A comparative studyiosrjce
- The document examines customer experience in supermarkets and hypermarkets in India through a survey of 418 customers.
- It finds that in supermarkets, previous experience, atmosphere, price, social environment and experience in other channels most influence customer experience, while in hypermarkets, previous experience, product assortment, social environment and experience in other channels are most influential.
- The study provides insights for retailers on key determinants of customer experience in each format to help them improve strategies and competitive positioning.
Social Media and Small Businesses: A Combinational Strategic Approach under t...iosrjce
IOSR Journal of Business and Management (IOSR-JBM) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of business and managemant and its applications. The journal welcomes publications of high quality papers on theoretical developments and practical applications inbusiness and management. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Secretarial Performance and the Gender Question (A Study of Selected Tertiary...iosrjce
IOSR Journal of Business and Management (IOSR-JBM) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of business and managemant and its applications. The journal welcomes publications of high quality papers on theoretical developments and practical applications inbusiness and management. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Implementation of Quality Management principles at Zimbabwe Open University (...iosrjce
This document discusses the implementation of quality management principles at Zimbabwe Open University's Matabeleland North Regional Centre. It begins with background information on ZOU and the importance of quality management in open and distance learning institutions. The study aimed to determine if quality management and its principles were being implemented at the regional centre. Key findings included that the centre prioritized customer focus and staff involvement. Decisions were made based on data analysis. The regional centre implemented a quality system informed by its policy documents. The document recommends ensuring staffing levels match needs and providing sufficient resources to the regional centre.
Organizational Conflicts Management In Selected Organizaions In Lagos State, ...iosrjce
IOSR Journal of Business and Management (IOSR-JBM) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of business and managemant and its applications. The journal welcomes publications of high quality papers on theoretical developments and practical applications inbusiness and management. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
The binding of cosmological structures by massless topological defectsSérgio Sacani
Assuming spherical symmetry and weak field, it is shown that if one solves the Poisson equation or the Einstein field
equations sourced by a topological defect, i.e. a singularity of a very specific form, the result is a localized gravitational
field capable of driving flat rotation (i.e. Keplerian circular orbits at a constant speed for all radii) of test masses on a thin
spherical shell without any underlying mass. Moreover, a large-scale structure which exploits this solution by assembling
concentrically a number of such topological defects can establish a flat stellar or galactic rotation curve, and can also deflect
light in the same manner as an equipotential (isothermal) sphere. Thus, the need for dark matter or modified gravity theory is
mitigated, at least in part.
The use of Nauplii and metanauplii artemia in aquaculture (brine shrimp).pptxMAGOTI ERNEST
Although Artemia has been known to man for centuries, its use as a food for the culture of larval organisms apparently began only in the 1930s, when several investigators found that it made an excellent food for newly hatched fish larvae (Litvinenko et al., 2023). As aquaculture developed in the 1960s and ‘70s, the use of Artemia also became more widespread, due both to its convenience and to its nutritional value for larval organisms (Arenas-Pardo et al., 2024). The fact that Artemia dormant cysts can be stored for long periods in cans, and then used as an off-the-shelf food requiring only 24 h of incubation makes them the most convenient, least labor-intensive, live food available for aquaculture (Sorgeloos & Roubach, 2021). The nutritional value of Artemia, especially for marine organisms, is not constant, but varies both geographically and temporally. During the last decade, however, both the causes of Artemia nutritional variability and methods to improve poorquality Artemia have been identified (Loufi et al., 2024).
Brine shrimp (Artemia spp.) are used in marine aquaculture worldwide. Annually, more than 2,000 metric tons of dry cysts are used for cultivation of fish, crustacean, and shellfish larva. Brine shrimp are important to aquaculture because newly hatched brine shrimp nauplii (larvae) provide a food source for many fish fry (Mozanzadeh et al., 2021). Culture and harvesting of brine shrimp eggs represents another aspect of the aquaculture industry. Nauplii and metanauplii of Artemia, commonly known as brine shrimp, play a crucial role in aquaculture due to their nutritional value and suitability as live feed for many aquatic species, particularly in larval stages (Sorgeloos & Roubach, 2021).
Current Ms word generated power point presentation covers major details about the micronuclei test. It's significance and assays to conduct it. It is used to detect the micronuclei formation inside the cells of nearly every multicellular organism. It's formation takes place during chromosomal sepration at metaphase.
Authoring a personal GPT for your research and practice: How we created the Q...Leonel Morgado
Thematic analysis in qualitative research is a time-consuming and systematic task, typically done using teams. Team members must ground their activities on common understandings of the major concepts underlying the thematic analysis, and define criteria for its development. However, conceptual misunderstandings, equivocations, and lack of adherence to criteria are challenges to the quality and speed of this process. Given the distributed and uncertain nature of this process, we wondered if the tasks in thematic analysis could be supported by readily available artificial intelligence chatbots. Our early efforts point to potential benefits: not just saving time in the coding process but better adherence to criteria and grounding, by increasing triangulation between humans and artificial intelligence. This tutorial will provide a description and demonstration of the process we followed, as two academic researchers, to develop a custom ChatGPT to assist with qualitative coding in the thematic data analysis process of immersive learning accounts in a survey of the academic literature: QUAL-E Immersive Learning Thematic Analysis Helper. In the hands-on time, participants will try out QUAL-E and develop their ideas for their own qualitative coding ChatGPT. Participants that have the paid ChatGPT Plus subscription can create a draft of their assistants. The organizers will provide course materials and slide deck that participants will be able to utilize to continue development of their custom GPT. The paid subscription to ChatGPT Plus is not required to participate in this workshop, just for trying out personal GPTs during it.
EWOCS-I: The catalog of X-ray sources in Westerlund 1 from the Extended Weste...Sérgio Sacani
Context. With a mass exceeding several 104 M⊙ and a rich and dense population of massive stars, supermassive young star clusters
represent the most massive star-forming environment that is dominated by the feedback from massive stars and gravitational interactions
among stars.
Aims. In this paper we present the Extended Westerlund 1 and 2 Open Clusters Survey (EWOCS) project, which aims to investigate
the influence of the starburst environment on the formation of stars and planets, and on the evolution of both low and high mass stars.
The primary targets of this project are Westerlund 1 and 2, the closest supermassive star clusters to the Sun.
Methods. The project is based primarily on recent observations conducted with the Chandra and JWST observatories. Specifically,
the Chandra survey of Westerlund 1 consists of 36 new ACIS-I observations, nearly co-pointed, for a total exposure time of 1 Msec.
Additionally, we included 8 archival Chandra/ACIS-S observations. This paper presents the resulting catalog of X-ray sources within
and around Westerlund 1. Sources were detected by combining various existing methods, and photon extraction and source validation
were carried out using the ACIS-Extract software.
Results. The EWOCS X-ray catalog comprises 5963 validated sources out of the 9420 initially provided to ACIS-Extract, reaching a
photon flux threshold of approximately 2 × 10−8 photons cm−2
s
−1
. The X-ray sources exhibit a highly concentrated spatial distribution,
with 1075 sources located within the central 1 arcmin. We have successfully detected X-ray emissions from 126 out of the 166 known
massive stars of the cluster, and we have collected over 71 000 photons from the magnetar CXO J164710.20-455217.
Travis Hills' Endeavors in Minnesota: Fostering Environmental and Economic Pr...Travis Hills MN
Travis Hills of Minnesota developed a method to convert waste into high-value dry fertilizer, significantly enriching soil quality. By providing farmers with a valuable resource derived from waste, Travis Hills helps enhance farm profitability while promoting environmental stewardship. Travis Hills' sustainable practices lead to cost savings and increased revenue for farmers by improving resource efficiency and reducing waste.
Remote Sensing and Computational, Evolutionary, Supercomputing, and Intellige...University of Maribor
Slides from talk:
Aleš Zamuda: Remote Sensing and Computational, Evolutionary, Supercomputing, and Intelligent Systems.
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Inter-Society Networking Panel GRSS/MTT-S/CIS Panel Session: Promoting Connection and Cooperation
https://www.etran.rs/2024/en/home-english/
When I was asked to give a companion lecture in support of ‘The Philosophy of Science’ (https://shorturl.at/4pUXz) I decided not to walk through the detail of the many methodologies in order of use. Instead, I chose to employ a long standing, and ongoing, scientific development as an exemplar. And so, I chose the ever evolving story of Thermodynamics as a scientific investigation at its best.
Conducted over a period of >200 years, Thermodynamics R&D, and application, benefitted from the highest levels of professionalism, collaboration, and technical thoroughness. New layers of application, methodology, and practice were made possible by the progressive advance of technology. In turn, this has seen measurement and modelling accuracy continually improved at a micro and macro level.
Perhaps most importantly, Thermodynamics rapidly became a primary tool in the advance of applied science/engineering/technology, spanning micro-tech, to aerospace and cosmology. I can think of no better a story to illustrate the breadth of scientific methodologies and applications at their best.
Purification of G-Protein Coupled Receptor from Membrane Cell of Local Strain of Saccharomyces cerevisiae
1. IOSR Journal of Applied Chemistry (IOSR-JAC)
e-ISSN: 2278-5736.Volume 8, Issue 11 Ver. I (Nov. 2015), PP 60-65
www.iosrjournals.org
DOI: 10.9790/5736-081116065 www.iosrjournals.org 60 |Page
Purification of G-Protein Coupled Receptor from Membrane Cell
of Local Strain of Saccharomyces cerevisiae
,Zeinab M. M. Al-Rubaei1
,Tariq A. Al-Hakeem1
,Rebah N.Algafari 2
. Noorhan
K. Shafeeq1
*
1
Chemistry Department, College of Education for pure Science, Baghdad University
2
Biotechnology Research Center ,Al-Nahria University
Abstract: The aim of this study to purify GPCR from a local strain of S. cerevisiae using gel filtration
chromatography techniques , by packing materials for columns which will be chosen of low cost comparing to
the already used in published researches, which depend on the costly affinity chromatography and other
expensive methods of purification. Local strain of S. cerevisiae chosen for extraction and purification of G-
protein coupled receptor (GPCR) .The strains were obtained from biology department in Al- Mosul University,
Iraq. The isolated colony was activated on Yeast Extract Pepton Dextrose Broth (YEPDB) and incubated at 30
C˚ for 24 h .Loop fully of the yeast culture was transferred to (10ml) of yeast extract peptone glucose agar
(YEPGA) slant , then incubated at 30C˚for 24h , after that it was stored at 4C˚ ,the yeast cultures were
reactivated and persevered after each two weeks period. S.cerevisiae was identified by morphological,
microscopic characterization and biochemical test . The GPCR that extract from membrane of S.cerevisiae was
purified by gel filtration chromatography in two steps using Sepharose 6B. The optical density for each fraction
was measured at 280 nm by UV-VS spectrophotometer then the GPCR concentration was determined by using
ELISA Kit . The fractions which gave the highest absorbance and concentration of GPCR were collected .The
molecular weight of GPCR was determined by gel filtration chromatography using blue dextrin solution.
Standard curve was plotted between log of molecular weight for standard protein and the ratio of Ve/Vo of
GPCR . The purity of the GPCR that extracted and purified from whole cell of S, cerevisiae were carried out by
using SDS-PAGE electrophoresis In the first step 5ml of crude extract was applied on sepharose 6B column
(1.6x 96 cm) which previously equilibrated with 50 mM phosphate buffer saline pH= 7.4 . Multiple proteins
peaks appeared after elution with elution buffer (PBS PH= 7.4 containing 0. 5 % DDM). One peak only give
positive result with GPCR assay, fractions representing GPCR were collected , pooled and concentrated by
sucrose. In the second step five active fractions from the previous step were collected and applied once again on
the same column and same conditions. This step gave a single peak that was identical with the peak of GPCR
concentration ,maximum concentration of GPCR that observed in the fractions (34-38) was 18.541 (ng/ml) . The
specific activity for these fractions was 261.14 (ng/mg) protein with yield of 47.717%. The present study a chive
a relatively high purification of GPCR from membrane fraction of a local strain S. cerevisiae with fold
purification 5.094 and a yield of 47.717%. and molecular weight about~55KD.
Key words: GPCR purification, S.Cerevisea, membrane cell
I. Introduction
G-protein coupled receptors (GPCRs) are integral membrane proteins characterized by seven
transmembrane helices and comprise one of the largest known superfamilies of receptors with in excess of 2000
genes identified across taxa [1]. They are attractive and proven drug targets in a wide range of therapeutic areas
due to their involvement in signaling and response to diverse external stimuli in nearly all human cell types [2].
Bacterial, yeast, and insect hosts have successfully been implemented for high-level expression of
soluble proteins, and similar approaches have been applied toward the expression of membrane proteins[3] .
Although all of these systems have proven useful for expression of some heterologous membrane
proteins, each host has advantages and disadvantages associated with its use. For instance, microbial hosts such
as E. coli and yeast offer well-understood genetics, low cost of culture, and relatively easy scale up. Insect and
mammalian cells can perform many more complicated protein processing and post-translational modifications
that may be necessary for proper function, but prove costly and time-intensive. However, even with several host
systems available, heterologous expression has not yet systematically allowed for highlevel expression of any
given GPCR of interest, and typically relies on trial-and-error methods. [4]
Even though all GPCRs share a commonality in their seven transmembrane domain segments and in
their ability to couple to trimeric G-proteins, they also display great diversity in their overall function, ligand
preference, tissue location, and physiological prevalence [5] .
Furthermore, significant differences exist in how proteins are expressed and processed in various
heterologous systems, which may have a direct impact upon folding and activity of heterologously expressed
2. Purification of G-Protein Coupled Receptor from Membrane Cell of Local Strain of ….
DOI: 10.9790/5736-081116065 www.iosrjournals.org 61 |Page
GPCRs. Given its ease of genetic manipulation, rapid growth, and eukaryotic secretory pathway, yeast are an
attractive host system for the development of a robust GPCR expression system. Yeast have been successfully
used for the heterologous expression of membrane proteins, specifically GPCRs. [6]
Three GPCRs are known in S. cerevisiae included a-factor receptor (Ste2), ∝- factor receptor (Ste3)
and Gpr1 . Although Ste2 and Ste3 are both coupled to Gpr1 and activate the mating pathway ,the sequence
similarity between them is limited [7]
Both haploid yeast cells types (∝ and a) express mating -type specific gene products such as the ∝ -
factor pheromone and the Ste2 in a- cells , and the ∝- factor and Set3 in ∝- cells. Pheromone binding to either
receptor stimulates the exchange of GDP to GTP on the protein Gpa1 , which in turn dissociates from the
dimmer , consisting of Ste4 and Ste18 The Ste 4 - Ste 18 dimmer transmits the signal to Ste 20 , the first
member of the activated protein kinase family , which activates a MAP -kinase cascade [8]
This process that is similar to hormone desensitization in mammalian cells . At the level of the receptor
Set2 and Set 3 are down regulated by hyperphosphorylation of several C-terminal residues , followed by
ubiquitylation , internalization and degradation . At the level of the G∝ protein ,GPa1, desensitization depends
on the GTPase stimulating protein which is a member of the RGS -protein family[9].
Astudy using a native S. cerevisiae expression system ,1 mg quantities of His-epitope tagged α-factor
receptor was purified on a Ni-NTA column and reconstituted in lipid vesicles. The binding activity of the
reconstituted receptor indicated that only 6 % of the receptor was capable of ligand binding, but the addition of
solubilized membranes from S. cerevisiae to the artificial membrane restored most of expected ligand binding
activity (at least 80 %). Nevertheless, the co-factors for this effect were not identified despite extensive efforts.
In another study, α-factor receptor was purified using a transient expression system in HEK293 EBNA cell line .
About 1 mg of Ste2p was purified per liter of culture with relatively high affinity to a fluorescently-labeled α-
factor. However, for large scale purification a stable expression system is required, and the heterogeneous
glycosylation of the recombinant protein in a mammalian system may interfere with the formation of
diffractable crystals. As almost all human GPCRs do not exist naturally in high abundance, heterologous
expression systems are required to achieve sufficient protein yields for structural characterization, generally at
the mg/L scale or greater. [10].
Recent expression of the human adenosine A2a receptor (hA2aR) in S. cerevisiae has yielded active
protein at greater than 10 mg/L of culture, which has facilitated its purification[11] Other studies have also cited
improper trafficking of recombinant membrane proteins in yeast [12]
The aim of this study to purify GPCR from a local strain of S. cerevisiae using gel filtration
chromatography techniques , by packing materials for columns which will be chosen of low cost comparing to
the already used in published researches, which depend on the costly affinity chromatography and other
expensive methods of purification.
II. Materials and Methods
Local strain of S. cerevisiae obtained from department of Biology / College of Science / AL Mosul
University. The isolated colony was activated on YEPGB and incubated at 30 C˚ for 24 h.
Loop fully of the yeast culture was transferred to (10ml) of YEPGA slant , then incubated at 30 C˚ for
24h , After that it was stored at 4C˚ .The yeast cultures were reactivated and persevered after each two weeks
period[13].
S.cerevisiae was identified by morphological, microscopical characterization and biochemical test.
The pellet cells were thawed and resuspended in solubilization buffer (50 mM PBS buffer , PH = 8.0 ,
100mM NaCl , 5mM MgCl2 , 1mM AEBSF) and protease inhibitor cocktail (us 10 µl for 1ml) with n-Dodecyl-
β-D-maltoside (DDM) (1%) and 5mM β-mercaptoethanol were added to the lysed sample with gentle swirling
on ice [10] . and agitation with sterilized beads (0.4-0.5 mµ) by using vortex mixer several cycles of agitation
(30-60 sec) was interspersed with cycles of cooling on ice[14] ,
The suspension was removed by pasteur pipte and suspension examined under light microscope (100X)
, the disruption of the cell wall was observed, the suspension was centrifuged again at 5000 rpm to remove
debris and remaining suspension was centrifuged at 25000 rpm for 120 min by ultra cooling centrifuge.
The Final membrane pellet was washed and resuspended in solubilization buffer (50 mM PBS buffer ,
PH = 8.0 , 100mM NaCl , 5mM MgCl2 , 1mM AEBSF) and stirred on ice for 1 hour and centrifuged at 14000
rpm([10] .
Gel filtration Chromatography was used in purification of GPCR from membrane fraction in two steps.
Sepharose 6B which washed several times with 50mM PBS (PH = 7.4) , degassed by vacuum pump to
remove the air bubbles and poured gradually in a column by using glass rod to avoid forming bubbles . Gel was
left to settle down and packed well to give column with (96 x 1.5) cm. The gel was equilibrated with 50 mM
PBS (PH = 7.4) .
3. Purification of G-Protein Coupled Receptor from Membrane Cell of Local Strain of ….
DOI: 10.9790/5736-081116065 www.iosrjournals.org 62 |Page
The GPCR crude that prepared by solubilization of membrane was applied to the sepharose 6B column
as a second step of purification that was pre equilibrated with 50 mM PBS PH = 7.4 , the GPCR crude was
eluted from the column by elution buffer( PBS PH=7.4 with 0.5%DDM). Aliquot of 5ml for each fraction was
collected with flow rate (0.5 ml/min).The fractions that gave the highest absorbance at 280 nm and GPCR
concentration were collected. Then ,these fractions are concentrated by sucrose using dialysis tube.
The concentrated GPCR from first step was applied to the sepharose 6B column that pre equilibrated
by 50 mM PBS (PH = 7.4) , and eluted with elution buffer . Aliquot of fraction were collected in each tube with
flow rate of 0.5 ml/min and optical density were determined for each collected fraction at 280 nm by UV-VIS
spectrophotometer. The fractions that gave the highest absorbance were collected , protein concentration
measured by using Bradford method . The GPCR concentration was determined by using ELISA Kit from
BlueGene Biotech, Shanghai, China for all steps of purification. The fractions which gave the highest
absorbance and concentration of GPCR were collected.
The molecular weight of GPCR was determined by gel filtration chromatography using blue dextrin
solution .Standard curve was plotted between log of molecular weight for standard protein and the ratio of
Ve/Vo of GPCR [15].
The purity of the GPCR that extracted and purified from cell membrane of S. cerevisiae were carried
out by using SDS-PAGE electrophoresis[16].
III. Result and discussion
The local strain of S.cerevisiae were identified by studying specific microscopically, morphological
and biochemical characterization[17].
The ability of isolate S.cerevisea for fermentation and assimilation was examined ,which glucose,
fructose, sucrose, galactose, lactose, maltose, raffinose were used. The results illustrated that glucose, fructose,
sucrose, galactose, , maltose, raffinose) were fermented and assimilated by the isolate strain while lactose was
not fermented and assimilated by this isolate. The isolate also show un ability to hydrolyzed urea and produce
ammonia. The characters are inagreement with previous study[18].
Yeast cells were thawed and resuspended in solubiziation buffer and transferred to tube containing
acid – washed , dried , and the cells were lyses by shaking with glass beads. All steps performed at 4C˚and
butters were supplemented with protease inhibitors cocktail in phosphate buffer[19].
GPCR extracted from membrane of S. cerevisiae was solubilized and purified by gel filtration
chromatography in two steps. In the first step 5ml of crude extract was applied on sepharose 6B column (1.5x
96 cm) which previously equilibrated with 50 mM phosphate buffer saline pH= 7.4.
Results in figure (1) illustrates multiple proteins peaks appeared after elution with elution buffer (PBS
PH= 7.4 containing 0. 5 % DDM). One peak only give positive result with GPCR assay. Fractions representing
GPCR were collected , pooled and concentrated by sucrose.
Figure( 1):Purification of GPCR by Gel Filtration Chromatography , first step.
In the second step five active fractions from the previous step were collected and applied once again on
the same column and same conditions. This step gave a single. peak that was identical with the peak of GPCR
concentration as shown in figure (2)
4. Purification of G-Protein Coupled Receptor from Membrane Cell of Local Strain of ….
DOI: 10.9790/5736-081116065 www.iosrjournals.org 63 |Page
Figure( 2):Purification of GPCR by Gel Filtration Chromatography, second step.
Table (1) represented volume ,protein concentration, GPCR concentration , specific activity
,purification fold and yield for all purification steps of GPCR from membrane of isolated S. cerevisiae The
maximum concentration of GPCR that observed in the fractions (34-38) was 18.541 (ng/ml) . The specific
activity for these fractions was 261.14 (ng/mg) protein with yield of 47.717%.
Table (1): Volume ,protein concentration, GPCR concentration , specific activity ,purification fold and yield
for all purification steps of GPCR from membrane of isolated S. cerevisiae
The molecular weight is determined by gel filtration in sepharose -6B based on the standared curve
made by standard protein figure (3). The Blue Dextran 2000 was used as a guide for assessing the correct
column packaging and the astimation of void volume ,In this study the curve plotted between absorbance at 600
nm fraction number and the resolution of one identical peak indicated the correct column packaging . Five ml
of the concentrated active fraction by sucrose obtained in the gel filtration monitored for absorbance at 280
and for GPCR concentration was used for estimation of molecular weight of GPCR by gel filtration
chromatography with the aid of fraction of standard protein, showed that the resolute protein(~54.950KD ) for
GPCR extract from membrane of S.cerevisiae.
The molecular weight and purity of GPCR was determined by sodium dodecyl sulfate -
polyacrylamide gel electrophoresis (SDS-PAGE) as shown in the figure (3). The molecular weight of GPCR
that extracted from solubilization membrane fraction of S.cerevisiae was( ~ 55 KD) with single band.
yield
(%)
Purification
fold
Specific
Activity
(ng/mg)
GPCR
Conc.GPCR
(ng/ml)
ProteinConc.
)mg/ml)
volume
(ml)
)
StepsSteps
100151.2635.370.6910
Crude GPCR
87.882.893148.3019.4280.13116
Gel filtration
First step
47.7175.094261.1418.5410.0718
Gel filtration
Second step
5. Purification of G-Protein Coupled Receptor from Membrane Cell of Local Strain of ….
DOI: 10.9790/5736-081116065 www.iosrjournals.org 64 |Page
Figure (3): Estimation of molecular weight of GPCR by gel filtration
Figure (4): Polyacrylamide gel electrophoresis of purified GPCR. crude( M): Membrane fraction
From previous reports, the isolated of membrane protein from S, cerevisiae cloned in eukaryotic
cell found to be easy extracted due to membrane proteins produced in S. cerevisiae is challenging, and the
limited number of membrane protein structure is due to difficulties encountered with production, solubilization
and purification of appropriate amounts of membrane proteins that are able to from crystals diffracting at a
high resolution [20,21].
6. Purification of G-Protein Coupled Receptor from Membrane Cell of Local Strain of ….
DOI: 10.9790/5736-081116065 www.iosrjournals.org 65 |Page
In previous study, the alfa – factor , which is a 13- amino acid residue- long peptide agonist of the
yeast pheromone response pathway, was successfully displayed on the yeast plasma membrane. Signal
activation was observed by employing a fluorescent reporter gene assay. Extended application of this system to
human GPCRs which comprise one of the most important types of drug targets[22].
The range of application of technology for displaying peptide ligands on yeast plasma membranes
namely" pep display" was extended in a recent study to activation human GPCR that was heterologous
produced in S. cerevisiae. The methodology presented in that study could be useful for identifying novel
peptide ligands for both liganded and orphan mammalian GPCRs .The authors suggest that plasma membrane is
more suitable than cell wall in terms of peptide- ligand display. The higher accessibility of membrane- displayed
ligands to GPCRs may be important for efficient activation[23]..
Other recent study demonstrated the production of large quantities of high quality eukaryotic
membrane proteins in S. cerevisiae , a high- copy vector was modified to express membrane proteins C-
terminally – fused to a tobacco protease detachable[24].
The conclusion from this study is The present study a chive a relatively high purification of GPCR
from cell membrane of S. cerevisiae with fold purification 5.094 and a yield of 47.717%.and molecular weight
about ~55KD .
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