Extraction and purification of product from fermentation is known as Downstream Processing ( DSP) or Product Recovery
It is an essential step in the manufacture of pharmaceuticals product
Cost of the product is determined by the DSP involved
INTRODUCTION
DEFINITION
HISTORY
TRANSGENIC FISH
METHODS OF GENE TRANSFER
HOW TO MAKE TRANSGENIC FISH
EXAMPLES
APPLICATIONS
TRANSGENIC BIRD
PRODUCTION METHOD
APPLICATIONS
CONCLUSION
REFRENCES
Science and technology of manipulating and improving microbial strains, in order to enhance their metabolic capacities for biotechnological applications, are referred to as strain improvement.
Process scale-up is a critical activity that enables a fermentation process achieved in research and development to operate at a commercially viable scale for manufacturing.
INTRODUCTION
DEFINITION
HISTORY
TRANSGENIC FISH
METHODS OF GENE TRANSFER
HOW TO MAKE TRANSGENIC FISH
EXAMPLES
APPLICATIONS
TRANSGENIC BIRD
PRODUCTION METHOD
APPLICATIONS
CONCLUSION
REFRENCES
Science and technology of manipulating and improving microbial strains, in order to enhance their metabolic capacities for biotechnological applications, are referred to as strain improvement.
Process scale-up is a critical activity that enables a fermentation process achieved in research and development to operate at a commercially viable scale for manufacturing.
Scale up means increasing the quantity or volume of cell culture. For animal cells, the scale up strategies are dependent upon cell types or i.e. whether the cells requires matrix for attachment and growth ( adherent cell culture) or grows freely in suspended form in aqueous media. The scaling up principle for adherent cells are just to increase surface area for attachment while for suspension culture is to increase culture volume. This presentation enlightens the reader about different methods of scaling up of cells culture. Readers are also provided with sample questions for better understanding
A knockout mouse is a mouse in which a specific gene has been inactivated or“knocked out” by replacing it or disrupting it with an artificial piece of DNA.
The loss of gene activity often causes changes in a mouse's phenotype and thus provides valuable information on the function of the gene.
Sheep named Dolly was cloned by transfer of a nucleus from a mammary (Udder) cell of an adult sheep into an egg cell.
mammary cell
Nucleus
insert into
a egg cell
First demonstration of pluripotency (totipotency) of a nucleus of a differentiated adult cell.
Cloning of dolly somatic cell nuclei
clone cattle, sheep, goats, pigs.
nuclear transfer procedures are similar.
Adult donor cells from a variety of cell types(mammary epithelial and ovarian cells, fibroblasts, lymphocytes) are isolated
Cultured and genetically modified methods.
individual donor cells are fused to an enucleated oocyte with short-duration electric pulse.
eg: two 2.5 kilovolt /cm pulses for 10microseconds
Used to fuse adult cattle fibroblasts with enucleated oocytes.
The pulses simultaneously induce cell fusion and oocyte activation.
Blastocyst stage before transferred into the uterus of a pseudopregant female.
Confirmed transgene at the time of birth
Surviving animals produced by nuclear transfer are healthy.
There, is a substantial loss of individual before and after birth some of the cloned animals display abnormalities.
Abnormlities such as increased birth weight.
Dna methylation and histone modification of the original donor cell is inappropriate maintained in the cells of the recipient animals.
Bacterias are fermented in optimal growth conditions in labs and at commercial places to extract various drugs and useful proteins which are genetically engineered or are present inside the bacterias.
to maximize the process certain things are to be considered, which are described in the slide
INTRODUCTION
HISTORY
NEED OF SYNCHRONIZATION
SYNCHRONOUS CULTURES CAN BE OBTAINED IN SEVERAL WAYS:
Physical fractionation .
Chemical appro ach
CENTRIFUGAL ELUTRIATION
Inhibition of DNA synthesis
Nutritional deprivation
SYNCHRONIZATION AT LOW TEMPERATURE
CELLULAR TOTIPOTENCY
SOME HIGHLIGHTS OF CELL SYNCHRONIZATION
REFERENCES
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
Cell culture based vaccine??
Cell cultures involve growing cells in a culture dish, often with a supportive growth medium. A primary cell culture consists of cells taken directly from living tissue, and may contain multiple types of cells such as fibroblasts, epithelial, and endothelial cells.
In the United States, 10 different vaccines for chicken pox, hepatitis A, polio, rabies, and rubella are cultured on aborted tissue from two fetal cell lines known as WI-38 and MRC-5. These vaccines are chicken pox, hep-A, hep-A, hep-A/hep-B, polio, rabies, rubella, measles/rubella, mumps/rubella, and MMR II (measles/mumps/rubella).
Introduction
Primary Culture
Steps In Primary Culture
Isolation Of Tissue
Dissection And/Or Disaggregation
Types Of Primary Culture
Primary Explant Culture
Enzymatic Disaggregation
Mechanical Disaggregation
Cell Line( Finite & Continuous)
Naming A Cell Line
Choosing A Cell Line
Maintenance Of Cell Line
Conclusion
reference
In shotgun sequencing the genome is broken randomly into short fragments (1 to 2 kbp long) suitable for sequencing. The fragments are ligated into a suitable vector and then partially sequenced. Around 400–500 bp of sequence can be generated from each fragment in a single sequencing run. In some cases, both ends of a fragment are sequenced. Computerized searching for overlaps between individual sequences then assembles the complete sequence.
Scale up means increasing the quantity or volume of cell culture. For animal cells, the scale up strategies are dependent upon cell types or i.e. whether the cells requires matrix for attachment and growth ( adherent cell culture) or grows freely in suspended form in aqueous media. The scaling up principle for adherent cells are just to increase surface area for attachment while for suspension culture is to increase culture volume. This presentation enlightens the reader about different methods of scaling up of cells culture. Readers are also provided with sample questions for better understanding
A knockout mouse is a mouse in which a specific gene has been inactivated or“knocked out” by replacing it or disrupting it with an artificial piece of DNA.
The loss of gene activity often causes changes in a mouse's phenotype and thus provides valuable information on the function of the gene.
Sheep named Dolly was cloned by transfer of a nucleus from a mammary (Udder) cell of an adult sheep into an egg cell.
mammary cell
Nucleus
insert into
a egg cell
First demonstration of pluripotency (totipotency) of a nucleus of a differentiated adult cell.
Cloning of dolly somatic cell nuclei
clone cattle, sheep, goats, pigs.
nuclear transfer procedures are similar.
Adult donor cells from a variety of cell types(mammary epithelial and ovarian cells, fibroblasts, lymphocytes) are isolated
Cultured and genetically modified methods.
individual donor cells are fused to an enucleated oocyte with short-duration electric pulse.
eg: two 2.5 kilovolt /cm pulses for 10microseconds
Used to fuse adult cattle fibroblasts with enucleated oocytes.
The pulses simultaneously induce cell fusion and oocyte activation.
Blastocyst stage before transferred into the uterus of a pseudopregant female.
Confirmed transgene at the time of birth
Surviving animals produced by nuclear transfer are healthy.
There, is a substantial loss of individual before and after birth some of the cloned animals display abnormalities.
Abnormlities such as increased birth weight.
Dna methylation and histone modification of the original donor cell is inappropriate maintained in the cells of the recipient animals.
Bacterias are fermented in optimal growth conditions in labs and at commercial places to extract various drugs and useful proteins which are genetically engineered or are present inside the bacterias.
to maximize the process certain things are to be considered, which are described in the slide
INTRODUCTION
HISTORY
NEED OF SYNCHRONIZATION
SYNCHRONOUS CULTURES CAN BE OBTAINED IN SEVERAL WAYS:
Physical fractionation .
Chemical appro ach
CENTRIFUGAL ELUTRIATION
Inhibition of DNA synthesis
Nutritional deprivation
SYNCHRONIZATION AT LOW TEMPERATURE
CELLULAR TOTIPOTENCY
SOME HIGHLIGHTS OF CELL SYNCHRONIZATION
REFERENCES
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
Cell culture based vaccine??
Cell cultures involve growing cells in a culture dish, often with a supportive growth medium. A primary cell culture consists of cells taken directly from living tissue, and may contain multiple types of cells such as fibroblasts, epithelial, and endothelial cells.
In the United States, 10 different vaccines for chicken pox, hepatitis A, polio, rabies, and rubella are cultured on aborted tissue from two fetal cell lines known as WI-38 and MRC-5. These vaccines are chicken pox, hep-A, hep-A, hep-A/hep-B, polio, rabies, rubella, measles/rubella, mumps/rubella, and MMR II (measles/mumps/rubella).
Introduction
Primary Culture
Steps In Primary Culture
Isolation Of Tissue
Dissection And/Or Disaggregation
Types Of Primary Culture
Primary Explant Culture
Enzymatic Disaggregation
Mechanical Disaggregation
Cell Line( Finite & Continuous)
Naming A Cell Line
Choosing A Cell Line
Maintenance Of Cell Line
Conclusion
reference
In shotgun sequencing the genome is broken randomly into short fragments (1 to 2 kbp long) suitable for sequencing. The fragments are ligated into a suitable vector and then partially sequenced. Around 400–500 bp of sequence can be generated from each fragment in a single sequencing run. In some cases, both ends of a fragment are sequenced. Computerized searching for overlaps between individual sequences then assembles the complete sequence.
Next generation biotherapeutics production system Trichoderma reeseiChristopher Landowski
The filamentous fungus Trichoderma reesei is an important production organism used by industrial enzyme companies world-wide. It is a low cost production system that secretes its native enzymes at levels exceeding 100 g/L of culture medium. Several T. reesei produced enzymes have obtained the generally recognized as safe status by the U.S. Food and Drug Administration. T. reesei has tremendous prospects to be a cost efficient and high yield system for producing therapeutic proteins. We have adapted the fungus to become more suitable for biotherapeutic production by reducing secreted protease activity and altering glycosylation pathways needed for adding mammalian glycoforms.
Expression strains for monoclonal antibodies, Fab antibody fragments, interferon alpha-2b, insulin-like growth factor 1, and fibroblast growth factor 21 were constructed, cultivated in bioreactors, and expression levels were measured from the culture medium. After deleting 13 of the most critical protease genes, the general secreted protease activity was reduced over 30-fold. Monoclonal antibodies could be produced up to 7.6 g/L, Fab antibody fragments up to 8.2 g/L, interferon alpha-2b at 7.9 g/L, and insulin-like growth factor fusion protein at 8 g/L. With protease inhibitor treatment interferon alpha-2b could be produced at over 10 g/L, insulin-like growth factor fusion protein at 19 g/L, and full length fibroblast growth factor 21 at 200 mg/L in addition to a shorter form at 3.5 g/L. Human glycoforms such as G0 and FG0 were produced on monoclonal antibodies.
Expression levels and product quality improved dramatically after multiple protease deletions and optimization of culture conditions. While the production levels achieved are already relatively high, the strains could be developed further to reach the 100 g/L potential of the organism. This study demonstrates the excellent prospects of T. reesei as a host for therapeutic protein production.
A reading report for <A Secreted Slit2 Fragment Regulates Adipose Tissue Ther...星云 王
A reading report for <A Secreted Slit2 Fragment Regulates Adipose Tissue Thermogenesis and Metabolic Function
>, only for private study use, please do not use it for profit or public.
Purification of protein involved in Mycobacterium tuberculosis as a potential...AmitSingh65788
Antibiotic resistance poses a significant challenge in the treatment of tuberculosis, requiring the development of new antibiotics with unique molecular mechanisms. In our research, we have identified a specific protein in Mycobacterium tuberculosis as a potential drug target. Let's explore our journey to synthesize and purify this protein.
In order to produce the protein of interest, we employ recombinant DNA technology. This involves isolating and cloning the coding sequence of the protein into an expression plasmid vector. Bacteria such as E. coli are commonly used for this purpose due to their simple manipulation and cost-effectiveness
This is an internship report on molecular biology techniques, which was performed at PERD center under the guidance of Dr. Anshu Srivastava. This pdf contains all the basic information which is a preliminary requisite to know while approaching the molecular biology experimentally.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
2. Introduction
2
Extraction and purification of product from
fermentation is known as Downstream
Processing ( DSP) or Product Recovery
It is an essential step in the manufacture of
pharmaceuticals product
Cost of the product is determined by the DSP
involved
3. [
3 Source - Glick
Upstrea
m
process
Downstream
process
7. Types of Filtration
7
Based on the particle sizes to be separated
Source – Nandakisor et.al.
8. Centrifugation
8
Principle - density differences between the
particles to be separated
Separating solid particles from liquid phase
Source – Nandakisor et.al.
11. Disadvantages
11
Whole cell contents are released out which
makes it difficult to separate out product of
interest from the mixture.
Cell lysis increases viscosity of the solution
making it difficult to process in the further steps.
Product released into harsh environment causing
the product to lose stability or activity.
Enzymatic cell-disruption in large scale can be
expensive
12. 3. Concentration
12
The commonly used techniques for concentrating
biological products are,
Evaporation
Liquid-liquid extraction
Membrane filtration
Precipitation
Adsorption
15. 5.Formulation
15
Maintenance of activity and stability of a
biotechnological products during storage and
distribution
Stabilizing additives - prolong the shelf life of
protein. (stabilizers include sugars , salts,
polymers and polyhydric alcohols)
Proteins may be formulated in the form of
solutions, suspensions or dry powders
17. Problems in DSP of rDNA
product
17
S.N
o
Problems Solutions
1 Protein stability - depends
on the susceptibility of the
protein for proteolytic
decomposition
•lon-minus mutants from E.Coli k12 strains
•Synthesis of fusion protein
2
Recombinant proteins are
often found as insoluble
aggregates in the
cytoplasm. These
accumulations of solid
insoluble proteins are
called inclusion bodies
•Force protein secretion
•Changing the specific properties of the target
protein
•Distribution of the charge
•Fusion of heterologous gene with soluble
protein
•Fusion of heterologous gene with chaperon
3
Separation of Cells and
Cell Disruption – loss of
the target protein
•kil-gene of the plasmid ColE1 may yield
complete lyses of the cells. kil-gene, under the
control of the lac-promoter may lyse the cells
18. Contd.
18
S.N
o
Problems Solutions
4 Localization – recombinant
protein may be lethal to the
host when overproduced in
cytoplasmic region
•Target protein coupled with Signal sequences
(malE, ompA ,phoA )
•Human growth hormone which accumulated
in the periplasm of E. coli was able to be
exported into the medium upon induction of
bacteriocin release protein BRP
5 Cleavage of fusion protein –
some recombinant protein
with fusion protein are toxic
to the cells
•Fusion protein has to be constructed with a
specific cleavage side. (cleavage proteins –
Thrombin, blood coagulation factor Xa,
enterokinase)
•Oligonucleotide linker
•Intein
6 Plasmid instability •Strong and inducible promoter
7 Modification of proteins for
improvement of separation
•Altering certain physico-chemical properties
of the target protein by means of sitedirected
19. Fusion protein
19
Fusion proteins - protects the cloned gene
product from attack by host cell proteases
Source - Glick
21. Cleavage of fusion protein
21
Oligonucleotide linker encoding the amino acid
sequence Ile-Glu-Gly-Arg can be joined to the
cloned gene.
Following synthesis and purification of the fusion
protein, a blood coagulation factor called Xa -
release the target protein
Source - Glick
22. 22
Additional purification
steps in order to
separate both the
protein and the fusion
protein from the
protein of interest.
This system has been
used to purify α-1-
antitrypsin and basic
human fibroblast
growth factor
Source - Glick
23. High recovery
23
Histidine-tagged protein - passed over an
affinity column of nickel–nitrilotriacetic acid -
eluted – imidazole
Greater than 90% recovery
24. 24
Human interleukin-2 gene - the marker peptide
sequence Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
Dual function of reducing the degradation of the
expressed interleukin-2 gene product and then
enabling the product to be purified.
Single step by immunoaffinity chromatography
Bovine intestinal enterokinase
26. Secretion
26
Directing a foreign
protein to the
periplasm or the
growth medium
makes its purification
easier and less costly
Secretion into the,
Periplasm
Medium
28. Secretion into the medium
28
Gram-negative bacteria can secrete a
bacteriocidal protein called a bacteriocin into the
medium.
A bacteriocin release protein activates
phospholipase A
Cleaves membrane phosopholipids so that both
the inner and outer membranes are
permeabilized.
Some cytoplasmic and periplasmic proteins are
released into the culture medium
31. Objective
31
In this paper they deal with different factors
influencing protein maturation and export, such
as
(i) the nature of the signal peptide (OmpA or PhoA),
(ii) the role of charge distribution near the leader
peptidase cleavage site,
(iii) the influence of chaperones (GroES and GroEL),
(iv) the incubation temperature and inducer
concentration
(v) the use of lysis proteins and
(vi) fusion to a known, secreted protein (preMBP)
34. Methods
34
The plasmid-containing strains were grown in rich
medium at 37 ˚C Gene expression was induced by the
addition of 01-1 mM IPTG
Cells in late exponential growth were harvested and
washed in TE
The cell pellet was gently resuspended
After 10 min at room temperature, the suspension was
centrifuged for 5 min at 6000g.
The sucrose solution was carefully drained from the
tube and the pellet was resuspended in a same volume
of cold water
The resuspended cells remained on ice for 10 min and
centrifuged at 15000 g for 10 min at 4 ˚C. The
35. 35
The pellet was resuspended, freeze-thawed six
times and centrifuged for 5 min at 15000 g.
The pellet and the supernatant are referred to as
the membrane and cytoplasmic fraction,
respectively. All samples were resuspended in
SDS loading buffer
Analyzed by 14%SDS PAGE
Visualized by immunodetection with monoclonal
antibodies directed against IL-2.
38. Reasons
38
The signal peptides may have become buried
very soon after synthesis and were unable to
direct the protein to the export machinery
Altered the charge distribution near the cleavage
site – Site-directed mutagenesis was performed
substitution lysines (K8/K9) of IL-2 by glutamic
acid (E), the K8E and substitution of cysteine
125 (C125) by alanine 125 (A125)
Early folding or aggregation of the precursor
leads to loss translocation - chaperone factors,
GroEL, GroES, DnaK and SecB appear to be
required to prevent early protein folding
40. IL-2 bioassay
40
Biological activity of human
IL-2 was tested using the
IL-2-dependent murine T
lymphocyte cell line CTLL-2
Both recombinant proteins
were found to be active in
the assay. Interestingly,
FXa cleavage yielded
aprotein with higher specific
activity which was very
Similar to that of a
preparation of Chinese
Hamster ovary-derived
recombinant IL-2
42. Introduction
42
Human G-CSF - single chain polypeptide
containing 174 amino acid residues
(MW=18.8kDa, pI=6.1)
One of the hemopoietic growth factors which
plays an important role in stimulating,
proliferation, differentiation, and functional
activation of blood cells.
It contains a free cysteine at position 17 and two
intramolecular disulfide bonds
43. Objective
43
To development of an efficient and scalable
procedure for production and purification of
recombinant human (rh-GCSF) of E. coli
48. Conclusion
48
2.2 g lˉ¹ rh-GCSF was produced in batch
cultivation with recovery yield about 40% and with
purity over than 99%.
The process established in this study may be
functional in the recovery of other proteins
expressed in E. coli as cytoplasmic IBs.
49. References
49
1. Erwin Flaschel et.al., 1993 Improvement of Downstream
Processing of Recombinant Proteins By Means of
Genetic Engineering Methods Vol. 11, Pp. 31-78
2. http://www.biologydiscussion.com/biotechnology/downstr
eam-processing/stages-in-downstream-processing-5-
stages/10160
3. Bernard R. Glick, Jack J. Pasternak, and Cheryl L.
Patten. Molecular biotechnology : principles and
applications of recombinant DNA
4. Gabrielhea Lfmann et.al., 1993, Targeting of interleukin-2
to the periplasm of Escherichia coli, Journal of General
Microbiology, 139, 2465-2473.
5. S. Abolghasemi Dehaghani et.al., June 2010 An efficient
purification method for high recovery of Recombinant
Human Granulocyte Colony Stimulating Factor from
recombinant E.coli International Journal of
Environmental Science and Development, Vol. 1, 111-