CHEMICALLY INDUCED DIMERIZATION (CID SYSTEM)
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This document discusses chemically induced dimerization (CID), where two proteins bind only in the presence of a small molecule or other agent. It provides the history of CID, noting the first system used FK-1012 to induce homo-dimerization of FKBP. The mechanism involves two proteins that do not interact binding when a dimerizing agent is present. Applications include regulating gene transcription, signal transduction, and post-translational modification to control protein localization and manipulate signaling pathways. Examples given are FK-506/immunophilin and cyclosporine A/cyclophilin interactions.
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host systems. In the present study, Pichia pastoris X-33strain was used as a host with pICZαA expression
vector to obtain extracellular expression of full length tPA gene. Specific primers were designed in such a
way to get native tPA protein sequence in subsequent purification steps. Further, construct pICZαA-tPA
was developed and electroporated into host to achieve stablert-PA gene by achieving genome integration. The transformants were screened for phenotypic characters.Mut+
phenotypic colony named Pichia tPA-3
showed expression of rt-PA at 66 kDa on SDS PAGE. Size Exclusion Chromatography (SEC) was
performed, resulting in product peak at same RT as reference standard. (alteplase).Cloning and expression
of rt-PA was successfully achieved in microbial system. Further process optimization at larger scales will surely provide cost effective alternative to mammalian system forrt-PA production
Cloning and extracellular expression of recombinant tissue plasminogen activa...
This document summarizes research conducted to clone and express recombinant tissue plasminogen activator (rt-PA) using the methylotrophic yeast Pichia pastoris. Specifically, the tissue plasminogen activator gene was cloned into the pICZαA expression vector. This construct was then transformed into Pichia pastoris X33 cells. One transformant, named Pichia tPA-3, showed expression of rt-PA at 66 kDa on SDS-PAGE. Size exclusion chromatography also showed a product peak at the same retention time as a reference tPA standard. Thus, the researchers were able to successfully clone and express rt-PA in the Pichia pastoris system.
Cloning and Extracellular Expression of Recombinant Tissue Plasminogen Activa...
This document summarizes research conducted to clone and express recombinant tissue plasminogen activator (rt-PA) using the methylotrophic yeast Pichia pastoris. Specifically, the full-length tPA gene was amplified by PCR and cloned into the pICZαA expression vector. This construct was then transformed into Pichia pastoris X33 cells. One transformant, named Pichia tPA-3, showed extracellular expression of rt-PA at 66 kDa on SDS-PAGE after induction with methanol over 144 hours. Size exclusion chromatography of samples from this transformant showed a product peak at the same retention time as a reference tPA standard. Thus, the researchers successfully achieved cloning and extracellular expression of
Cloning and Extracellular Expression of Recombinant Tissue Plasminogen Activa...
Tissue plasminogen activator (tPA) has noteworthy application in treatment of acute myocardial
infarctions. This study focuses on expression of rt-PA using microbial systems in order to reduce cost
without compromising on quality as an alternative to commercial (rt-PA)produced by using mammalian
host systems. In the present study, Pichia pastoris X-33strain was used as a host with pICZαA expression
vector to obtain extracellular expression of full length tPA gene. Specific primers were designed in such a
way to get native tPA protein sequence in subsequent purification steps. Further, construct pICZαA-tPA
was developed and electroporated into host to achieve stablert-PA gene by achieving genome integration.
The transformants were screened for phenotypic characters.Mut+
phenotypic colony named Pichia tPA-3
showed expression of rt-PA at 66 kDa on SDS PAGE. Size Exclusion Chromatography (SEC) was
performed, resulting in product peak at same RT as reference standard. (alteplase).Cloning and expression
of rt-PA was successfully achieved in microbial system. Further process optimization at larger scales will
surely provide cost effective alternative to mammalian system forrt-PA production.
CLONING AND EXTRACELLULAR EXPRESSION OF RECOMBINANT TISSUE PLASMINOGEN ACTIVA...
Tissue plasminogen activator (tPA) has noteworthy application in treatment of acute myocardial infarctions. This study focuses on expression of rt-PA using microbial systems in order to reduce cost
without compromising on quality as an alternative to commercial (rt-PA)produced by using mammalian host systems. In the present study, Pichia pastoris X-33strain was used as a host with pICZαA expression vector to obtain extracellular expression of full length tPA gene. Specific primers were designed in such a way to get native tPA protein sequence in subsequent purification steps
Plasma derived chemical mediators of inflammation
This document summarizes the four interlinked systems - kinin, clotting, fibrinolytic, and complement - that are involved in the chemical mediators of inflammation. It describes how activation of factor XII leads to activation of these four systems through a series of reactions. The end products generated by these systems - such as bradykinin, fibrin, fibrin split products, and anaphylatoxins - act as chemical mediators that increase vascular permeability, cause smooth muscle contraction and vasodilation, and recruit leukocytes to sites of inflammation.
Protein and peptide drug delivery system
This document discusses protein and peptide drug delivery systems. It begins with an introduction to proteins and peptides, noting they are composed of amino acids and can have various properties depending on their sequence. New biotechnologies like recombinant DNA and hybridoma technology allow production of protein and peptide drugs to treat cancers, neurological disorders, and other conditions. Such drugs are often delivered via injection but systems are being developed using polymers, liposomes, and other methods to provide controlled release and improve stability and bioavailability through non-parenteral routes. Analytical techniques to characterize these complex drug products are also discussed.
Copia de presentacion seminario biomol
The document summarizes a study investigating the effects of PKI-402, a dual PI3K/mTOR inhibitor, on ovarian cancer cell lines. The study used various molecular biology techniques like western blotting, RNA extraction, immunoprecipitation, and immunofluorescence to analyze the mechanism by which PKI-402 suppresses cell growth. The results showed that PKI-402 not only inhibited proliferation in cisplatin-sensitive and resistant cell lines, but also promoted degradation of Mcl-1 protein through both proteasome and autophagy-lysosome pathways. In conclusion, the different methods demonstrated the effects of inhibitors on ovarian cancer cell lines and how molecular biology techniques can evaluate cellular activity and different pathologies.
PROTEIN DEGRADATION
The document summarizes protein degradation. It discusses that protein half-lives vary from minutes to infinity, and enzymes have particularly short half-lives ranging from 11 minutes to 2 hours. The two main pathways of protein degradation are the ubiquitin-proteasome pathway, which degrades the majority of intracellular proteins, and lysosomal degradation, which degrades extracellular and some intracellular proteins. The ubiquitin-proteasome pathway involves ubiquitination of proteins by E1-E3 enzymes, recognition and degradation by the proteasome, and deubiquitination.
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Cloning and extracellular expression of recombinant tissue plasminogen activa...
Cloning and extracellular expression of recombinant tissue plasminogen activa...
Cloning and extracellular expression of recombinant tissue plasminogen activa...
Cloning and extracellular expression of recombinant tissue plasminogen activa...
Cloning and Extracellular Expression of Recombinant Tissue Plasminogen Activa...
Cloning and Extracellular Expression of Recombinant Tissue Plasminogen Activa...
Cloning and Extracellular Expression of Recombinant Tissue Plasminogen Activa...
Cloning and Extracellular Expression of Recombinant Tissue Plasminogen Activa...
CLONING AND EXTRACELLULAR EXPRESSION OF RECOMBINANT TISSUE PLASMINOGEN ACTIVA...
CLONING AND EXTRACELLULAR EXPRESSION OF RECOMBINANT TISSUE PLASMINOGEN ACTIVA...
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TAY-SACHS DISEASE
Tay-Sachs disease is a rare genetic disorder caused by mutations in the HEXA gene resulting in reduced or absent levels of the enzyme beta-hexosaminidase A. This enzyme normally helps break down a fatty substance called GM2 ganglioside in neurons. Without enough enzyme activity, GM2 ganglioside accumulates toxically in the brain. There are three main types classified by age of neurological symptom onset - infantile, juvenile, and adult-onset. Currently there is no cure, so treatment aims to manage symptoms and provide comfort through physical therapy, feeding tubes, medication, etc. Potential future treatments include gene therapy and enzyme replacement research.
CYSTIC FIBROSIS
Cystic fibrosis is an inherited disorder that affects the cells that produce mucus, sweat and digestive juices. It is caused by a mutation in the CFTR gene that results in a misfolded CFTR protein. This prevents proper water flow and leads to thick, dehydrated mucus. The buildup of mucus in the lungs and pancreas can cause life-threatening lung infections and problems with digestion. Cystic fibrosis is typically diagnosed through newborn screening, genetic testing, sweat tests, imaging and lung function tests. While there is no cure, treatment focuses on loosening mucus, treating infections, improving nutrition and managing symptoms with medications like antibiotics, enzymes and airway clearance techniques.
CLINICAL DIAGNOSTIC TESTS
This document provides an overview of common diagnostic tests and activities performed in medical laboratories. It describes tests in various sections including serology, clinical chemistry, haematology, parasitology, and microbiology. Precise procedures and appropriate equipment are needed for accurate testing and results that clinicians rely on for 70% of medical decisions. Proper laboratory layout, trained personnel, quality control, and sterile techniques are crucial for reliable diagnostic results.
URINALYSIS AND RPFT
Renal function tests evaluate kidney health. This document discusses urinalysis and renal plasma flow tests. Urinalysis involves macroscopic, chemical, and microscopic examination of urine to detect compounds and diagnose kidney or urinary tract disorders. Renal plasma flow is measured using the para-amino hippurate test, which determines the volume of plasma filtered by the kidneys. Both tests provide information about kidney function and help diagnose underlying health conditions.
ACIDIC PHOSPHATASE ENZYME (ACP)
Acidic phosphatase is an enzyme found primarily in the prostate gland. It catalyzes the hydrolysis of phosphate esters. There are two types based on pH and tissue source. Prostate acid phosphatase is important clinically as increased levels indicate prostate damage or cancer. Diagnosis involves quantitative analysis of plasma acid phosphatase levels along with scans of affected areas. Precautions must be taken due to lability and presence in red blood cells. Other conditions like Gaucher's disease can also increase levels.
LIPID METABOLISM DISORDERS
The document discusses lipid metabolism disorders related to carnitine. It describes how carnitine transports long-chain fatty acids into mitochondria for breakdown (β-oxidation), a process called the carnitine shuttle. Deficiencies in carnitine can cause metabolic disorders by inhibiting fatty acid breakdown. The primary types are genetic deficiencies of carnitine palmitoyltransferases I and II, resulting in hypoglycemia, liver disease, and muscle issues. Secondary deficiencies occur with liver disease, malnutrition, or other medical conditions increasing carnitine needs. Diagnosis involves carnitine level testing, and treatment is carnitine supplementation.
GLANDULAR EPITHELIUM-Microscopic slides
This document discusses glandular epithelium microscopic slides and is reaching out to biotechnologists and medical laboratory technologists. The sender, Sir. Stymass kasty from Sokoine University of Agriculture, has glandular epithelium slides available and is asking recipients to contact him via email if interested in the slides for research purposes.
AMINOACIDURIA
Aminoaciduria is a protein metabolism disorder where excess amino acids are present in the urine. It occurs when more than 5% of filtered amino acids are excreted in the urine instead of being reabsorbed by the kidneys. Aminoaciduria can be caused by defects in renal reabsorption, increased amino acid levels in the blood overwhelming reabsorption capacity, or abnormal excretion of amino acid byproducts. Treatment depends on the underlying cause but may include dietary restrictions, increased fluid intake, or medications to help the kidneys clear excess amino acids.
GENE REGULATION
Prokaryotes regulate protein synthesis in response to nutrient availability through induction and repression of operons. An operon contains genes that are coordinately controlled, with one polycistronic mRNA transcribed. Repression occurs when a repressor binds the operator to prevent transcription. Induction occurs when an inducer binds the repressor, inhibiting it and allowing transcription. The lac and trp operons demonstrate repression and induction in response to lactose/glucose and tryptophan availability, respectively. Catabolite repression prioritizes glucose utilization by inhibiting cAMP levels and thus transcription of certain operons.
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CHEMICALLY INDUCED DIMERIZATION (CID SYSTEM)
- 1. CHEMICALLY INDUCED DIMERIZATION (FK- 506/cyclosporin A) Sir. Stymass Kasty SOKOINE UNIVERSITY OF AGRICULTURE MOROGORO-TANZANIA April 26, 2018 CID SYSTEM 1
- 2. INTRODUCTION Meaning Chemically Induced Dimerization (CID).;Refer to a biological mechanism in which two proteins binds only in the presence of a certain small molecule, enzyme or any other dimerizing agent. History First small molecule CID system was developed in 1993 and used FK-1012, a derivative of the drug Tacrolimus(FK- 506), to induce homo-dimerization of FK506-binding protein (FKBP) in vivo. April 26, 2018 CID SYSTEM 2
- 3. MECHANISM Two proteins which do not interact, binds in the presence of Dimerizing agent. April 26, 2018 CID SYSTEM 3
- 4. MECHANISM April 26, 2018 CID SYSTEM 4
- 5. APPLICATIONS The genetically engineered CID systems has been used to regulate and monitor :- • Gene transcription. • Signal transduction. • Post translational modification in proteins. All in all to control protein localization, to manipulate signalling pathways and induce protein activation. Cyclosporine A and Tacrolimus are used in the immunosuppression process after organ transplant to inhibit graft rejection i.e. to induce interaction between proteins of interest from different origin. It have been also Used to create a basic component of bio-computers, logic gates from genetically manipulated cells i.e. Cell ruffling (Two CID system, Plant protein and Bacterial protein expressed in the same cell). April 26, 2018 CID SYSTEM 5
- 6. EXAMPLES FK-506 - which interacts with immunophilin. Cyclosporine A - which interacts with cyclophilin. GAL4 (DNA binding protein)- which binds to the promoter without activating transcription, and HSV vp16 (activator) which can not bind to promoter; when they interact, the combination of the two cause activation of transgenic expression. April 26, 2018 CID SYSTEM 6
- 7. Email. Stymass1@gmail.com Contact.+255755527871 THANK YOU…! !.<YOUR WELCOME>.! April 26, 2018 CID SYSTEM 7