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rdna technology evaluating purify desired gene and some of medical diagnostics

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  • 1. SEMINAR ON1EVALUATION OF RECOMBINANT PROTEINSPresented by,D. Pranitha,M. Pharmacy 2sem,Pharmaceutics.

2. CONTENTSIntroductionGene expressionProtein Expression and PurificationProduction of Recombinant ProteinsApplicationsConclusionReferences2 3. Introduction Proteins are the most abundant organic molecules of the livingsystem. They have significant role in structural and functionalorganisation of the cell. Proteins that result from the expression of recombinant DNA withinliving cells are termed recombinant proteins. Recombinant DNA technology involves taking genetic material fromone source and recombining it in vitro with another source followedby introducing of recombined material into host cell. Once a Recombinant DNA is inserted into bacteria, these bacteriawill make protein based on this rDNA.This protein is know asRecombinant Protein.3 4. Protein4rDNA 5. Gene ExpressionDNARNAPROTEIN5TranscriptionTranslation 6. Protein Expression and Purification Isolation of genes. Insertion of isolated gene to expression vector. Transfer of recombinant vector into host cell throughTransformation. Identification and isolation of cells containing recombinantvector. Growth of cells through fermentation. Isolation and purification of protein.6 7. Production of Recombinant protein There are basically two methods for producing recombinantproteins. One is the molecular Cloning a laboratory method used to makerecombinant DNA. The other method is the Polymerase chain reaction used toproceed the replication of any specific DNA sequence selected . The basic difference between the two methods is that molecularcloning incorporates the replication of the DNA within a livingcell, whereas PCR replicates DNA in the test tube, withoutliving cells.7 8. Cloning process Gene of interest is cut out withrestriction enzymes (RE) Host plasmid (circularchromosome) is cut with sameRE Gene is inserted into plasmid andligated with ligase. New (engineered) plasmidinserted into bacterium(transform)8 9. Vectors Self-replicating DNA molecules used to transfer foreign DNAsegments between host cells. An ideal vector should be small in size, with single restrictionendonuclease site. Three types of vectors9PlasmidsBacteriophagesCosmids 10. Plasmids Bacteria contain extrachromosomal molecules of DNAcalled plasmids which are circular. pBR322 of E.coli is most popular and widely used plasmidvector. Bacteriophages Bacteriophages or simply phages are the viruses thatreplicate within the bacteria. Phages can accept foreign DNA fragments of 10-20 kblength. Cosmids These are specialized plasmids containing DNA sequencenamely cos sites. Cosmids can carry larger fragments of foreign DNAcompared to plasmids .i.e 20-50kb.10... 11. Polymerize chain reaction A method for amplifying DNA segments using cycles ofdenaturation, annealing to primers, and DNA polymerase-directedDNA synthesis PCR copies a DNA molecule without restriction enzymes, vectors,or host cells . Faster and easier than conventional cloning. First Step in PCR: Denaturation1. DNA is heated to break the hydrogen bonds between the twopolynucleotide strands. Two single-stranded DNA molecules serve as templates.11 12. Second Step in PCR: Annealing2. Short nucleotide sequences (primers for DNA replication) aremixed with the DNA and bind to complementary regions onsingle-stranded DNA . Takes place at lower temperature. Primers are 20-30 nucleotides long, synthesized in thelaboratory. Third Step in PCR: DNA Synthesis3. The enzyme Taq polymerase is added to synthesize acomplementary DNA strand. Taq is a DNA polymerase from a bacterium found in hotsprings. These three steps make up one PCR cycle .12 13. Production of recombinant Insulin Insulin Insulin is produced by cells of islets of Langerhans ofpancreas. Human insulin contains 51 amino acids ,arranged in twopolypeptide chains. The chain A has 21 amino acids while chain B has 30amino acids both are held together by disulfide bonds. 14. Insulin Production14 15. APPLICATIONS Several proteins are created from recombinant DNA(recombinant proteins) and are used in medical applications. Hematopoietic growth factor. Interferons Hormones Recombinant protein vaccines Tissue/bone growth factors and clotting factors Biological response modifiers Monoclonal/Diagnostic/Therapeutic antibodies Recombinant proteins is extensively used in biotechnology,medicine and research.15 16. Hematopoietic growth factor Product of blood cells in bone marrow of central axial skeletonis referred to as medullary hematopoiesis. While the mechanism of early stages of lineage commitment bybone marrow to particular type of blood cells remains elusive,the later stages of this process is driven by hematopoieticgrowth factor List of factors of recombinant origin16Product Company IndicationThrombopoietin Phamacia ThrombocytopeniaErythropoietin Amgen AnaemiaAncestim Amgen Blood cell transplantation 17. Antibody StructureAntibodies are immune system-related proteins calledimmunoglobulins. Each antibody consists of fourpolypeptides two heavy chains and two light chainsjoined to form a "Y" shaped molecule.The amino acid sequence in the tips of the "Y" variesgreatly among different antibodies. This variableregion, composed of 110-130 amino acids, give theantibody its specificity for binding antigen. Thevariable region includes the ends of the light andheavy chains. Treating the antibody with a proteasecan cleave this region, producing Fab or fragmentantigen binding that include the variable ends of anantibody.The constant region determines the mechanism usedto destroy antigen. Antibodies are divided into fivemajor classes, IgM, IgG, IgA, IgD, and IgE, based ontheir constant region structure and immune function.17 18. Genetically engineered humanized ab Genetically engineered chimeric ab18 19. Interferons In 1957 it was noted that infected by viruses produces protein calledInterferon ,viral resistance to native cells. Inability to produce sufficient quality and inadequate purity, limits theclinical use of interferons. The problem of both purity and quality were resolved usingrecombinant DNA technology.IFN IFN IFN IFN IFN IFN IFN- 1 IFN- 2 IFN-1a IFN- 1bIFN- 2a IFN-2b19Types of recombinant IFN 20. Hormones Initially peptide and hormones used clinically were extractedand purified from animal or human source. Since these extraction may also contain animal proteins orpeptides their use can lead to development of antibodies. There were limitations on use due to scarcity, contaminationwith other peptides and in some cases ineffectiveness. By introduction of recombinant DNA techniques,synthesizing of proteins and peptides takes place. In 1982 first recombinant protein hormone , insulin wasintroduced in USA.20 21. List of hormones of recombinant origin21Hormones Company IndicationHuman chronic gonadotropin Sereno Breast cancerLeptin Amgen Diabetes mellitusThyroid stimulating hormone Genzyme Recurrent thyroidcancer 22. Electrophoresis Most often used technique forprotein products is sodium dodecylsulphate polyacrylamide gelelectrophoresis . Proteins are denatured by boiling inthe SDS solution. All charges ofprotein are masked by negativecharge of dodecyl sulphate. Thus protein moves onpolyacrylamide gel strictly on basisof size of protein molecule. This technique is useful fordetermining molecular weight ofproteins. For visualization of proteins on thegel reagents used are silver nitrate,coomassie brilliant blue dye.22 23. List of products of recombinant originProduct Company IndicationAlpha-glucosidase Genzyme Pompes diseaseInterleukin-4 receptor Immunex AsthmaTumor necrosis factor receptor Immunex Rheumatoid arthritisVascular endothelial growthfactorGenvec CardiovasculardisordersHIV vaccine Chiron AIDSProstvac Therion Prostate cancerNeurex Xoma Cystic fibrosis23 24. CONCLUSION Recombinant technology is mostly used in production ofinsulin, human growth hormone, vaccines ,Interferons etc. Recombinant proteins are used in medical applications,particularly as medications and vaccines. Development of improved drug delivery system. Recombinant technology is that it allows introduction ofmodifications into proteins at desired positions.24 25. REFERENCES Daan J.A.Crommelin, Robert D.Sindelar. PharmaceuticalBiotechnology: An Introduction for Pharmacists and PharmaceuticalScientists. 2nd Edition. London: Taylor & Francis Routledge; pg no.5-18 U.Satyanarayana. Biotechnology ,pg no.75-85;189-198 WEBSITES http://www.innovateus.net/science/what-are-recombinant-proteins . http://en.wikipedia.org/wiki/Recombinant_DNA . http://www.accessexcellence.org/RC/VL/GG/plasmid.php25 26. 26

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