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Recombinant vectors are valuable tools in the biopharmaceutical industry with a number of novel vectors being developed every day. These Antibodies are often expressed in mammalian cells by co-transfecting light chain and heavy chain containing plasmids. However, co-transfection can lead to variable in the copy number of both heavy chain and light chain, there by affecting the protein productivity. Double gene expression vectors can overcome these problems. In the current design The first transcriptional unit comprises sequences for the CMV promoter, multiple cloning site (MCS) and a Polyadenylation Sequence and the second transcriptional unit comprises sequences for the SV40 promoter, MCS and polyadenylation sequence. The double gene expression vector (pUB-C-S) was constructed by ligating the BglII and BamHI fragment (974bp) form pSI plasmid with BglII digested pCI plasmid. The presence of two independent transcriptional units in the recombinant plasmid was confirmed by colony PCR. Key Words:Recombinant vectors, biopharmaceutical industry, co-transfection, promoter, multiple cloning site. REFERENCES Johnston K., Clements A., Venkataramani. R.N., Trienvel R.C., and Marmorstein.R., 2000. Coexpression of proteins in bacteria using T7-based expression plasmids: Expression of heteromeric cell-cycle and transcriptional regulatory complexes. Protein Expr. Purif. 20: 435-443. Rucker. P., Torti F.M., and Torti S.V., 1997. Recombinant ferritin: Modulation of subunit stoichiometry in bacterial expression system. Protein Eng. 10: 967-973. Susan K. Eszterhas1 Eric E. Bouhassira,2 David I. K. Martin,3 and Steven Fiering1*, Transcriptional Interference by Independently Regulated Genes Occurs in Any Relative Arrangement of the Genes and Is Influenced by Chromosomal Integration Position. Mol Cell Biol. 2002 January; 22(2): 469–479. Bizily SP, Rugh CL, Meagher RB.,Phytodetoxification of hazardous organomercurials by genetically engineered plants., Nat Biotechnol. 2000 Feb;18(2):213-7.. Lapierre C, Pollet B, Petit-Conil M, Toval G, Romero J, Pilate G, Leple JC, Boerjan W, Ferret V V, De Nadai V, Jouanin L., Structural alterations of lignins in transgenic poplars with depressed cinnamyl alcohol dehydrogenase or caffeic acid O-methyltransferase activity have an opposite impact on the efficiency of industrial kraft pulping. Plant Physiol. 1999 Jan;119(1):153-64. Chen L, Marmey P, Taylor NJ, Brizard JP, Espinoza C, D'Cruz P, Huet H, Zhang S, de Kochko A, Beachy RN, Fauquet CM., Expression and inheritance of multiple transgenes in rice plants., Nat Biotechnol. 1998 Nov;16(11):1060-4. Goderis IJ, De Bolle MF, François IE, Wouters PF, Broekaert WF, Cammue BP., A set of modular plant transformation vectors allowing flexible insertion of up to six expression units. Plant Mol Biol. 2002 Sep;50(1):17-27. Ashton, G. (2001) Growing pains for biopharmaceuticals. Nature Biotechnology 19, 307-311 Walsh, G. (2004) Second-generation biopharmaceuticals. European Journal of Pharmaceutics &Biopharmaceutics58, 185-196 Current protocols in molecular biology, vol 1, 2005 Sambrook J and Russell D W, Molecular cloning a laboratory manual, Vol 1, 2001. Baneyx, F. (1999) Recombinant protein expression in Escherichia coli. Current Opinion in Biotechnology 10, 411-421 Swartz, J. R. (2001) Advances in Escherichia coli production of therapeutic proteins. Current Opinion in Biotechnology 12, 195-201 Kjaerulff, S. and Jensen, M. R. (2005) Comparison of different signal peptides for secretion of heterologous proteins in fission yeast. Biochemical & Biophysical Research Communications 336, 974-982 Daly, R. and Hearn, M. T. W. (2005) Expression of heterologous proteins in Pichiapastoris: a useful experimental tool in protein engineering and production. Journal of Molecular Recognition 18, 119-138 Blanchard V, Gadkari RA, George AV, Roy S, Gerwig GJ, Leeflang BR, Dighe RR, Boelens R, Kamerling JP., High-level expression of biologically active glycoprotein hormones in Pichiapastoris strains-selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated (15)N-labeled phCG. Glycoconj J. 2008 Feb 15; [Epub ahead of print] Forsburg SL., Overview of Schizosaccharomycespombe. CurrProtocMol Bio, 2003, Chapter 13, unit 13.14. Rodney E. Kellems, Gene amplification in mammalian cells. 1992. Paulina Balbas and Francisco Bolivar, Design and Construction of Expression Plasmid Vectors in Escherichacoli.., Methods in Enzymology, 185:14-37, 1990. Makrides, S.C. (1996). Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60, 512-538. Lehman IR, DNA ligase: Structure, mechanism and function. Vol. 186, no. 4166, pp. 790-797, 1974. |
