Expression of recombinant proteins in E. coli

This procedure is used routinely for expression of protein from pRSET, pGEX and pET vectors (see Table 3.1). 

Plasmid/company Tag Matrix Tag removal In vitro labelling Promoter 

2 x YT, 16 g/1 Tryptone, 10 g/1 yeast extract, 5 g/1 NaCl (add 2% glucose when using GEX plasmids). 

37°C incubator and a 2.5 1 flask Tubes and rotor for harvesting of cells 

Pick a single colony of E. coli cells and inoculate 10-50 ml of 2 x YT medium containing 50 pg/ml ampicillin and let it grow overnight at 37°C with vigorous shaking.  

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Peleg Y, Unger T (2008) Application of high-throughput methodologies to the expression of recombinant proteins in E. coli. Methods Mol Biol 426 197-208... 

Valdez-Cruz, N.A., Caspeta, L., Perez, N.O., Ramirez, O.T., Trujillo-Rolddn, M.A., 2010. Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters. Microb. Cell Factories 9, 1-16. 

The expression of recombinant proteins in cells in which they do not naturally occur is termed heterologous protein production (Chapter 3). The first biopharmaceutical produced by genetic engineering to gain marketing approval (in 1982) was recombinant human insulin (tradename Humulin ), produced in E. coli. An example of a more recently approved biopharmaceutical that is produced in E. coli is that of Kepivance, a recombinant keratinocyte growth factor used to treat oral mucositis (Chapter 10). Many additional examples are provided in subsequent chapters. 

The success of Chapman and co-workers in expression of flavocytochrome 2 in E. coli [23] is encouraging in its impUcations for future expression of flavoproteins in this host because, in their experience both the flavin and heme groups are incorporated into the recombinant protein. Moreover, the bacterial expression system produces the protein 500-1000 fold more efficiently than the yeast from which it was cloned. The enzyme produced in E. coli, however, lacks the first five amino acid residues at its amino terminus, a result which presumably reflects subtle differences in protein synthesis between the two organisms. 

Any plasmid designed to express recombinant proteins in E. coli under the control of a bacteriophage RNA polymerase promoter can be used in an E. coli cell-free system. As a mle of thumb, constructs that express weU in vivo tend to express well in vitro. T7 RNA polymerase is often considered intrinsically more efficient than other RNA polymerases (see Part IV, Chapter 12). However, this differential efficiency can often be attributed to differential sensitivity to salt concentration. Because of the relative robustness and efficiency of the T7 polymerase, plasmids under the control of a T7 promoter are almost exclusively used in E. coli cell-free systems. 

Alternative methods have been developed to obtain virus stocks without plaque purification for expression of recombinant proteins in infected insect cells. The Bac-to-Bac technology (Invitrogen, Thermo Fisher Scientific) avoids homologous recombination in insect cells by using site-specific transposition in E. coli. With this technology, recombinant bacmid-DNA is generated that is used to transfect insect cells. 

Expression of Recombinant Antibody Fusion Proteins in E. coli... 

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