Heterologous Gene Expression in E.coli

Volume 705 of the series Methods in Molecular Biology pp 211-224


Periplasmic Chaperones Used to Enhance Functional Secretion of Proteins in E. coli

  • Martin SchlapschyAffiliated withLehrstuhl für Biologische Chemie, Technische Universität München
  • , Arne SkerraAffiliated withLehrstuhl für Biologische Chemie, Technische Universität München Email author 

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While Escherichia coli is in wide use as a host organism for preparative protein production, problems with the folding of the recombinant gene product as well as protein aggregation, i.e., formation of inclusion bodies, are frequently encountered. This is particularly true for proteins that carry structural disulfide bonds, including antibody fragments, cytokines, growth factors, and extracellular fragments of eukaryotic cell surface receptors. In these cases, secretion into the oxidizing milieu of the bacterial periplasm in principle enables disulfide bond formation, resulting in a correctly folded and soluble protein. However, this process often occurs at low efficiency, depending on the nature of the recombinant gene product. Therefore, we have developed the helper plasmid pTUM4, which effects overexpression of four established periplasmic chaperones and/or folding catalysts: the thiol-disulfide oxidoreductases DsbA and DsbC, which catalyze the formation and isomerization of disulfide bridges, and two peptidyl-prolyl cis/trans isomerases with chaperone activity, FkpA and SurA. Here, we present a detailed protocol how to use this system for the bacterial secretion of recombinant proteins, including human EGF as a new example, and we give hints on optimization of the expression procedure.

Key words

Chaperone disulfide isomerase DsbA DsbC FkpA folding catalyst peptidyl-prolyl cis/trans isomerase SurA