Examination of calf prochymosin accumulation in Escherichia coli: disulphide linkages are a structural component of prochymosin-containing inclusion bodies
Schoemaker, J.M.; Brasnett, A.H.; Marston, F.A.
EMBO Journal 4(3): 775-780
1985
ISSN/ISBN: 0261-4189 PMID: 3924595 Document Number: 250711
Recent reports have shown that synthesis of certain recombinant proteins in E. coli results in the production of intracellular inclusion bodies. These studies have not analyzed the structure of the inclusion body, especially regarding the intermolecular forces holding it together. Structural aspects of inclusion bodies made in E. coli as a result of high level expression of the eukaryotic protein, calf prochymosin, are examined here. Prochymosin is a monomeric protein containing 3 disulfide bridges. It was expressed at up to 20% of cell protein from a plasmid containing the E. coli tryptophan promoter, operator and ribosome binding site. Proteins in the inclusion bodies were analyzed by Western blotting of sodium dodecyl sulfate-polyacrylamide gels. When experiments were done using conditions which preserved the in vitro state of thiol groups, inclusions were shown to be composed of multimers of prochymosin molecules which were interlinked partly by disulfide bonds. The inclusion bodies also contained a high concentration of reduced prochymosin. The presence of intermolecular disulfides probably contributes to the difficulty of solubilizing recombinant prochymosin during its purification from E. coli.