Summary
The human pancreatic prokallikrein gene has been fused to the DNA sequence coding for the signal peptide of the Escherichia coli major outer membrane protein F (OmpF) and expressed under the control of tac promoter in E. coli. By induction with isopropyl-β-d-thiogalactopyranoside, the cells produced prokallikrein very efficiently. The fused OmpF signal peptide was verified as being processed correctly at the cleavage site of the OmpF signal peptide, and the N-terminal amino acid sequence of the product was found to be identical to that of native human prokallikrein. However, the prokallikrein produced by E. coli formed insoluble aggregates and was always collected in the insoluble fraction. An electron micrograph of prokallikrein-producing cells indicated that the prokallikrein was secreted into the periplasmic space and formed insoluble inclusion bodies there. By treating the insoluble inclusion bodies with oxidized and reduced glutathione in 1 M guanidine-HCl solution, a portion of them could be solubilized in water and showed kallikrein activity of 8 units (approx. 264 μg kallikrein) per litre of culture by trypsin activation.
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Shibui, T., Matsui, R., Uchida-Kamizono, M. et al. Periplasmic production of human pancreatic prokallikrein in Escherichia coli . Appl Microbiol Biotechnol 31, 253–258 (1989). https://doi.org/10.1007/BF00258405
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DOI: https://doi.org/10.1007/BF00258405