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Applied Microbiology and Biotechnology

, Volume 31, Issue 3, pp 253–258 | Cite as

Periplasmic production of human pancreatic prokallikrein in Escherichia coli

  • Tatsurou Shibui
  • Rie Matsui
  • Michiru Uchida-Kamizono
  • Hiroko Okazaki
  • Jun Kondo
  • Kenji Nagahari
  • Shigetada Nakanishi
  • Yutaka Teranishi
Applied Genetics and Regulation

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.

Keywords

Glutathione Outer Membrane Cleavage Site Reduce Glutathione Outer Membrane Protein 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • Tatsurou Shibui
    • 1
  • Rie Matsui
    • 1
  • Michiru Uchida-Kamizono
    • 1
  • Hiroko Okazaki
    • 2
  • Jun Kondo
    • 1
  • Kenji Nagahari
    • 1
  • Shigetada Nakanishi
    • 3
  • Yutaka Teranishi
    • 1
  1. 1.Biosciences Laboratory, Research CenterMitsubishi Kasei CorporationYokohama-shi, KanagawaJapan
  2. 2.Toxicology Laboratory, Research CenterMitsubishi Kasei CorporationYokohama-shi, KanagawaJapan
  3. 3.Institute for ImmunologyKyoto University Faculty of MedicineKyotoJapan

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