Applied Microbiology and Biotechnology

, Volume 62, Issue 4, pp 369–373

Genetic system constructed to overproduce and secrete proinsulin in Bacillus subtilis

Original Paper

DOI: 10.1007/s00253-003-1289-4

Cite this article as:
Olmos-Soto, J. & Contreras-Flores, R. Appl Microbiol Biotechnol (2003) 62: 369. doi:10.1007/s00253-003-1289-4

Abstract

The first amino acid residue from a proinsulin gene was fused in frame with the last amino acid residue of the aprE signal peptide sequence from Bacillus subtilis, using an overlapping PCR methodology. For expression of the fused DNA the aprE regulatory region (aprERR) was used. A six-protease-deficient strain of B. subtilis with the hpr2 and degU32 mutations was constructed for overproduction of the recombinant protein. The production of proinsulin was carried out in a mineral medium which facilitated the purification of proinsulin. Samples were taken during growth and analyzed by RIA and Western blot. Proinsulin was overproduced (1 mg ml−1) and 90% was secreted into the culture medium 1 h after stationary phase began.

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  1. 1.Biotecnología MarinaSan DiegoUSA
  2. 2.Marine Biotechnology Department, Molecular Microbiology LaboratoryCentro de Investigación Científica y de Educación Superior de Ensenada (CICESE)Mexico

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