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.
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Acknowledgements
We thank Jose Maria Dominguez and Francisco Ponce for assistance with the figures and Judy Swan for English language assistance. This research was supported by Grant 225080-5-4301 PB, from the Consejo Nacional de Ciencia y Tecnología (CONACyT), Mexico.
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Olmos-Soto, J., Contreras-Flores, R. Genetic system constructed to overproduce and secrete proinsulin in Bacillus subtilis . Appl Microbiol Biotechnol 62, 369–373 (2003). https://doi.org/10.1007/s00253-003-1289-4
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DOI: https://doi.org/10.1007/s00253-003-1289-4