Abstract
The construction of a gene encoding Lys-human proinsulin, its direct expression inE. coli, and the simple purification procedure are described here. The temperature inducible promotor was employed for induction in a very short time. The expression level could reach 20–30%. After simple downstream processing and only one step of Sephadex G50 purification, 150 mg recombinant Lys-human proinsulin with a purity of up to 90% could be obtained easily from 1L of high density fermentation medium. The obtained product is in the form of Met-Lys-human proinsulin because of the failure of the bacterial host to remove the initiator methionine residue. The Lys-human proinsulin could be changed into human insulin by trypsin and carboxypeptidase B treatment in later steps. After separation with DEAE-Sephadex A25, human insulin with expected amino acid composition and full native biological activity could be obtained with a yield of 50 mg/L of fermentation medium.
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Chen, JQ., Zhang, HT., Hu, MH. et al. Production of human insulin in anE. coli system with Met-Lys-human proinsulin as the expressed precursor. Appl Biochem Biotechnol 55, 5–15 (1995). https://doi.org/10.1007/BF02788744
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DOI: https://doi.org/10.1007/BF02788744