Production of human insulin in an E. coli system with Met-Lys-human proinsulin as the expressed precursor.
The construction of a gene encoding Lys-human proinsulin, its direct expression in E. 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 1 L 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.
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007328:Insulin / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011384:Proinsulin / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017421:Sequence Analysis
D007328:Insulin; D011994:Recombinant Proteins; D011384:Proinsulin