Selection for signal sequence mutations that enhance production of secreted human proinsulin by Escherichia coli.
This paper describes a method for the positive selection of signal sequence mutations that result in enhanced production of secreted human proinsulin by Escherichia coli. Coding sequences for the structural portion of beta-lactamase (EC 3.5.2.6) were substituted for those of the C terminus of proinsulin in a plasmid that normally directs the synthesis and secretion of proinsulin. The resulting plasmid directed the synthesis of a proinsulin/beta-lactamase fusion protein that was secreted into the periplasmic space and conferred resistance to low levels of ampicillin (Ap). Beneficial changes to the signal sequence were selected by the host's ability to grow on high levels of Ap. The beta-lactamase coding sequences were then replaced with those of human proinsulin, resulting in plasmids which directed enhanced production of secreted proinsulin.
Stahl
S J
SJ
Department of Molecular Biology, Biogen S.A., Geneva, Switzerland.
Christiansen
L
L
D000595:Amino Acid Sequence; D001483:Base Sequence; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011384:Proinsulin / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics*; D001618:beta-Lactamases / Q000235:genetics
D004274:DNA, Recombinant; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D011384:Proinsulin; D001618:beta-Lactamases