Secretion and export of IGF-1 in Escherichia coli strain JM101.
The processing of LamB-IGF-1 fusion protein and the export of processed IGF-1 (insulin-like growth-factor-1) into the growth medium was examined in the Escherichia coli host strain, JM101. Several strain or plasmid modifications were tried to increase export of periplasmic (processed) IGF-1 into the growth medium of JM101. These included: (1) use of a lon null mutant strain to increase accumulation levels of unprocessed LamB-IGF-1 fusion protein; (2) use of an alternative drug resistance marker on the expression plasmid rather than beta-lactamase, thereby reducing any competition for processing of LamB-IGF-1 by signal peptidase; (3) examination of whether phage M13 gene III protein expression caused more periplasmic IGF-1 to be exported into the growth medium due to increased outer membrane permeability; and (4) examination of the effect of E. coli or yeast optimized IGF-1 codons. None of these strain or plasmid modifications caused any significant increase in export of IGF-1 into the growth medium of JM101. Solubility studies of LamB-IGF-1 and processed IGF-1 showed that virtually all of the LamB-IGF-1 and IGF-1 remaining within the cell after a 2 h induction period was insoluble. This implied that only soluble LamB-IGF-1 was processed to IGF-1 and that only soluble IGF-1 was exported into the growth medium. Taken together, the results indicated that LamB-IGF-1 and IGF-1 solubility were the limiting factors in secretion of IGF-1 into the periplasm and export of IGF-1 into the growth medium.
Obukowicz
M G
MG
Department of Biological Sciences, Monsanto Company, St. Louis, MO 63198.
Turner
M A
MA
Wong
E Y
EY
Tacon
W C
WC
D000595:Amino Acid Sequence; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013002:Somatomedins / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics
D003062:Codon; D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013002:Somatomedins; D014764:Viral Proteins; D007334:Insulin-Like Growth Factor I; D005954:Glucosephosphate Dehydrogenase; D001618:beta-Lactamases