Recombinant coho salmon insulin-like growth factor I. Expression in Escherichia coli, purification and characterization.
Recombinant coho salmon insulin-like growth factor I (rsIGF-I) was produced in Escherichia coli, purified and characterized. The rsIGF-I expression vector was constructed by polymerase chain reaction and cloning into a plasmid containing a phage T7 RNA polymerase promoter. The rsIGF-I was recovered from bacterial inclusion bodies, solubilized under reducing conditions, immediately refolded, then fractionated by a two-step ion-exchange chromatography on DEAE-52 and Mono-S columns. It was further purified by HPLC on a reverse-phase Asahi-Pak C4P-50 C4 column. Purification of rsIGF-I was monitored by SDS/PAGE and immunoblot with anti-[human somatomedin C (SM C)/IGF-I] serum. The rsIGF-I appeared as a single band with molecular mass of 7 kDa, the same size as recombinant human IGF-I (rhIGF-I) and cross-reacted with anti-(human SM C/IGF-I) serum. The amino acid sequence of rsIGF-I contained an NH2-terminal methionine residue followed by the sequence predicted for mature sIGF-I. At concentrations in the range 3.9-250 ng/ml, rsIGF-I significantly stimulated sulfate uptake by the cultured branchial cartilage of coho salmon. The stimulatory effect of rsIGF-I was concentration dependent and slightly more potent than that of rhIGF-I at the highest concentration tested.
Moriyama
S
S
School of Fisheries HF-15, University of Washington, Seattle 98195.
Duguay
S J
SJ
Conlon
J M
JM
Duan
C
C
Dickhoff
W W
WW
Plisetskaya
E M
EM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007334:Insulin-Like Growth Factor I / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D017684:Oncorhynchus kisutch; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I