High-level expression and simple purification of recombinant human insulin-like growth factor I.
Human insulin-like growth factor I (IGF-I) was expressed in Escherichia coli as a truncated beta-galactosidase-IGF-I fusion protein. The Lac Z" gene was truncated by removal of a 490 bp fragment which encoded 163 N-terminal residues of beta-galactosidase and was connected to the IGF-I cDNA by a linker encoding hydroxylamine cleavage recognition sequence. By truncating Lac Z" gene, the overall yield and purification procedures of IGF-I from fusion protein have been improved. The fusion protein was produced in the form of insoluble inclusion bodies with isopropyl-1-thio-beta-D-galactoside (IPTG) induction. After cleavage of the fusion protein with hydroxylamine, the released IGF-I was purified to homogeneity by a cation exchange chromatography, refolding and reverse-phase high performance liquid chromatography (rp-HPLC). The purified IGF-I was found to be indistinguishable from the native IGF-I by N-terminal amino acid sequence, SDS-polyacrylamide gel electrophoresis, and rp-HPLC and by biological activities such as thymidine uptake, protein synthesis and receptor binding. These results suggest that the expression and simple purification of recombinant human IGF-I described in this paper may be useful for large scale production of IGF-I.
Kim
S O
SO
Molecular Biomedicine Research Group, Korea Institute of Science and Technology, Taejon, Korea.
Lee
Y I
YI
D000818:Animals; D001709:Biotechnology; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D007763:Lac Operon; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D001616:beta-Galactosidase / Q000235:genetics / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D007334:Insulin-Like Growth Factor I; D001616:beta-Galactosidase