Secretion in Escherichia coli and phage-display of recombinant insulin-like growth factor binding protein-2.
Insulin-like growth factors (IGFs) promote cell growth and differentiation. Their actions are regulated by six different, but related, binding proteins (IGFBPs). To investigate the molecular interactions between IGFs and IGFBPs, an Escherichia coli based production method and a phage display system has been developed. The cDNA for bovine IGFBP-2 was inserted between regions coding for the pelB signal sequence and geneIII product, g3p, of bacteriophage fd in a phagemid vector to generate pGF14. The coding sequences of IGFBP-2 and g3p were separated by an amber stop codon and a flexible linker containing the cleavage recognition site for H64A subtilisin. Using this system in BL21, a non-supE strain lacking ompT, most product, approximately 4 mg 1(-1) of IGFBP-2, was obtained in the growth medium. The bacterially derived IGFBP-2 had a correct N-terminal sequence, molecular mass on SDS-PAGE and the same affinity for IGF-1 and IGF-II as IGFBP-2 from mammalian cells. In a supE strain of E. coli, IGFBP-2 was produced as an IGF-binding fusion to g3p. Procedures for display and approximately 10000 fold enrichment of IGFBP-2 bearing phage using adsorption to IGF-II coated microtitre plates were developed. Thus IGFBP-2 can be secreted in E. coli and displayed on filamentous phage. These can be selectively enriched by binding to immobilised IGF-II.
Lucic
M R
MR
Department of Biochemistry, University of Adelaide, Australia.
Forbes
B E
BE
Grosvenor
S E
SE
Carr
J M
JM
Wallace
J C
JC
Forsberg
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D019556:COS Cells; D002417:Cattle; D003090:Coliphages / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D005821:Genetic Techniques; D005822:Genetic Vectors; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D018971:Insulin-Like Growth Factor Binding Protein 2; D011993:Recombinant Fusion Proteins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II