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Type: Journal article
Title: Secretion in Escherichia coli & phage-display of recombinant insulin-like growth factor binding protein-2
Author: Lucic, M.
Forbes, B.
Grosvenor, S.
Carr, J.
Wallace, J.
Forsberg, G.
Citation: Journal of Biotechnology, 1998; 61(2):95-108
Publisher: Elsevier BV
Issue Date: 1998
ISSN: 0168-1656
Abstract: 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.
Keywords: COS Cells
Escherichia coli
Insulin-Like Growth Factor I
Insulin-Like Growth Factor II
Insulin-Like Growth Factor Binding Protein 2
Recombinant Fusion Proteins
DNA, Complementary
Genetic Techniques
Polymerase Chain Reaction
Gene Expression
Amino Acid Sequence
Base Sequence
Genetic Vectors
Molecular Sequence Data
DOI: 10.1016/S0168-1656(98)00012-1
Appears in Collections:Aurora harvest 2
Biochemistry publications

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