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Expression, purification, and in vitro characterization of recombinant salmon insulin-like growth factor-II

Citation

Wilkinson, Ryan and Elliott, P and Carragher, JF and Francis, G, Expression, purification, and in vitro characterization of recombinant salmon insulin-like growth factor-II, Protein Expression and Purification, 35, (2) pp. 334-343. ISSN 1046-5928 (2004) [Refereed Article]

DOI: doi:10.1016/j.pep.2004.02.014

Abstract

The insulin-like growth factors, IGF-I and IGF-II, are single chain polypeptides, which are structurally related to proinsulin and promote proliferation and differentiation of cells in many vertebrate species. Previous attempts to produce recombinant salmon IGF-II (rsIGF-II) were compromised by low expression levels and co-purification of incorrectly cleaved protein with the authentic recombinant product. In this study, a gene containing the coding region for Atlantic salmon (Salmo salar) IGF-II was cloned into a modified pET32a expression vector and transformed into Escherichia coli BL21 trxB (DE3) cells. Upon growth and induction (with IPTG) of the transformant, recombinant salmon IGF-II (rsIGF-II) was expressed as an insoluble, 28 kDa thioredoxin.sIGF-II fusion protein linked by a protease cleavage motif (trx.FAHY.sIGF-II) in inclusion bodies. The inclusion bodies were subsequently solubilized and the fusion protein was purified by Ni-affinity chromatography. Recombinant IGF-II (7.8 kDa) was then released from the fusion partner using H64A subtilisin BPN protease and purified by reversed-phase HPLC. Homogeneity of the final recombinant product was confirmed by N-terminal amino acid sequencing, ion-spray mass spectrometry, SDS-polyacrylamide gel electrophoresis, and analytical reversed-phase HPLC. The biological activity of rsIGF-II was demonstrated in cultured rat L6 myoblasts and was found to be approximately 9- and 5-fold less potent than recombinant human IGF-I and recombinant salmon IGF-I, respectively, a result similar to that demonstrated previously with other recombinant fish IGF-II's in non-homologous cell lines.

Item Details

Item Type:Refereed Article
Research Division:Biological Sciences
Research Group:Biochemistry and Cell Biology
Research Field:Biochemistry and Cell Biology not elsewhere classified
Objective Division:Animal Production and Animal Primary Products
Objective Group:Fisheries - Aquaculture
Objective Field:Aquaculture Fin Fish (excl. Tuna)
Author:Wilkinson, Ryan (Dr Ryan Wilkinson)
ID Code:63064
Year Published:2004
Web of Science® Times Cited:11
Deposited By:NC Marine Conservation and Resource Sustainability
Deposited On:2010-04-12
Last Modified:2010-05-04
Downloads:0

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