University of Tasmania
Browse

File(s) not publicly available

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

journal contribution
posted on 2023-05-17, 02:20 authored by Wilkinson, Ryan, Elliott, P, Carragher, JF, Francis, G
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Œ 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.

History

Publication title

Protein Expression and Purification

Volume

35

Pagination

334-343

ISSN

1046-5928

Department/School

Institute for Marine and Antarctic Studies

Publisher

Academic Press Inc Elsevier Science

Place of publication

525 B St, Ste 1900, San Diego, USA, Ca, 92101-4495

Repository Status

  • Restricted

Socio-economic Objectives

Aquaculture fin fish (excl. tuna)

Usage metrics

    University Of Tasmania

    Exports

    RefWorks
    BibTeX
    Ref. manager
    Endnote
    DataCite
    NLM
    DC