Expression, purification, and in vitro characterization of recombinant salmon insulin-like growth factor-II
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...
Published in: | Protein Expression and Purification |
---|---|
Main Authors: | , , , |
Format: | Article in Journal/Newspaper |
Language: | English |
Published: |
Academic Press Inc Elsevier Science
2004
|
Subjects: | |
Online Access: | https://doi.org/10.1016/j.pep.2004.02.014 http://www.ncbi.nlm.nih.gov/pubmed/15135411 http://ecite.utas.edu.au/63064 |
id |
ftunivtasecite:oai:ecite.utas.edu.au:63064 |
---|---|
record_format |
openpolar |
spelling |
ftunivtasecite:oai:ecite.utas.edu.au:63064 2023-05-15T15:32:40+02:00 Expression, purification, and in vitro characterization of recombinant salmon insulin-like growth factor-II Wilkinson, Ryan Elliott, P Carragher, JF Francis, G 2004 https://doi.org/10.1016/j.pep.2004.02.014 http://www.ncbi.nlm.nih.gov/pubmed/15135411 http://ecite.utas.edu.au/63064 en eng Academic Press Inc Elsevier Science http://dx.doi.org/10.1016/j.pep.2004.02.014 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] http://www.ncbi.nlm.nih.gov/pubmed/15135411 http://ecite.utas.edu.au/63064 Biological Sciences Biochemistry and Cell Biology Biochemistry and Cell Biology not elsewhere classified Refereed Article PeerReviewed 2004 ftunivtasecite https://doi.org/10.1016/j.pep.2004.02.014 2019-12-13T21:33:06Z 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. Article in Journal/Newspaper Atlantic salmon Salmo salar eCite UTAS (University of Tasmania) Protein Expression and Purification 35 2 334 343 |
institution |
Open Polar |
collection |
eCite UTAS (University of Tasmania) |
op_collection_id |
ftunivtasecite |
language |
English |
topic |
Biological Sciences Biochemistry and Cell Biology Biochemistry and Cell Biology not elsewhere classified |
spellingShingle |
Biological Sciences Biochemistry and Cell Biology Biochemistry and Cell Biology not elsewhere classified Wilkinson, Ryan Elliott, P Carragher, JF Francis, G Expression, purification, and in vitro characterization of recombinant salmon insulin-like growth factor-II |
topic_facet |
Biological Sciences Biochemistry and Cell Biology Biochemistry and Cell Biology not elsewhere classified |
description |
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. |
format |
Article in Journal/Newspaper |
author |
Wilkinson, Ryan Elliott, P Carragher, JF Francis, G |
author_facet |
Wilkinson, Ryan Elliott, P Carragher, JF Francis, G |
author_sort |
Wilkinson, Ryan |
title |
Expression, purification, and in vitro characterization of recombinant salmon insulin-like growth factor-II |
title_short |
Expression, purification, and in vitro characterization of recombinant salmon insulin-like growth factor-II |
title_full |
Expression, purification, and in vitro characterization of recombinant salmon insulin-like growth factor-II |
title_fullStr |
Expression, purification, and in vitro characterization of recombinant salmon insulin-like growth factor-II |
title_full_unstemmed |
Expression, purification, and in vitro characterization of recombinant salmon insulin-like growth factor-II |
title_sort |
expression, purification, and in vitro characterization of recombinant salmon insulin-like growth factor-ii |
publisher |
Academic Press Inc Elsevier Science |
publishDate |
2004 |
url |
https://doi.org/10.1016/j.pep.2004.02.014 http://www.ncbi.nlm.nih.gov/pubmed/15135411 http://ecite.utas.edu.au/63064 |
genre |
Atlantic salmon Salmo salar |
genre_facet |
Atlantic salmon Salmo salar |
op_relation |
http://dx.doi.org/10.1016/j.pep.2004.02.014 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] http://www.ncbi.nlm.nih.gov/pubmed/15135411 http://ecite.utas.edu.au/63064 |
op_doi |
https://doi.org/10.1016/j.pep.2004.02.014 |
container_title |
Protein Expression and Purification |
container_volume |
35 |
container_issue |
2 |
container_start_page |
334 |
op_container_end_page |
343 |
_version_ |
1766363165016195072 |