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Production of two recombinant insulin-like growth factor binding protein-1 subtypes specific to salmonids.
- Source :
-
General and comparative endocrinology [Gen Comp Endocrinol] 2020 Dec 01; Vol. 299, pp. 113606. Date of Electronic Publication: 2020 Sep 03. - Publication Year :
- 2020
-
Abstract
- Salmonids have four subtypes of insulin-like growth factor binding protein (IGFBP)-1, termed -1a1, -1a2, -1b1 and 1b2, owing to teleost- and a lineage-specific whole-genome duplications. We have previously produced recombinant proteins of masu salmon IGFBP-1a1 and -1b2 and conducted functional analysis. To further characterize salmonid-specific IGFBP-1s, we cloned cDNAs encoding mature proteins of IGFBP-1a2 and -1b1 from the liver of masu salmon (Oncorhynchus masou). IGFBP-1a2 and -1b1 shared a 56% amino acid sequence homology whereas their homologies with their counterparts (i.e. -1a1 and -1b2) were 77% and 82%, respectively. We next expressed recombinant masu salmon (rs) IGFBP-1a2 and -1b1 with fusion partners thioredoxin (Trx) and a His-tag using the pET-32a(+) vector system in Escherichia coli. Trx.His.rsIGFBP-1s were detected in the insoluble faction, solubilized in a buffer containing urea, and isolated by Ni-affinity chromatography. They were refolded by dialysis and cleaved from the fusion partners by enterokinase. rsIGFBP-1a2 and -1b1 were purified by reversed-phase high performance liquid chromatography. Purified rsIGFBP-1a2 and -1b1 had the ability to bind digoxigenin-labeled human IGF-I on ligand blotting. We then examined the effects of rsIGFBP-1a1, -1a2, -1b1 and -1b2 in combination with human IGF-I on growth hormone (GH) release from cultured pituitary cells of masu salmon. IGF-I alone reduced GH release while the addition of rsIGFBP-1a1, -1b1 or -1b2, but not rsIGFBP-1a2, diminished the suppressive effect of IGF-I. Addition of rsIGFBP-1s without IGF-I had no effect on GH release. These results show that rsIGFBP-1b1, along with rsIGFBP-1a1 and -1b2, inhibits IGF-I action on the pituitary in masu salmon. The lack of the effect by rsIGFBP-1a2 suggests that salmon IGFBP-1 subtypes underwent subfunction partitioning and have different degrees of IGF-inhibitory action.<br /> (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Subjects :
- Animals
Humans
Insulin-Like Growth Factor Binding Protein 1 genetics
Protein Isoforms
Recombinant Proteins genetics
Salmonidae metabolism
Sequence Homology, Amino Acid
Human Growth Hormone metabolism
Insulin-Like Growth Factor Binding Protein 1 metabolism
Insulin-Like Growth Factor I metabolism
Pituitary Gland metabolism
Recombinant Proteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1095-6840
- Volume :
- 299
- Database :
- MEDLINE
- Journal :
- General and comparative endocrinology
- Publication Type :
- Academic Journal
- Accession number :
- 32890480
- Full Text :
- https://doi.org/10.1016/j.ygcen.2020.113606