Expression, purification and characterization of the structure and disulfide linkages of insulin-like growth factor binding protein-4
in Journal of EndocrinologySearch for other papers by D Chelius in
Current site
Google Scholar
PubMed
Search for other papers by MA Baldwin in
Current site
Google Scholar
PubMed
Search for other papers by X Lu in
Current site
Google Scholar
PubMed
Search for other papers by EM Spencer in
Current site
Google Scholar
PubMed
Insulin-like growth factor binding protein-4 (IGFBP-4), like the other five IGFBPs, is a critical regulator of the activity of insulin-like growth factor (IGF)-I and IGF-II. However IGFBP-4 seems to be the only IGFBP with no potential to enhance the mitogenic actions of the IGFs. IGFBP-1 to -3 and -5 each contain 18 conserved cysteine residues, IGFBP-6 lacks two of the twelve N-terminal cysteines, while IGFBP-4 has two additional cysteines in the central region. A plasmid was constructed to express rat IGFBP-4 as a thioredoxin fusion protein that included a hexahistidine sequence to permit affinity purification. The fusion protein was expressed in E.coli, purified using nickel-chelate affinity chromatography and cleaved by tobacco etch virus (TEV) protease to produce mature rat IGFBP-4 with an additional glycine residue at the N-terminus. Final purification was achieved by further nickel affinity chromatography and reverse phase HPLC. The isoelectric points of the recombinant IGFBP-4 were the same as those of the non-glycosylated isoforms of IGFBP-4 in rat serum. The binding affinities of the recombinant protein and IGFBP-4 secreted by rat cells to IGF-I were compared using a newly developed binding assay. No significant difference could be detected, consistent with proper folding of the recombinant protein. This indicates that glycosylation of IGFBP-4 does not affect its binding to IGF-I. Using mass spectrometry and tandem mass spectrometry no differences between authentic and recombinant IGFBP-4 could be detected. Eight of the ten disulfide linkages have been determined, including linkages of conserved cysteine residues not previously identified in other IGFBPs. Numbering the cysteine residues sequentially from the N-terminus only the disulfide connectivity of C1, C2, C5 and C6 could not be determined. However, C1 is not linked to C1 and C5 is not linked to C6. The established linkages were C3 to C8, C4 to C7, C9 to C 11, and C10 to C12. The two cysteines in the non-conserved mid-region unique to IGFBP-4 (C13 and C14) are linked together. Linkage of the C-terminal cysteine residues is identical to that of IGFBP-2, -5 and -6 (C15 to C16, C17 to C18 and C19 to C20). The central flexible core of IGFBP-4, containing two additional cysteines may contribute to its unique biological action.
Journal of Endocrinology is committed to supporting researchers in demonstrating the impact of their articles published in the journal.
The two types of article metrics we measure are (i) more traditional full-text views and pdf downloads, and (ii) Altmetric data, which shows the wider impact of articles in a range of non-traditional sources, such as social media.
More information is on the Reasons to publish page.
| Sept 2018 onwards | Past Year | Past 30 Days | |
|---|---|---|---|
| Full Text Views | 286 | 165 | 18 |
| PDF Downloads | 151 | 66 | 2 |
Online ISSN: 1479-6805
Print ISSN: 0022-0795
Strengthening biomedical communities
to advance science and health
CONTACT US
Bioscientifica Ltd | Starling House | 1600 Bristol Parkway North | Bristol BS34 8YU | UK
Bioscientifica Ltd | Registered in England no 3190519