Expression, purification, and refolding of recombinant collagen alpha1(XI) amino terminal domain splice variants.
The amino terminal domain of collagen type XI alpha1 chain is a noncollagenous structure that is essential for the regulation of fibrillogenesis in developing cartilage. The amino terminal domain is alternatively spliced at the mRNA level, resulting in proteins expressed as splice variants. These splice variants, or isoforms, have unique distribution in growing tissues, alluding to distinct roles in development. We report here a rapid and straightforward method for expression, purification and in vitro folding of recombinant collagen XI isoforms alpha1(XI) NTD[p7] and alpha1(XI) NTD[p6b+7]. The recombinant isoforms were expressed in Escherichia coli as bacterial inclusion bodies. Unfolded carboxy terminal polyhistidine tagged proteins were purified via nickel affinity chromatography and refolded with specific protocols optimized for each isoform. Purity was assessed by SDS-PAGE and correct secondary structure by a comparison of circular dichroism data with that obtained for Npp. Protein expression and purification of the recombinant collagen XI splice variants will allow further studies to elucidate the structure and molecular interactions with components of the extracellular matrix. This research will clarify the mechanism of collagen XI mediated regulation of collagen fibrillogenesis.
Warner
Lisa R
LR
Department of Materials Science and Engineering, Boise State University, Boise, ID, USA.
Blasick
Christina M
CM
Brown
Raquel J
RJ
Oxford
Julia Thom
JT
D017398:Alternative Splicing / Q000502:physiology*; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D024161:Collagen Type XI / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D024161:Collagen Type XI; D011994:Recombinant Proteins