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Article
Expression, Purification, and Refolding of Recombinant Collagen α1(Xi) Amino Terminal Domain Splice Variants
Protein Expression and Purification
  • Lisa R. Warner, Boise State University
  • Christina M. Blasick, Boise State University
  • Raquel J. Brown, Boise State University
  • Julia Thom Oxford, Boise State University
Document Type
Article
Publication Date
4-1-2007
DOI
http://dx.doi.org/10.1016/j.pep.2006.10.016
Disciplines
Abstract

The amino terminal domain of collagen type XI α1 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 α1(XI) NTD[p7] and α1(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.

Citation Information
Lisa R. Warner, Christina M. Blasick, Raquel J. Brown and Julia Thom Oxford. "Expression, Purification, and Refolding of Recombinant Collagen α1(Xi) Amino Terminal Domain Splice Variants" Protein Expression and Purification (2007)
Available at: http://works.bepress.com/julia_oxford/26/