Expression, purification, and refolding of recombinant collagen α1(XI) amino terminal domain splice variants

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.

Biomineralization of bone: a fresh view of the roles of non-collagenous proteins

“…In contrast, another splice variant, 8, while expressed by UMR106-01 osteoblastic cells as a full length 105 kDa alpha 1 chain, is not enriched within BMF (Figure 2). Both the 6b and 8 sequences are part of the N-terminal domain of the type XI alpha 1 chain (58) (Figure 3). The charge character of the NTD can be changed dramatically by alternative splicing since the 6b sequence has an isoelectric point of 11.3 and unusual arginine triplet repeat sequences.…”

“…Model of type XI collagen structure [adapted from Warner et al (58)]. A, Proposed trimeric structure showing large non-helical N-terminal domain for alpha 1 chain.…”

Differential Expression of Collagen Type V and XI α-1 in Human Ascending Thoracic Aortic Aneurysms

“…This differential regulation has been previously demonstrated in smooth muscle cells in the tunica media of ATAAs [19, 20] and in aortic smooth muscle cells in culture [17], and has been suggested as a possible mechanism modulating ECM remodeling. Recent studies have shown that the amino-terminal propeptide of collagen α1(XI) contains a well-characterized heparin binding domain [21, 22]. This heparin binding domain has been shown to interact with specific integrin receptors that promote the regulation of expression and activity of matrix metalloproteinases (MMPs) [23].…”

Collagen 11a1 is indirectly activated by lymphocyte enhancer-binding factor 1 (Lef1) and negatively regulates osteoblast maturation

“…Recombinant proteins were purified, refolded and characterized as previously described (Warner et al 2007). …”