Preparation and characterization of monomers to tetramers of a collagen-like domain fromStreptococcus pyogenes

Abstract

Abbreviations: V, non-collagenous N-terminal domain of the S. pyogenes collagen-like protein; CL, collagenous domain of the S.pyogenes collagen-like protein; VCL, full collagen-like protein structureThe collagen like domain Scl2 from Streptococcus pyogenes has been proposed as a potential biomedical material. It is non-cytotoxic and non-immunogenic and can be prepared in good yield in fermentation. The Scl2 collagen domain is about a quarter of the length, 234 residues, of the main collagen type, mammalian type I collagen (1014 residues) that is currently used in biomedical devices. In the present study we have made constructs comprising 1 to 4 copies of the Scl2 collagen domain, plus these same constructs with a CysCys sequence at the C-terminal, analogous to that found in mammalian type III collagens. The yields of these constructs were examined from 2 L fermentation studies. The yields of both series declined with increasing size. Circular dichroism showed that the addition of further collagen domains did not lead to a change in the melting temperature compared to the monomer domain. Addition of the CysCys sequence led to a small additional stabilization of about 2-3 C for the monomer construct when the folding (V) domain was present.

Engineering specific chemical modification sites into a collagen‐like protein from Streptococcus pyogenes

“…The yield was similar to that found in other shake flask trials, based on SDS‐PAGE gel staining, but full determination of yield would need studies in bioreactors as previously described . The stability of the product, demonstrated by a resistance to proteolysis and determined by CD, was slightly reduced at 36.5°C as compared to the 37.6°C for unmodified protein or 37.5°C for a construct where there are two Cys residue inclusions, adjacent to each other, were at the C‐terminal of the protein . If further Cys residues were included that led to a more problematic loss in stability, then changes to other residues in the triple helix to enhance stability are possible .…”

“…Reduction of purified proteins to eliminate disulfide bonds used 5 m M tris(2‐carboxyethyl)phosphine (TCEP) prior to modification reactions. Protein stability was examined by susceptibility to proteolysis and CD spectroscopy …”

“…The construct used in this study to illustrate potential chemical reactions was based around a single, modified bacterial collagen structure, although other bacterial collagen options and Cys modification numbers and locations are also possible. The DNA sequence for the fragment of the scl2.28 allele (Q8RLX7) encoding the combined globular and collagen‐like portions of the Scl2.28 protein, but lacking the C‐terminal attachment domain was used (GenBank record: AY069936.1.).…”

Routes towards Novel Collagen-Like Biomaterials

“…Nevertheless, since these proteins circumvent all the problems associated with the production of eukaryotic collagens in bacterial hosts mentioned earlier, they are strong candidates for future materials based thereon; however, their low melting point of 36-38 • C requires the presence of a non-collagenous folding domain which has been shown to be strongly immunogenic [63] and necessitates other means of stabilization, such as glutaraldehyde crosslinking, once this domain has been removed. Increasing the stability of the Scl2 protein by multimerization does not significantly increase its melting point [68]. Therefore, while the low temperature stability of the Scl proteins is of no consequence for the production of three-dimensional scaffolds [69][70][71], it hinders the spinning of stable collagen fibers.…”

Biomedical applications of collagens

“…This approach also allows dissection of sequence hot spots from animal sequences where several overlapping functional sites are present . Further one can make new molecules, such as dimers and multimers and play “mix and match” with sequences from different species . These various products can be potentially used singly, or in combination with other modified forms.…”