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Supporting Text

Recombinant Expression of the Collagen Peptides in Escherichia coli.

The bacterial expression vector pHisMfCol13Cys₂ encoding the fusion protein consisting of the His-Tag sequence, the mini-fibritin domain, the thrombin cleavage site, and the collagen fragment Col13Cys₂ was used for the preparation of (Col13Cys₂)₃. The cDNA encoding the a 1 chain of human type III collagen was a gift from Takako Sasaki (Max-Planck-Institut für Biochemie, Martinsried, Germany). The cDNA was used as a template to prepare the gene encoding the type III collagen fragment Col49Cys₂, spanning residues GS-GYP-G874-G1023-G (numbering of mature human type III collagen) by PCR using two oligonucleotides:

5'-GACGGATCCGGCTATCCGGGCCCACCTGGTCCTGTCGGTCCAGCTGGAAA-3' (forward primer, with the BamHI site underlined) and 5'-CGAATTCTTAGCCACCACCACAGCAAGGACCAGGGGC-3' (reversed primer, with the EcoRI site underlined). The amplified gene was cloned into the BamHI/EcoRI site of the bacterial expression vector pHisMf . The plasmid pHisMf was designed to express a 6´ His-tag (amino acid sequence MHHHHHH), a short linker (GSG), the mini-fibritin domain (T2-L109), a second linker (GSSGS), thrombin cleavage site (GLVPRGS), and the insert of interest, which could be cloned into the BamHI/EcoRI site. Recombinant insert DNA was verified by Sanger dideoxy DNA sequencing. Two additional residues, Gly and Ser, at the N terminus are necessary for the thrombin cleavage site and were retained after the proteolysis. The GYP triplet was introduced for concentration determination. This sequence deviates from the human sequence, but a GYP triplet is present at the corresponding position in the chicken sequence.

The recombinant proteins were expressed in E. coli JM109(DE3) host strain after IPTG induction (final concentration 1 mM). Purification of the 6´ His-tagged fusion proteins by immobilized metal affinity chromatography on Ni²⁺-Sepharose (Apbiotech, Piscataway, NJ) and separation of the collagen fragment from the 6´ His-tagged carrier protein after thrombin cleavage were carried out as described in ref. 1. Col49Cys₂ was additionally subjected to two cycles of purification on a Macro-Prep Ceramic hydroxyapatite column (Bio-Rad). The protein sample was dialyzed against 50 mM Hepes and 10 mM potassium phosphate (pH 8.0) at 4° C. Before loading the column, the sample was heated for 15 min at 32° C (and completely unfolded), chilled down to 4° C, and kept at this temperature for 3 h to allow the collagen to refold. After that, the sample was loaded on the column (all steps at 4° C) and was step-wise eluted by increasing concentration of potassium phosphate complemented with 50 mM Hepes (pH 8.0). The non-cross-linked and shortened fragments were eluted at 10, 25, and 50 mM potassium phosphate, while the cross-linked material was eluted at 200 mM. The majority (» 90%) of contaminants was removed after the first cycle. Without prior unfolding and refolding, the sample was eluted with all contaminants in 200 mM potassium phosphate showing the tight interaction of collagen triple helix with hydroxyapatite. Thus, only single unfolded or misfolded collagen chains could be eluted at low potassium concentrations. It is known that the refolding of single long collagen chains is very slow and leads to misaligned aggregates; this observation was used to purify cross-linked (Col49Cys₂)₃. The total yield of the purified (Col49Cys₂)₃ out of 1 liter of a bacterial culture medium was 0.5 mg.

The concentrations of proteins were determined by tyrosine absorption in 6 M guanidine hydrochloride.