Expanding the family of collagen proteins: recombinant bacterial collagens of varying composition form triple-helices of similar stability

Abstract

The presence of the (Gly-Xaa-Yaa)(n) open reading frames in different bacteria predicts the existence of an expanded family of collagen-like proteins. To further explore the triple-helix motif and stabilization mechanisms in the absence of hydroxyproline (Hyp), predicted novel collagen-like proteins from Gram-positive and -negative bacteria were expressed in Escherichia coli and characterized. Soluble proteins capable of successful folding and in vitro refolding were observed for collagen proteins from Methylobacterium sp 4-46, Rhodopseudomonas palustris and Solibacter usitatus . In contrast, all protein constructs from Clostridium perfringens were found predominantly in inclusion bodies. However, attachment of a heterologous N-terminal or C-terminal noncollagenous folding domain induced the Clostridium perfringens collagen domain to fold and become soluble. The soluble constructs from different bacteria had typical collagen triple-helical features and showed surprisingly similar thermal stabilities despite diverse amino acid compositions. These collagen-like proteins provide a resource for the development of biomaterials with new properties.

Figure 1. Pie chart representation of the non-Gly residues composition of the bacterial collagen-like domains

Amino acids only at Xaa and Yaa positions have been considered. The following groups are shown: Polar (Ser, Thr, Cys, Asn, Gln), Charged (Asp, Glu, Lys, Arg, His), Small (Gly and Ala), Pro and Hydrophobic (Val, Ile, Leu, Met, Phe, Trp, Cys). For comparison, the amino acid composition of the αI (I) chain of human type I collagen is shown.

Figure 2. Schematic diagram of recombinant proteins with bacterial collagen-like domains, constructed for the expression in E coli

Numbers, length of each domain in AA; black filled boxes, CL domains of corresponding proteins; empty boxes, N- and C-terminal domains; vertical empty box, collagen-like sequence interruption; boxes with line dashed patterns, V domain from Scl2 of S. pyogenesis (V_Sp) and C-terminal V domain from R. palustris (V_Rp). Also shown are the expression levels (Ex) and solubility (Sol), determined by fractionation and SDS PAGE.

Figure 3. Expression of recombinant proteins in E. coli and resistance to trypsin digestion

Recombinant plasmids were transfected in BL21 strain. Protein expression was induced by 1 mM isopropyl-D-thiogalactopyranoside overnight at 20°C. Proteins were purified on an Ni-NTA agarose (QIAGEN) column and dialyzed against Na-phosphate buffer, pH 8.6 with 50 mM glycine. To test for sensitivity of the recombinant proteins to trypsin digestion, they were digested at room temperature with trypsin at ratio 1:1000 (protein:enzyme) for one hour (or different period of time where shown) and efficiency of the digestion was checked by electrophoresis. Arrows indicate trypsin resistant species.

Figure 4. Thermal stability of the recombinant bacterial collagen-like domains

CD thermal transitions of the corresponding CL domains at pH 8.6 (filled squares, continuous lines) and pH 2.2 (empty circles, dotted lines) were monitored at 220 nm. CD spectra of proteins are shown as insets.

Figure 5. Thermal transitions of the recombinant proteins determined by the monitoring CD signal at 220 nm

The arrow indicates the direction of temperature change with →, for the unfolding curve with increasing temperature and ← for the refolding curve with decreasing temperature. The heating rates were ~0.1°C/min in both directions. CD spectra of each protein are shown as an inset.