Collagen is the most abundant structural protein in vertebrates being a substantial part of the extracellular matrix. The protein is built of three polyproline-II-like polypeptides which super-coil forming a triple-helix. These triple-helices form bundles and fibres to form larger tissue structures.1
Collagen is used in a variety of medical products, but there is significant concern about possible transfer of diseases due to extraction from animal sources. Cost-effective production of recombinant collagen is desirable.2 For animal collagens, the requirement of co-expression of prolyl-4-hydroxylase (P4H), which enables secondary modification of certain proline residues to give hydroxyproline, makes fermentation cost intensive and complex.2 An alternative approach uses stable collagen-like triple-helical sequences, with glycine as every third residue and high proline content, which have been found in various bacteria, and which do not require co-expression with P4H.3
The bacterial collagen V-CL(V: globular domain, CL: collagen-like domain) examined in this work was fermented giving yields up to 19 g/L lacking biological activity, making it a versatile template for further modifications.4 To enhance the properties of this collagen-like protein, expressed protein ligation (EPL) is used. This method enables a number of novel materials to be produced. A key aspect of this approach is the possibility to modify the protein after expression by functionalising its C-terminal in many potential ways only at one specific site. For example, new functionality can be easily introduced by ligation of biologically active peptides with different cell specificities. In particular, this allows the introduction of unusual amino acids, cyclic peptides, sugars, molecules to enhance solubility, cross-linking, etc., that cannot normally be introduced by recombinant approaches. Alternatively, constructs bearing an N-terminal Cys residue can be ligated to the protein via its C-terminal thioester yielding a native peptide bond and forming dimers or higher polymers.