Engineered recombinant bacterial collagen as an alternative collagen-based biomaterial for tissue engineering.

Abstract

The key structural and signaling roles of collagen in the extracellular matrix (ECM) make it an attractive biomaterial for tissue engineering, but there are limitations in the standardization and purity of natural collagen sources currently available for such applications (Ruggiero and Koch, 2008; Werkmeister and Ramshaw, 2012). Significant effort has been made to produce human collagen in recombinant systems, such as yeast, insect cells and plants (Ruggiero et al., 2000; Myllyharju, 2009). However, the requirement for post-translational proline hydroxylation has proven to be a significant obstacle in achieving large scale production. Recent findings of collagen-like proteins in bacteria suggest these may represent alternative biosynthetic collagen materials which may complement current sources. Over the past 10 years, collagen-like proteins have been identified from numerous bacterial genomes database based on the signature (Gly-Xaa-Yaa)n repeating amino acid sequence characteristic of the collagen triple-helix (Rasmussen et al., 2003; Yu et al., 2014). Some of these collagen-like molecules may function as virulence factors by bacteria to evade the immune system of higher animals or to interact with surface receptors or with other ECM molecules necessary to promote host cell invasion (Humtsoe et al., 2005). More than 100 putative collagen-like proteins have been identified in bacterial genomes, of which eight have been recombinantly expressed in Escherichia coli (see Yu et al., 2014 for review). All eight expressed bacterial collagens were shown to form stable triple-helices with Tm~35–39°C. E. coli and most bacteria lack prolyl hydroxylase, so this high stability is attained in the absence of hydroxyproline (Hyp), a post-translationally modified amino acid known to be critical to the thermal stability of mammalian collagens. Initial interest in bacterial collagen-like proteins focused on their roles in pathogenesis. However, recent work has focused on one specific bacterial collagen protein, designated Scl2, to demonstrate the utility of recombinant bacterial collagen as a tool for defining collagen sequence/structure/function relationships and for establishing a class of novel collagen-based biomaterials. The gram positive bacterium Streptococcus pyogenes contains two collagen-like proteins, Scl1 and Scl2, which have been well characterized in terms of structure and functional properties (Lukomski et al., 2001; Xu et al., 2002; Mohs et al., 2007; Caswell et al., 2008). The Scl2 protein includes an N-terminal globular trimerization domain adjacent to a (Gly-Xaa-Yaa)79 core collagen-like domain. It has been possible to generate constructs in a recombinant E. coli system with various sequence modifications of Scl2 and to establish large scale production methods. Based on recent progress, we suggest that the Scl2 recombinant bacterial collagen system has advantages compared to recombinant human collagen strategies for large scale production and biomedical applications, and may serve as a prototype for engineering novel collagen-based biomaterials.