# Routes towards Novel Collagen-Like Biomaterials
## 2 Collagen as a Biomaterial
### Collagen-Analogues as Basis for Future Biomaterials
Since most collagen sequences are strictly water soluble but tend to form gels at higher molecular weights, peptide synthesis suffers from problems with limited solubility and aggregation as the molecular weight (MW) increases. Native chemical ligation has been used to increase the chain length after synthesis [52], which increased the thermal stability of the product but reduced the MW homogeneity of the resulting proteins. Biotechnological collagen production is non-trivial and suffers from the complex requirements of collagen synthesis (Figure 2a). In vivo, human type I collagen is assembled within fibroblasts from two pro-\(\alpha\)1(I) and one pro-\(\alpha\)2(I) procollagen chains, both around 1400 amino acids long, to form a procollagen triplehelix. Both the \(\alpha\)-collagens as well as the procollagen get enzymatically modified by prolyl-3-, prolyl-4- and lysyl-oxidase, after which the N- and C-terminal telopeptides are cleaved by specific proteases to yield the 300 nm long tropocollagen triplehelix. These tropocollagen trimers can then assemble into collagen fibrils during secretion and further form macroscopic collagen fibers with varying degrees of chemical crosslinking [53; 54]. The most common bacterial production host, _E. coli_, is unable to produce full-length collagens due to size constraints, but has successfully been utilized in the production of \"collagen-like proteins\" [55], which are truncated proteins derived from human collagen. Nevertheless, _E. coli_ has no intrinsic apparatus to catalyze the aforementioned secondary modifications, which therefore result in unstable products, mostly due to the lack of 4-hydroxyproline. A newly discovered bacterial P4H found in _Bacillus anthracis_[56; 57], might be able to overcome this limitation in future experiments. In addition, recent advances have been made in coexpressing eukaryotic proline-4-hydroxylase in origami-type _E. coli_[58], which provides conditions similar to that of the endoplasmic reticulum (ER) within  
Figure 2: (**a**) Biosynthesis of fibrillar collagens within the ECM. After translation, the nascent \(\alpha\)-chains assemble into procollagen within the endoplasmic reticulum. During and after secretion, the propeptides are cleaved by specific enzymes, yielding the soluble tropocollagen molecule, which quickly self-assembles into collagen fibrils. These fibrils can then form collagen fibers; (**b**) transmission electron micrograph of recombinantly produced bacterial \(\mathrm{ScI}1\) collagen, showing the globular terminal V-domain and the rod-like collagen helix. Bacterial collagen does not assemble into fibrils. Modified with permission from [53] (**a**) (Portland Press 2012) and [21] (**b**) (American Society for Biochemistry and Molecular Biology, 2002). its cytosol and therefore allow the activity of the transgenic P4H-complex, as well as the yeast _P. pastoris_[59; 60] which, being an eukaryote, has the necessary organelles for early-stage collagen assembly and the ability to produce high MW proteins, although only with poor yields and varying degrees of hydroxylation [61; 62]. Interestingly, bacterial collagen-like proteins have been identified [21], which function as a virulence factor in _Streptococcus pyogenes_: The Scl1 and Scl2 proteins are secreted and carboxymethyl anchored within the cell wall of the bacterium, where a terminal domain induces trimerization and, thereby, nucleation resulting in the formation of stable triple helical collagen rods (Figure 2b). The proteins then attach to collagen-binding proteins in the extracellular matrix and shield the bacterium from the immune system of e.g. vertebrates. On a structural basis, these proteins are remarkable, because they contain no non-canonical amino acids, and instead of utilizing hydroxproline, stabilize the collagen helix by forming salt bridges via charged amino acids between the \(\alpha\)-chains [22].