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  • The present study has shown that additional protein

    2019-07-15

    The present study has shown that additional protein stability can be achieved (Fig. 6). The 0.6 to 1.0 °C increases that were observed were not large compared, for example, to the ∼5 °C increase that has been reported using sequence modifications [22]. This may reflect that the Hyp enters both the Xaa and Yaa positions in the triple-helix and that those in the Xaa position may be slightly destabilising [37]. The method of incorporation means that the Hyp residues are probably present in both the Xaa and Yaa positions as Pro is found in both locations (Fig. 7). The sequence data show that in the CL domain Pro is found in the Xaa position predominantly in the first, N-terminal third of the sequence while Pro in the Yaa position is found predominantly in the C-terminal third of the sequence (Fig. 7). The stabilising effect of Hyp in the Yaa position is well established from studies on full-length collagens [6] as well as peptides studies, for example using the peptide (Pro-Hyp-Gly)10[38]. However, the effect on stability of 4-Hyp in the Xaa position has been less certain. Initially, studies on the peptide (Hyp-Pro-Gly)10 showed that it did not form a stable triple helix [39], suggesting that 4-Hyp in the Xaa position destabilised the triple helix. Subsequently, however, studies on a different polypeptide, (Gly-Hyp-Thr(β-D-Gal))10[40], which does form a stable triple helix suggested that 4-Hyp in the Xaa position, albeit in an unusual glycosylated construct, may not necessarily be destabilising, with the repetitive peptide (Hyp-Pro-Gly)10 being an anomaly. Further studies, using a host-guest system in which single substitutions are made into a constant baseline structure (Gly-Pro-Hyp)8[41], showed that a single 4-Hyp in the Xaa position did not lead to stabilisation like 4-Hyp in the Yaa position [37]. The fairly small increase in Tm when Hyp is incorporated reflects in part that there are approximately twice as many Pro residues in the Xaa than the Yaa position (Fig. 7). If there is a preference for only having Hyp in the Yaa position then an alternative approach is needed. For example, this could be a system such as non-canonical amino Natural Product Library mutagenesis, where the cellular protein synthesis is ‘evolved’ so that the tRNA and tRNA synthase systems are adapted to allow Hyp incorporation [42], [43]. Alternatively, sequence modifications could be made [22]. The samples from the experiments with higher amounts of added NaCl, >200 mM, had much lower yields, in part because they were also aggregating. Normally, bacterial collagens which lack any Hyp do not seem to form aggregates, for example by the lateral associations that are normal for animal interstitial (fibrillar) collagens [44]. Recombinant animal collagens that have been produced without any prolyl hydroxylation also do not form aggregated fibrillar structures [10]. Together, both these observations indicate the key role of Hyp in forming and stabilising higher order structures. In the present study, it may be that inclusion of Hyp is leading to aggregates, which may affect extraction of the recombinant product as well as leading to loss of yield during purification. The low yield of soluble protein from experiments where >200 mM NaCl has been added suggests that practically, considering the yield versus the extent of modification, a preferred value for NaCl inclusion would be no more than 200 mM NaCl.
    Introduction Collagen is the most abundant protein in animals, and maintains the structural integrity of various connective tissues. The overproduction of collagen is associated with fibrotic diseases, including myocardial infarction, stroke, peripheral vascular disease, diabetes, and severe anemias. The stability of collagen relies on catalysis by collagen prolyl 4-hydroxylase (P4H). This enzyme is located in the lumen of the endoplasmic reticulum, where it catalyzes a remarkable post-translational modification—the conversion of (2S)-proline residues (Pro) to (2S,4R)-4-hydroxyproline residues (Hyp) in protocollagen strands. The human enzyme consists of an α2β2 tetramer in which each α subunit contains an active site. P4H is essential for animals,4, 5, 6 as the conformational stability of mature collagen relies on its hydroxylation.7, 8, 9 Moreover, P4H is a validated target for the treatment of fibrotic diseases.