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  • br Discussion The use of

    2020-07-27


    Discussion The use of co-translational incorporation during fermentation [27] to introduce Hyp residues instead of Pro residues in a bacterial collagen has been demonstrated. Inclusion of NaCl in the medium further increases the level of Hyp incorporation. It has been proposed [27] that this is due to hyperosmotic shock that increases the intracellular availability and proportion of Hyp relative to Pro, with the Hyp being incorporated using the standard Pro codons through their broad specificity. Both amino Pefloxacin sale analysis and mass spectrometry confirmed the incorporation of the Hyp. The amino acid analysis data suggested a larger incorporation than the mass spectrometry, which may be due to limited resolution of the deconvoluted mass spectra obtained from the intact protein. The use of the pCold vector system seems an ideal choice for this approach as high cell densities can be achieved prior to addition of Hyp and optionally NaCl to the medium prior to induction. After induction, this system almost exclusively produces the recombinant protein, so it is probable that a high efficiency in Hyp incorporation is being achieved [31]. Bacterial collagens do not contain any Hyp in their natural state [15] and can be readily expressed in excellent yield in E. coli[23]. While these proteins are stable without the Hyp that is an essential factor in the stability of animal collagens, the opportunity to include Hyp can assist with further enhancing the thermal stability of the proteins which may assist in their utility as a biomedical material [14], [29], [36]. Also, it could allow better activity to be achieved for binding domains that preferably require Hyp to be present. For example, binding to α2β1 integrin is reduced and receptor activation is absent for discoidin domain receptor when proline hydroxylation is absent [12], [13]. 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 acid 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].