Mass spectrometry has previously been used to
Mass spectrometry has previously been used to examine the presence of Hyp in recombinant collagen, using hydrolysates to identify the individual imino acid, equivalent to an amino doxercalciferol analysis , and also by analysis of mass changes to tryptic peptides . The present study, however, has examined intact protein and has identified Hyp incorporation into the collagen samples (Fig. 3, Fig. 4). This was evident by additional peaks (some are shown with asterisks) with mass increases of +16 Da due to the additional oxygen atom in the Hyp relative to Pro. This is illustrated readily by a “butterfly plot” comparing test samples to a control sample where there was no added Hyp nor any added NaCl in the expression medium. These data show that more than one Hyp was incorporated in each sample. The mass spectrometry data also showed an exponential drop in the observed intensity of collagen ions for samples with increasing NaCl concentration in the expression buffer, as well as an increase in the relative amount of Hyp incorporation into the collagen protein as NaCl concentration (Fig. 5).
The effect of inclusion of Hyp in the collagen structure was examined by circular dichroism spectroscopy (Fig. 6). The Tm values were averaged from 3 determinations for each sample type. The observed variation between determinations was 0.2 °C or better for each sample type. The extent of the data that could be collected was limited to the control sample, with no Hyp inclusion, and samples obtained after addition of 100 and 200 mM NaCl to the protein expression. At higher NaCl concentrations the protein yield became significantly reduced, and, furthermore, the samples that were obtained had very low solubility and had a tendency to aggregate. Nevertheless, the data obtained using a 1 °C/min temperature ramp in all cases indicated that the thermal stability of both of the samples that included Hyp were slightly greater than the control sample by about 0.6 and 1.0 °C (Fig. 6). Changes in the rate of heating lead to changes in the observed the Tm values obtained for a single sample , but should have less effect on comparisons between samples examined under the same conditions, as in the present study.
Discussion The use of co-translational incorporation during fermentation  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  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 acid 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 . Bacterial collagens do not contain any Hyp in their natural state  and can be readily expressed in excellent yield in E. coli. 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 , , . 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 , .