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  • In the present study we have examined an alternative approac


    In the present study, we have examined, an alternative approach for introduction of Hyp into bacterial, non-animal collagens. Previously, Gruskin and colleagues [27] had shown that Hyp could be introduced into recombinant mammalian collagen sequences in E. coli through a mechanism of co-translational incorporation during fermentation using the codons for Pro in the gene construct [27]. Addition of NaCl increased the level of incorporation [27] through hyperosmotic shock increasing the intracellular availability of Hyp from the medium [27], [28]. We have expressed the Scl2 collagen from Streptococcus pyogenes in the pColdIII vector, as previously described [29], [30], using shaker flasks. The medium was changed immediately prior to induction to include Hyp and various amounts of NaCl. Changing the medium between the cell expansion phase and the production phase (see Material and Methods) avoids incorporation of Hyp into the host proteins during the expansion phase, as this could be detrimental. Also, addition of NaCl only during production avoids stressing the cells during the cell expansion phase. The use of the pColdIII vector [31] means that post-induction, almost all protein synthesis activity is focused on the expressed product, and so limits any changes to critical host proteins, while reducing or eliminating any intracellular Pro synthesis. The extent of Hyp incorporation was examined under the different NaCl conditions along with the thermal stability of the products.
    Materials and methods
    Results The modification to the fermentation protocol to include Hyp in the medium during expression of recombinant Scl2 protein from S. pyogenes led to the successful incorporation of Hyp into the protein product. This was shown, for example, by amino Proteinase K and analysis (Fig. 1) where the presence of Hyp in purified protein was seen. Hyp was incorporated even when no added NaCl was present in the medium. However, the amount of Hyp incorporated increased further with addition of NaCl to the medium (Fig. 1). Comparisons have been made to the Ala content where there are 15 residues within the collagenous domain, suggesting that even when no NaCl has been added about 6 equivalents of Hyp inclusion had occurred, with about 10–11 equivalents when 200 mM NaCl had been added. At 600 mM NaCl addition, although the protein yield was very low, the protein contained about 16–17 equivalents of Hyp, out of the total Pro content of 25 residues in a complete sequence of ∼234 residues. Although Hyp incorporation increased with NaCl addition, the overall yields of protein decreased with increasing NaCl concentrations (Fig. 2). Reasonable yields were obtained for additions up to 200 mM NaCl, but at higher concentrations a major loss in yield was seen (Fig. 2). At 700 mM and 800 mM NaCl additions (data not shown) the cell growth was particularly adversely affected and little cell growth was seen and protein yields were too low for analyses. Mass spectrometry analyses indicate the sites of the pepsin cleavage of the expressed protein products (Fig. 3). In this example, the pepsin cleavage had not reached completion, as several cleavage points were observed. Two major cleavage points were observed, between Leu and Asp and between Asp and Leu, with a further peak at a lower yield between Leu and Val (Fig. 3). Additional cleavage points were also observed that gave higher mass fragments, those between Gly and Ile and between Asp and His. It is probable that with longer pepsin treatment some of the multiple peaks would be lost. The present data are consistent with the previous determination of the amino acid sequence after pepsin digestion. Automated Edman degradation indicated that the cleavage site between Asp and Leu was a significant pepsin cleavage site, and an N-terminal sequence starting Leu-Val-Pro-Xaa-Gly-Ser-Pro-Gly- was observed [30]. The overall stability of the CL domain to pepsin confirms that the inclusion of Hyp has not adversely affected the triple-helical structure or stability.