Expression of an engineered form of recombinant procollagen in mouse milk

Abstract

We have examined the suitability of the mouse mammary gland for expression of novel recombinant procollagens that can be used for biomedical applications. We generated transgenic mouse lines containing cDNA constructs encoding recombinant procollagen, along with the α and β subunits of prolyl 4-hydroxylase, an enzyme that modifies the collagen into a form that is stable at body temperature. The lines expressed relatively high levels (50–200 μg/ml) of recombinant procollagen in milk. As engineered, the recombinant procollagen was shortened and consisted of a proα2(I) chain capable of forming a triple-helical homotrimer not normally found in nature. Analysis of the product demonstrated that (1) the proα chains formed disulphide-linked trimers, (2) the trimers contained a thermostable triple-helical domain, (3) the N-propeptides were aligned correctly, and (4) the expressed procollagen was not proteolytically processed to collagen in milk.

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Figure 1: Transgene constructs and recombinant protein schematic diagram.
Figure 2: Southern and northern blot analysis of transgenic lines.
Figure 3: Recombinant procollagen expression in the milk of transgenic mice.
Figure 4: Chymotrypsin-trypsin treatment—helix thermostability determination.
Figure 5: Expressed procollagens are trimers composed of intermolecularly disulphide-bonded monomers.
Figure 6: N-proteinase cleavage of recombinant procollagen.

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Acknowledgements

The authors wish to thank Yvonne Gibson and the PPL small animal unit for the production of transgenic mice. K.E.K. is a recipient of a Senior Research Fellowship from the Wellcome Trust (019512). N.J.B. is a recipient of a Senior Research Fellowship from the Royal Society. An MRC-LINK cell engineering grant (G9623358) generously supported the work.

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Correspondence to Neil J. Bulleid.

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John, D., Watson, R., Kind, A. et al. Expression of an engineered form of recombinant procollagen in mouse milk. Nat Biotechnol 17, 385–389 (1999). https://doi.org/10.1038/7945

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