Genetically modified human type II collagen for N- and C-terminal covalent tagging

Wieczorek A, Chan CK, Kovacic S, Li C, Dierks T, Forde NR (2018)
CANADIAN JOURNAL OF CHEMISTRY 96(2): 204-211.

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Abstract
Collagen is the predominant structural protein in vertebrates, where it contributes to connective tissues and the ECM; it is also widely used in biomaterials and tissue engineering. Dysfunction of this protein and its processing can lead to a wide variety of developmental disorders and connective tissue diseases. Recombinantly engineering the protein is challenging due to post-translational modifications generally required for its stability and secretion from cells. Introducing end labels into the protein is problematic, because the N- and C-termini of the physiologically relevant tropocollagen lie internal to the initially flanking N-and C-propeptide sequences. Here, we introduce mutations into human type II procollagen in a manner that addresses these concerns and purify the recombinant protein from a stably transfected HT1080 human fibrosarcoma cell line. Our approach introduces chemically addressable groups into the N-and C-telopeptide termini of tropocollagen. Simultaneous overexpression of formylglycine generating enzyme (FGE) allows the endogenous production of an aldehyde tag in a defined, substituted sequence in the N terminus of the mutated collagen, whereas the C-terminus of each chain presents a sulfhydryl group from an introduced cysteine. These modifications are designed to enable specific covalent end-labelling of collagen. We find that the doubly mutated protein folds and is secreted from cells. Higher order assembly into well-ordered collagen fibrils is demonstrated through transmission electron microscopy. Chemical tagging of thiols is successful; however, background from endogenous aldehydes present in wild-type collagen has thus far obscured the desired specific N-terminal labelling. Strategies to overcome this challenge are proposed.
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Wieczorek A, Chan CK, Kovacic S, Li C, Dierks T, Forde NR. Genetically modified human type II collagen for N- and C-terminal covalent tagging. CANADIAN JOURNAL OF CHEMISTRY. 2018;96(2):204-211.
Wieczorek, A., Chan, C. K., Kovacic, S., Li, C., Dierks, T., & Forde, N. R. (2018). Genetically modified human type II collagen for N- and C-terminal covalent tagging. CANADIAN JOURNAL OF CHEMISTRY, 96(2), 204-211. doi:10.1139/cjc-2017-0335
Wieczorek, A., Chan, C. K., Kovacic, S., Li, C., Dierks, T., and Forde, N. R. (2018). Genetically modified human type II collagen for N- and C-terminal covalent tagging. CANADIAN JOURNAL OF CHEMISTRY 96, 204-211.
Wieczorek, A., et al., 2018. Genetically modified human type II collagen for N- and C-terminal covalent tagging. CANADIAN JOURNAL OF CHEMISTRY, 96(2), p 204-211.
A. Wieczorek, et al., “Genetically modified human type II collagen for N- and C-terminal covalent tagging”, CANADIAN JOURNAL OF CHEMISTRY, vol. 96, 2018, pp. 204-211.
Wieczorek, A., Chan, C.K., Kovacic, S., Li, C., Dierks, T., Forde, N.R.: Genetically modified human type II collagen for N- and C-terminal covalent tagging. CANADIAN JOURNAL OF CHEMISTRY. 96, 204-211 (2018).
Wieczorek, Andrew, Chan, Clara K., Kovacic, Suzana, Li, Cindy, Dierks, Thomas, and Forde, Nancy R. “Genetically modified human type II collagen for N- and C-terminal covalent tagging”. CANADIAN JOURNAL OF CHEMISTRY 96.2 (2018): 204-211.
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