bioRxiv Biochemistry
bioRxiv Biochemistry
5 years ago

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

Kovacic, Wieczorek, Li, Dierks, C. K., Forde, Chan, S., T., N. R., A., C.
Collagen is the predominant structural protein in vertebrates, where it contributes to connective tissues and the extracellular matrix; 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 posttranslational 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 address 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, while 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, while 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 wildtype collagen has thus far obscured the desired specific N-terminal labelling. Strategies to overcome this challenge are proposed.

Publisher URL: http://biorxiv.org/cgi/content/short/145136v1

DOI: 10.1101/145136

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