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.
An optimized polyamine moiety boosts the potency of human type II topoisomerase poisons as quantified by comparative analysis centered on the clinical candidate F14512