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Development and Characterization of a Eukaryotic Expression System for Human Type II Procollagen

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BackgroundTriple helical collagens are the most abundant structural protein in vertebrates and are widely used as biomaterials for a variety of applications including drug delivery and cellular and tissue engineering. In these applications, the mechanics of this hierarchically structured protein play a key role, as does its chemical composition. To facilitate investigation into how gene mutations of collagen lead to disease as well as the rational development of tunable mechanical and chemical properties of this full-length protein, production of recombinant expressed protein is required.ResultsHere, we present a human type II procollagen expression system that produces full-length procollagen utilizing a previously characterized human fibrosarcoma cell line for production. The system exploits a non-covalently linked fluorescence readout for gene expression to facilitate screening of cell lines. Biochemical and biophysical characterization of the secreted, purified protein are used to demonstrate the proper formation and function of the protein. Assays to demonstrate fidelity include proteolytic digestion, mass spectrometric sequence and posttranslational composition analysis, circular dichroism spectroscopy, single-molecule stretching with optical tweezers, atomic-force microscopy imaging of fibril assembly, and transmission electron microscopy imaging of self-assembled fibrils.ConclusionsUsing a mammalian expression system, we produced full-length recombinant human type II procollagen. The integrity of the collagen preparation was verified by various structural and degradation assays. This system provides a platform from which to explore new directions in collagen manipulation.
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Wieczorek A, Rezaei N, Chan CK, Xu C, Panwar P, Brömme D, Merschrod S EF, Forde NR. Development and characterization of a eukaryotic expression system for human type II procollagen. BMC Biotechnol. 2015 Dec 15;15(1):112. doi: 10.1186/s12896-015-0228-7.
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BMC Biotechnol
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Development and Characterization of a Eukaryotic Expression System for Human Type II Procollagen
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