Abstract
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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0228-7) contains supplementary material, which is available to authorized users.
Highlights
Triple 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
The demonstrated fidelity of Results and discussion To produce post-translationally modified type II human procollagen, HT1080 human fibrosarcoma cells were used as the host cell line
This cell line was chosen for the transfection and expression of the recombinant protein because its endogenous expression of collagen IV provides the requisite enzymes for correct posttranslational modification and secretion of the recombinant type II procollagen [17]
Summary
Triple 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. Alterations in collagens’ composition, resulting from genetic modifications, aging, and diabetes, have been identified with an extensive list of diseases [1, 2] Due to their natural role as the structural component in the extracellular matrix, collagens have found widespread use in biomaterials, used for cellular and tissue. To surmount issues arising from variability of tissue-derived collagen, an alternative strategy employs
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