Development and characterization of a eukaryotic expression system for human type II procollagen

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.

Intact Telopeptides Enhance Interactions between Collagens

“…Our study extends previous applications of microrheology, which examined interactions of cross-linking proteins with filamentous actin substrates, by focusing on interactions intrinsic to the self-assembling proteins themselves. Extensions of this work could examine how collagen associations may be altered in disease, using recombinantly expressed proteins to test sequence-function hypotheses (42), and could explore sequence-dependent protein-protein interactions important for nucleating higher-order structure formation in other self-assembling and/or pathogenic aggregating protein systems.…”

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

“…Mutations were introduced at the plasmid level (Finnzymes Phusion sitedirected mutagenesis kit, New England Biolabs) into the COL2A1-containing modified pYIC vector previously developed for stable expression of wild-type human procollagen. [18] Primers used to alter the N-telopeptide sequence (LGVMQ into LCTPSR) were 5'-CTG TGC ACC CCC AGC CGG GGA CCA ATG GGC CCC ATG GGA CCT CGA GG-3' and 5'-CTG GGC GCC ACC AGC CTT TTC ATC AAA TCC TCC-3'. In the C-telopeptide, the Ser1221Cys mutation was introduced using primers 5'-CCT GGC ATC GAC ATG TGC GCC TTT GCT GG-3' and 5'-GCC AGG GGG ACC TGG AGG ACC AGG GG-3'.…”

“…[18] In this work, an HT1080 fibrosarcoma cell line stably overexpressing the enzyme formylglycine generating enzyme (FGE) [19][20] was utilized for stable expression of the mutated procollagen.…”

“…[18,21] To ensure stable retention of both introduced constructs (FGE and collagen), the HT1080 FGE-overexpressing cells (originally selected by G418 resistance) [19][20] were co-transfected with a linear puromycin resistance marker (Clontech 631626) and a linearized version of the plasmid DNA from which the neomycin selection was removed. After transfection, cells were selected in the presence of D r a f t 6 400 µg/ml G418 and 0.25 µg/ml puromycin; surviving cells were screened for blue fluorescence indicating the expression of procollagen.…”

“…[5][6][7][8][9][10] While such studies have traditionally focussed on high-level collagen structures, in recent years there has been increasing focus on understanding collagen's mechanics at the molecular level. [11][12][13][14][15][16][17][18] Collagen molecules can be subjected to strain, and their resultant response characterized, by stretching these individual proteins by their ends.…”