title: Development and characterization of a eukaryotic expression system for human type II procollagen.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  Here, 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.
  
  
  CONCLUSIONS
  Using 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.
authors.affiliation: Department of Physics, Simon Fraser University, 8888 University Drive, Burnaby, BC, V5A 1S6, Canada.
authors.forename: Andrew
authors.initials: A
authors.lastname: Wieczorek
authors.affiliation: Department of Physics, Simon Fraser University, 8888 University Drive, Burnaby, BC, V5A 1S6, Canada.
authors.forename: Naghmeh
authors.initials: N
authors.lastname: Rezaei
authors.affiliation: Department of Physics, Simon Fraser University, 8888 University Drive, Burnaby, BC, V5A 1S6, Canada.|Present Address: Department of Bioengineering, University of California at Los Angeles, Los Angeles, USA.
authors.forename: Clara K
authors.initials: CK
authors.lastname: Chan
authors.affiliation: Department of Chemistry, Memorial University, St. John's, NL, A1B 3X7, Canada.|Present Address: Green Innovative Technologies R&D Centre Ltd, Vancouver, Canada.
authors.forename: Chuan
authors.initials: C
authors.lastname: Xu
authors.affiliation: Faculty of Dentistry, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.
authors.forename: Preety
authors.initials: P
authors.lastname: Panwar
authors.affiliation: Faculty of Dentistry, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.|Department of Biochemistry, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.
authors.forename: Dieter
authors.initials: D
authors.lastname: Brömme
authors.affiliation: Department of Chemistry, Memorial University, St. John's, NL, A1B 3X7, Canada.
authors.forename: Erika F
authors.initials: EF
authors.lastname: Merschrod S
authors.affiliation: Department of Physics, Simon Fraser University, 8888 University Drive, Burnaby, BC, V5A 1S6, Canada. nforde@sfu.ca.
authors.forename: Nancy R
authors.initials: NR
authors.lastname: Forde
mesh_terms: D056657:Cathepsin K / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D002942:Circular Dichroism; D002999:Clone Cells; D024043:Collagen Type II / Q000096:biosynthesis* / Q000235:genetics*; D056890:Eukaryota / Q000235:genetics* / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D005354:Fibrosarcoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D006801:Humans; D018625:Microscopy, Atomic Force; D052898:Optical Tweezers; D011347:Procollagen / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D024043:Collagen Type II; D011347:Procollagen; D011994:Recombinant Proteins; D056657:Cathepsin K