Recombinant Collagen Trimers from Insect Cells and Yeast

  • Johanna Myllyharju
Part of the Methods in Molecular Biology™ book series (MIMB, volume 139)

Abstract

At least 19 proteins have now been defined as collagens (1,2), but many of those recently discovered are present in tissues in such small amounts that their isolation for characterization at the protein level has so far been impossible. Some of the fibril-forming collagens are now in medical use, in applications ranging from the use of type I collagen as a biomaterial and a delivery system for several drugs to trials for the potential of type II collagen as an oral tolerance-inducing agent for the treatment of rheumatoid arthritis (3,4). An efficient recombinant expression system for collagens can thus be expected to have numerous scientific and medical applications. The systems commonly used for expressing other proteins in lower organisms are not suitable as such for the production of recombinant collagens, however, as bacteria and yeast have no prolyl 4-hydroxylase activity and insect cells have insufficient levels of it. Prolyl 4-hydroxylase, an α2β2 tetramer in vertebrates, plays a central role in the synthesis of all collagens, as 4-hydroxyproline-deficient collagen polypeptide chains cannot form triple helices that are stable at 37°C (5,6). All attempts to assemble an active prolyl 4-hydroxylase tetramer from its subunits in vitro have been unsuccessful, but active recombinant human prolyl 4-hydroxylase has been produced in insect cells and yeast by coexpression of its α- and β-subunits (7,7). We have recently shown that recombinant human type III collagen with a stable triple helix can be efficiently produced in insect and yeast cells by simultaneous coexpression with the recombinant human prolyl 4-hydroxylase (8,9). This chapter describes detailed procedures for the production of stable recombinant human type III collagen in insect cells and in the yeast Pichia

Keywords

Insect Cell Translation Initiation Codon Baculovirus Expression Vector Recombinant Collagen Recombinant Human Type 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc. 2000

Authors and Affiliations

  • Johanna Myllyharju
    • 1
  1. 1.Collagen Research UnitUniversity of OuluOulu

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