title: Production of recombinant human type I procollagen trimers using a four-gene expression system in the yeast Saccharomyces cerevisiae.
abstract: The expression of stable recombinant human collagen requires an expression system capable of post-translational modifications and assembly of the procollagen polypeptides. Two genes were expressed in the yeast Saccharomyces cerevisiae to produce both propeptide chains that constitute human type I procollagen. Two additional genes were expressed coding for the subunits of prolyl hydroxylase, an enzyme that post-translationally modifies procollagen and that confers heat (thermal) stability to the triple helical conformation of the collagen molecule. Type I procollagen was produced as a stable heterotrimeric helix similar to type I procollagen produced in tissue culture. A key requirement for glutamate was identified as a medium supplement to obtain high expression levels of type I procollagen as heat-stable heterotrimers in Saccharomyces. Expression of these four genes was sufficient for correct assembly and processing of type I procollagen in a eucaryotic system that does not produce collagen.
authors.affiliation: Cohesion Technologies, Palo Alto, California 94303, USA. dtoman@cson.com
authors.forename: P D
authors.initials: PD
authors.lastname: Toman
authors.forename: G
authors.initials: G
authors.lastname: Chisholm
authors.forename: H
authors.initials: H
authors.lastname: McMullin
authors.forename: L M
authors.initials: LM
authors.lastname: Giere
authors.forename: D R
authors.initials: DR
authors.lastname: Olsen
authors.forename: R J
authors.initials: RJ
authors.lastname: Kovach
authors.forename: S D
authors.initials: SD
authors.lastname: Leigh
authors.forename: B E
authors.initials: BE
authors.lastname: Fong
authors.forename: R
authors.initials: R
authors.lastname: Chang
authors.forename: G A
authors.initials: GA
authors.lastname: Daniels
authors.forename: R A
authors.initials: RA
authors.lastname: Berg
authors.forename: R A
authors.initials: RA
authors.lastname: Hitzeman
mesh_terms: D001704:Biopolymers; D003470:Culture Media; D006801:Humans; D011347:Procollagen / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*
chemical_list: D001704:Biopolymers; D003470:Culture Media; D011347:Procollagen; D011994:Recombinant Proteins