Production of recombinant human type I procollagen trimers using a four-gene expression system in the yeast Saccharomyces cerevisiae.
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.
Toman
P D
PD
Cohesion Technologies, Palo Alto, California 94303, USA. dtoman@cson.com
Chisholm
G
G
McMullin
H
H
Giere
L M
LM
Olsen
D R
DR
Kovach
R J
RJ
Leigh
S D
SD
Fong
B E
BE
Chang
R
R
Daniels
G A
GA
Berg
R A
RA
Hitzeman
R A
RA
D001704:Biopolymers; D003470:Culture Media; D006801:Humans; D011347:Procollagen / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D001704:Biopolymers; D003470:Culture Media; D011347:Procollagen; D011994:Recombinant Proteins