High-level production of human type I collagen in the yeast Pichia pastoris.
Four human genes, two of them encoding the proalpha1 and proalpha2 chains of type I procollagen and two of them the two types of subunit of prolyl 4-hydroxylase (4-PH), were integrated into the genome of Pichia pastoris. The proalpha1 and proalpha2 chains expressed formed type I procollagen molecules with the correct 2:1 chain ratio, and the 4-PH subunits formed an active enzyme tetramer that fully hydroxylated the proalpha chains. Chains lacking their N but not C propeptides formed pCcollagen molecules with the 2:1 chain ratio and, surprisingly, the expression levels of pCcollagen were 1.5-3-fold relative to those of procollagen. Both types of molecule could be converted by pepsin treatment to collagen molecules that formed native-type fibrils in vitro. The expression levels obtained for the pCcollagen using only single copies of each of the four genes and a 2 l fermenter ranged up to 0.5 g/l, indicating that it should be possible to optimize this system for high-level production of recombinant human type I collagen for numerous medical applications.
Nokelainen
M
M
Collagen Research Unit, Biocenter and Department of Medical Biochemistry, PO Box 5000, FIN-90014 University of Oulu, Finland.
Tu
H
H
Vuorela
A
A
Notbohm
H
H
Kivirikko
K I
KI
Myllyharju
J
J
D000596:Amino Acids / Q000032:analysis; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods; D024042:Collagen Type I / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000648:ultrastructure; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D010843:Pichia / Q000235:genetics*; D011347:Procollagen / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011393:Procollagen-Proline Dioxygenase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000648:ultrastructure
D000596:Amino Acids; D024042:Collagen Type I; D011347:Procollagen; D011993:Recombinant Fusion Proteins; D011393:Procollagen-Proline Dioxygenase