Expression of recombinant human type I-III collagens in the yeast pichia pastoris.
An efficient expression system for recombinant human collagens will have numerous scientific and medical applications. However, most recombinant systems are unsuitable for this purpose, as they do not have sufficient prolyl 4-hydroxylase activity. We have developed methods for producing the three major fibril-forming human collagens, types I, II and III, in the methylotrophic yeast Pichia pastoris. These methods are based on co-expression of procollagen polypeptide chains with the alpha- and beta-subunits of prolyl 4-hydroxylase. The triple-helical type-I, -II and-III procollagens were found to accumulate predominantly within the endoplasmic reticulum of the yeast cells and could be purified from the cell lysates by a procedure that included a pepsin treatment to convert the procollagens into collagens and to digest most of the non-collagenous proteins. All the purified recombinant collagens were identical in 4-hydroxyproline content with the corresponding non-recombinant human proteins, and all the recombinant collagens formed native-type fibrils. The expression levels using single-copy integrants and a 2 litre bioreactor ranged from 0.2 to 0.6 g/l depending on the collagen type.
Myllyharju
J
J
Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, P.O. Box 5000, FIN-90014 Oulu, Finland. johanna.myllyharju@oulu.fi
Nokelainen
M
M
Vuorela
A
A
Kivirikko
K I
KI
D000595:Amino Acid Sequence; D000818:Animals; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D003094:Collagen / Q000096:biosynthesis* / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010843:Pichia / Q000378:metabolism*; D011347:Procollagen / Q000096:biosynthesis; D011393:Procollagen-Proline Dioxygenase / Q000096:biosynthesis / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry
D010455:Peptides; D011347:Procollagen; D011994:Recombinant Proteins; D003094:Collagen; D011393:Procollagen-Proline Dioxygenase