title: Expression of recombinant human type I-III collagens in the yeast pichia pastoris.
abstract: 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.
authors.affiliation: Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, P.O. Box 5000, FIN-90014 Oulu, Finland. johanna.myllyharju@oulu.fi
authors.forename: J
authors.initials: J
authors.lastname: Myllyharju
authors.forename: M
authors.initials: M
authors.lastname: Nokelainen
authors.forename: A
authors.initials: A
authors.lastname: Vuorela
authors.forename: K I
authors.initials: KI
authors.lastname: Kivirikko
mesh_terms: 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
chemical_list: D010455:Peptides; D011347:Procollagen; D011994:Recombinant Proteins; D003094:Collagen; D011393:Procollagen-Proline Dioxygenase