Selective expression of nonsecreted triple-helical and secreted single-chain recombinant collagen fragments in the yeast Pichia pastoris.
High-level recombinant expression systems for the production of stable triple-helical human collagens and collagen fragments have been developed in the yeast Pichia pastoris. Collagen fragments are secreted as single-chain polypeptides by the yeast alpha-mating factor pre-pro sequence, but secretion of full-length triple-helical procollagen molecules has not been achieved despite the use of the same secretory signal. We studied here the effects of the secretory signal and the conformation and size of the collagen polypeptide on its secretion in P. pastoris. Unlike the collagen signal sequence, the alpha-mating factor pre-pro sequence led to efficient secretion of single-chain 45 and 9 kDa type I collagen fragments. The efficiency was dependent on the length of the collagen polypeptide, as secretion of single-chain full-length 90 kDa alpha1(I) polypeptides was less efficient than that of the 45 kDa fragment. Furthermore, the conformation of the collagen polypeptides had a marked effect on secretion, as induction of trimerization of the 45 and 9 kDa fragments by either the C propeptide or the small trimerizing domain foldon led to an accumulation of triple-helical molecules inside the cells despite the presence of the alpha-mating factor pre-pro sequence. Our results show that P. pastoris is a suitable host for the development of tailored expression systems aimed at selective production of nonsecreted triple-helical and secreted single-chain collagen fragments of varying lengths for specific purposes.
Pakkanen
Outi
O
Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, P.O. Box 5000, University of Oulu, FIN-90014 Oulu, Finland.
Pirskanen
Asta
A
Myllyharju
Johanna
J
D003094:Collagen / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D012680:Sensitivity and Specificity
D010446:Peptide Fragments; D003094:Collagen