1. Production of recombinant hydroxylated human type III collagen fragment in Saccharomyces cerevisiae.
- Author
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Vaughn PR, Galanis M, Richards KM, Tebb TA, Ramshaw JA, and Werkmeister JA
- Subjects
- Cloning, Molecular, Collagen chemistry, Escherichia coli genetics, Escherichia coli metabolism, Galactose pharmacology, Humans, Hydroxylation, Hydroxyproline metabolism, Macromolecular Substances, Peptide Fragments chemistry, Plasmids, Procollagen biosynthesis, Promoter Regions, Genetic, Protein Structure, Secondary, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Saccharomyces cerevisiae genetics, Collagen biosynthesis, Peptide Fragments biosynthesis, Procollagen-Proline Dioxygenase biosynthesis, Saccharomyces cerevisiae metabolism
- Abstract
A recombinant hydroxylated fragment of human type III collagen has been produced in Saccharomyces cerevisiae by coordinated coexpression of a collagen gene fragment together with both the alpha- and beta-subunit genes for prolyl-4-hydroxylase (EC 1.14.11.2). The collagen fragment consisted of 255 residues of the helical domain and the complete C-telopeptide and C-propeptide domains. It was inserted under the control of the ethanol-inducible ADH2 promoter in a multicopy, TRP1-selectable, yeast expression vector, YEpFlag1. The prolyihydroxylase subunit genes were cloned on either side of a bidirectional galactose-inducible promoter in a low-copy minichromosome yeast expression vector, pYEUra3, which is URA3 selectable. Coordinated expression of the three different gene products after cotransformation into S. cerevisiae was detected by immunoblotting. Amino acid analysis of an immunoreactive collagen fraction demonstrated the presence of hydroxyproline, while the presence of a triple-helical domain in the collagen fragment was demonstrated by its resistance to pepsin proteolysis.
- Published
- 1998
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