Production of synthetic spider dragline silk protein in Pichia pastoris

• Published in: Applied microbiology and biotechnology Vol. 47; no. 1; pp. 33 - 39
• Main Authors: Fahnestock, S.R, Bedzyk, L.A
• Format: Journal Article
• Language: English
• Published: Berlin Springer 01.01.1997; Springer Nature B.V
• Subjects: animal breeding; animal genetics; Animals; Araneae; Araneidae; arthropods; Biological and medical sciences; Biological Sciences; Biotechnology; E coli; entomology; Escherichia coli; Fibroins; Fundamental and applied biological sciences. Psychology; Gene Expression; Genes; Genes, Synthetic; Genetic aspects; Genetic engineering; Genetic technics; Methods. Procedures. Technologies; Microbial genetics; Modification of gene expression level; molecular sequence data; Nephila clavipes; nonfood animal products; Physiological aspects; Pichia - genetics; Pichia pastoris; Polymers; Protein Biosynthesis; Proteins; Proteins - genetics; Proteins - isolation & purification; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Silk; Solubility; Spiders - chemistry; Transformation, Genetic; Yeast fungi; Yeasts; United States; Yeast; Copy number; Gene product; Araneida; Gene expression; Nephila clavipes; Fungi; Proteins; Arachnida; Silk; Synthetic product; Gene; Arthropoda; Production; Ascomycetes; Invertebrata; Chemical synthesis; Araneidae; Thallophyta
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s002530050884

The methylotrophic yeast Pichia pastoris was tested as a host for the production of long, repetitive protein polymers. Synthetic genes for a designed analog of a spider dragline silk protein were readily expressed at high levels under control of the methanol-inducible AOX1 promoter. Transformants containing multiple gene copies produced elevated levels of silk protein, but of a variety of altered sizes as a result of gene rearrangements at the time of transformation. Genes up to 3000 codons in length or longer could be expressed with no evidence of the prevalent truncated synthesis observed for similar genes in Escherichia coli, though genes longer than 1600 codons were expressed less efficiently than shorter genes. Silk-producing P. pastoris strains were stable without selection for at least 100 doublings.