title: Expression of recombinant vitellogenin in the yeast Pichia pastoris.
abstract: Vitellogenin (Vtg) plays vital roles as precursor to the yolk proteins and as carrier for lipids, carbohydrates, phosphates, metal ions, vitamins, and hormones into the oocytes during the massive deposition of yolk nutrients for subsequent nourishment of the developing embryos. Reproductive success is highly sensitive to the nutritional quality of the broodstock diet, which greatly affects the egg and larval viability. We present a novel strategy for genetically engineering a Pichia pastoris yeast strain that constitutively produces recombinant Vtg (rVtg), for application as an enriched feed. The tilapia Oreochromis aureus Vtg (OaVtg) cDNA (5.3 kb) was cloned into a nonsecretory pGAPZA vector. Clones containing up to 31 copies of glyceraldehyde-3-phosphate dehydrogenase (GAP)-promoter-driven Vtg expression cassettes were isolated. These clones expressed a membrane-associated intracellular rVtg protein of 194 kDa, constituting up to 1.16% of total protein. To facilitate future purification of rVtg, we explored the possibility of secreting rVtg using the native Vtg secretion signal and the alpha-factor secretion signal of Saccharomyces cerevisiae. However, neither signal promoted the secretion of rVtg. The clones maximally expressed rVtg at 23 degrees C, reaching a peak at 22 h in shake flasks and 16 h in a fermentor. The clones exhibited a significant increase in essential amino acids and long-chain polyunsaturated fatty acids, which are important for its application as a high-quality nutrient feed.
authors.affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore.
authors.forename: J L
authors.initials: JL
authors.lastname: Ding
authors.forename: E H
authors.initials: EH
authors.lastname: Lim
authors.forename: H F
authors.initials: HF
authors.lastname: Li
authors.forename: J K
authors.initials: JK
authors.lastname: Kumar
authors.forename: S L
authors.initials: SL
authors.lastname: Lee
authors.forename: T J
authors.initials: TJ
authors.lastname: Lam
mesh_terms: D000601:Amino Acids, Essential / Q000096:biosynthesis; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D005231:Fatty Acids, Unsaturated / Q000096:biosynthesis; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics* / Q000378:metabolism*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017210:Tilapia / Q000235:genetics / Q000378:metabolism; D014819:Vitellogenins / Q000096:biosynthesis* / Q000235:genetics*
chemical_list: D000601:Amino Acids, Essential; D005231:Fatty Acids, Unsaturated; D011994:Recombinant Proteins; D014819:Vitellogenins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases