PMID: 8611177Feb 1, 1996Paper

Expression and secretion of recombinant ovine beta-lactoglobulin in Saccharomyces cerevisiae and Kluyveromyces lactis

The Biochemical Journal
T L RochaL A Fothergill-Gilmore

Abstract

High expression and secretion of recombinant ovine beta-lactoglobulin has been achieved in the yeast Kluyveromyces lactis. The yield of beta-lactoglobulin is 40-50 mg per litre of culture supernatant and accounts for approx. 72% of the total secreted protein. Constitutive expression is under the control of the Saccharomyces cerevisiae phosphoglycerate kinase promoter from an intronless version of the beta-lactoglobulin gene. Secretion is specified by the ovine protein's own signal sequence. this system, coupled to an efficient and novel recovery protocol, allows 30 mg of pure protein to be isolated from a typical 1 litre culture. The protein is virtually indistinguishable from beta-lactoglobulin conventionally purified from sheep milk by its behaviour in native PAGE and SDS/PAGE, reactivity to antibodies, CD, fluorescence spectroscopy and N-terminal sequencing. Attempts to achieve a similar expression and secretion system in the yeast S. cerevisiae met with only limited success, although it was found that heat-shock treatment modestly increased the yield up to approx. 3-4 mg per litre of culture supernatant. Site-directed mutagenesis showed that secretion in S. cerevisiae depended upon correct formation of the two disulphide bo...Continue Reading

Citations

Oct 4, 2011·Biotechnology Advances·Eda Celik, Pınar Calık
Apr 25, 2006·FEMS Yeast Research·Albert J J van OoyenChristopher H Taron
Sep 22, 2005·Applied Microbiology and Biotechnology·Francesca Salani, Michele M Bianchi
Jun 4, 2005·Applied and Environmental Microbiology·Paul A ColussiChristopher H Taron
May 7, 2004·Applied and Environmental Microbiology·Claudia DonniniClaudio Palleschi
Dec 5, 2006·Applied and Environmental Microbiology·Andrea CamattariLuca Brambilla
Oct 19, 2000·Fungal Genetics and Biology : FG & B·R Schaffrath, K D Breunig
Oct 6, 1999·Current Opinion in Biotechnology·G P Cereghino, J M Cregg
Jun 23, 2001·Journal of Chromatography. B, Biomedical Sciences and Applications·A R LorenzS Vieths

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