Expression and secretion of recombinant ovine beta-lactoglobulin in Saccharomyces cerevisiae and Kluyveromyces lactis.
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 bonds present in beta-lactoglobulin.
Rocha
T L
TL
Edinburgh Centre for Molecular Recognition, University of Edinburgh, Scotland, U.K.
Paterson
G
G
Crimmins
K
K
Boyd
A
A
Sawyer
L
L
Fothergill-Gilmore
L A
LA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004274:DNA, Recombinant / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D015870:Gene Expression; D007716:Kluyveromyces / Q000235:genetics*; D007782:Lactoglobulins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012756:Sheep
D004274:DNA, Recombinant; D004220:Disulfides; D007782:Lactoglobulins; D011994:Recombinant Proteins