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 -lactogobulin is 40-50 mg per litre of culture supernatant and account s for approx. 72% of the total secreted protein. Constitutive expressi on is under the control of the Saccharomyces cerevisiae phosphoglycera te kinase promoter from an intronless version of the beta-lactoglobuli n gene. Secretion is specified by the ovine protein's own signal seque nce. This system, coupled to an efficient and novel recovery protocol, allows 30 mg of pure protein to be isolated from a typical 1 litre cu lture. The protein is virtually indistinguishable from beta-lactoglobu lin conventionally purified from sheep milk by its behaviour in native PAGE and SDS/PAGE, reactivity to antibodies, CD, fluorescence spectro scopy and N-terminal sequencing. Attempts to achieve a similar express ion and secretion system in the yeast S. cevevisiae met with only limi ted success, although it was found that heat-shock treatment modestly increased the yield up to approx. 3-4 mg per litre of culture supernat ant. Site-directed mutagenesis showed that secretion in S. cerevisiae depended upon correct formation of the two disulphide bonds present in beta-lactoglobulin.