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

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 bonds present in beta-lactoglobulin.

Genetics and Molecular Physiology of the Yeast Kluyveromyces lactis

“…Strong transcriptional activation is obtained by both constitutive and conditional promoter systems (van den Bui et al, 1996;Ferminan and Dominguez, 1998;Saliola et al, 1999;Mustilli et al, 1999;Hsieh and Da Silva, 2000). As far as posttranslational modifications are concerned, recombinant ␤-lactoglobulin secreted from K. lactis has been shown to be virtually indistinguishable from the native protein based on PAGE analysis, reactivity to antibodies, CD, fluorescence spectroscopy, and N-terminal sequencing (Rocha et al, 1996).…”

Induction by Hypoxia of Heterologous-Protein Production with the Kl PDC1 Promoter in Yeasts

“…In addition, genetic manipulations are facilitated by the availability of integrative and episomal vectors and by the publication of its entire genome (7). Considering its good secretion ability, K. lactis constitutes a valid alternative to the classical baker's yeast, Saccharomyces cerevisiae, for the expression of heterologous products, often being the best producer when a direct comparison has been attempted between these two yeasts (25). This good biotechnological potential is confirmed by the expression of several proteins for industrial and medical use (reviewed in reference 29).…”

Improved Production of Heterologous Proteins by a Glucose Repression-Defective Mutant of Kluyveromyces lactis

“…The yeast K. lactis is one of the model systems utilized for heterologous protein production in the food and pharmacology industries (18,19,35,39,44,51). In this work, we reported the characterization of a non-glucose-repressible mutant of K. lactis to be used as a host for recombinant protein production.…”

“…This ability could be due to the residual hexokinase activity, which might dimin- ish the intracellular accumulation of unphosphorylated glucose that is regarded as deleterious for the cells (35). The reduced mannose extension of N-and O-glycoproteins observed for dgr151-1 cells could be ascribed to a reduced availability of GDP-mannose, the substrate for the glycosylation reactions that occur in the Golgi apparatus (46).…”