Protein expression in yeast; comparison of two expression strategies regarding protein maturation

The driving force for the modification of existing, or the development of n ew, protein expression systems lies in the identification of a tremendous n umber of potential novel drug targets through recent genomics approaches. S accharomyces cerevisiae as a host for recombinant protein expression, offer s many advantages, as its biosynthetic pathways resemble higher eukaryotic cells in many aspects. Two yeast vectors were compared to evaluate the vers atility of this organism for expression of recombinant proteins. One expres sion Vector enables the secretion of the recombinant protein into the cultu re medium through fusion with the leader sequence of the mating-type pherom one a; the other directs the expression product into the cytoplasm of the y east cell through fusion with ubiquitin. To facilitate immunological detect ion and purification, proteins were expressed as fusions to an octapeptide, the so-called Flag-tag, which is recognised by a monoclonal antibody in th e presence of Ca2+. We chose 20 functionally different cDNAs to compare the efficiency of both expression systems. All cDNAs could be expressed at the correct size but at varying yields and purity. Both expression systems dif fered greatly in the degree of glycosylation and other, not further analyse d, post-translational modifications. Secretion of all model proteins into t he cell culture supernatant could be accomplished if membrane domains or si gnal sequences were absent, but many proteins were heavily glycosylated as demonstrated by lectin mapping or enzymatical deglycosylation. Some protein s, however, were expressed as homogenous products, and could be easily puri fied for further functional studies. Further investigations on the expressi on biology of yeast are required, in order to optimise the conditions of fe rmentation which may finally lead to more homogeneous expression products. (C) 2000 Elsevier Science B.V. All rights reserved.