Protein expression in yeast; comparison of two expression strategies regarding protein maturation.
The driving force for the modification of existing, or the development of new, protein expression systems lies in the identification of a tremendous number of potential novel drug targets through recent genomics approaches. Saccharomyces cerevisiae as a host for recombinant protein expression, offers many advantages, as its biosynthetic pathways resemble higher eukaryotic cells in many aspects. Two yeast vectors were compared to evaluate the versatility of this organism for expression of recombinant proteins. One expression vector enables the secretion of the recombinant protein into the culture medium through fusion with the leader sequence of the mating-type pheromone alpha; the other directs the expression product into the cytoplasm of the yeast cell through fusion with ubiquitin. To facilitate immunological detection and purification, proteins were expressed as fusions to an octapeptide, the so-called Flag-tag, which is recognised by a monoclonal antibody in the 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 differed greatly in the degree of glycosylation and other, not further analysed, post-translational modifications. Secretion of all model proteins into the cell culture supernatant could be accomplished if membrane domains or signal sequences were absent, but many proteins were heavily glycosylated as demonstrated by lectin mapping or enzymatical deglycosylation. Some proteins, however, were expressed as homogenous products, and could be easily purified for further functional studies. Further investigations on the expression biology of yeast are required, in order to optimise the conditions of fermentation which may finally lead to more homogeneous expression products.
Schuster
M
M
Novartis Forschungsinstitut, Brunnerstrasse 59, 1235 Vienna, Austria.
Einhauer
A
A
Wasserbauer
E
E
Süssenbacher
F
F
Ortner
C
C
Paumann
M
M
Werner
G
G
Jungbauer
A
A
D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors / Q000138:chemical synthesis; D006031:Glycosylation; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic; D014452:Ubiquitins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins