Heterologous expression of L. major proteins in S. cerevisiae: a test of solubility, purity, and gene recoding.
High level expression of many eukaryotic proteins for structural analysis is likely to require a eukaryotic host since many proteins are either insoluble or lack essential post-translational modifications when expressed in E. coli. The well-studied eukaryote Saccharomyces cerevisiae possesses several attributes of a good expression host: it is simple and inexpensive to culture, has proven genetic tractability, and has excellent recombinant DNA tools. We demonstrate here that this yeast exhibits three additional characteristics that are desirable in a eukaryotic expression host. First, expression in yeast significantly improves the solubility of proteins that are expressed but insoluble in E. coli. The expression and solubility of 83 Leishmania major ORFs were compared in S. cerevisiae and in E. coli, with the result that 42 of the 64 ORFs with good expression and poor solubility in E. coli are highly soluble in S. cerevisiae. Second, the yield and purity of heterologous proteins expressed in yeast is sufficient for structural analysis, as demonstrated with both small scale purifications of 21 highly expressed proteins and large scale purifications of 2 proteins, which yield highly homogeneous preparations. Third, protein expression can be improved by altering codon usage, based on the observation that a codon-optimized construct of one ORF yields three-fold more protein. Thus, these results provide direct verification that high level expression and purification of heterologous proteins in S. cerevisiae is feasible and likely to improve expression of proteins whose solubility in E. coli is poor.
Quartley
Erin
E
Center for Pediatric Biomedical Research, University of Rochester Medical School, Rochester, NY 14642, USA.
Alexandrov
Andrei
A
Mikucki
Maryann
M
Buckner
Frederick S
FS
Hol
Wim G
WG
DeTitta
George T
GT
Phizicky
Eric M
EM
Grayhack
Elizabeth J
EJ
D000818:Animals; D003001:Cloning, Molecular; D003062:Codon / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018320:Leishmania major / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics*; D015202:Protein Engineering; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D012995:Solubility
D003062:Codon; D015800:Protozoan Proteins
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