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Heterologous expression of L. major proteins in S. cerevisiae: a test of solubility, purity, and gene recoding

  • Published:
Journal of Structural and Functional Genomics

Abstract

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.

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Abbreviations

LIC:

Ligation independent cloning

PSI:

Protein structure initiative

PDB:

Protein database

L. major :

Leishmania major

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Acknowledgments

We thank M. Dumont for advice and comments. This work was supported by National Institutes of Health (NIH) Grant NIH 1 U54 GM074899 establishing the Center for High Throughput Structural Biology.

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Author notes

  1. Andrei Alexandrov

    Present address: Yale University School of Medicine, HHMI, Rm 135 BCMM, 295 Congress Ave, New Haven, CT, 06536-0812, USA

Authors and Affiliations

  1. Center for Pediatric Biomedical Research, University of Rochester Medical School, Rochester, NY, 14642, USA

    Erin Quartley, Maryann Mikucki, Eric M. Phizicky & Elizabeth J. Grayhack

  2. Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington School of Medicine, Seattle, WA, 98195, USA

    Frederick S. Buckner

  3. Department of Biochemistry, University of Washington School of Medicine, Seattle, WA, 98195, USA

    Wim G. Hol

  4. Hauptman-Woodward Medical Research Institute, 700 Ellicott St., Buffalo, NY, 14203, USA

    George T. DeTitta

  5. Department of Structural Biology, SUNY at Buffalo, 700 Ellicott St., Buffalo, NY, 14203, USA

    George T. DeTitta

  6. Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave, Rochester, NY, 14642, USA

    Andrei Alexandrov, Eric M. Phizicky & Elizabeth J. Grayhack

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  1. Erin Quartley
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Electronic supplementary material

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Supplementary Table 1

L major ORF targets: Putative Function and Expression in E. coli (DOC 142 kb)

Supplementary Figure 1

Evaluation of soluble protein expression based on affinity purification of L. major ORFs on IgG sepharose. Purification of of L. major ORF-fusions 8264, 4486 and 6593 on IgG sepharose. Proteins were bound to IgG Sepharose and washed, and bound protein was eluted after cleavage of the ZZ tag with 3C protease, lanes a–e, of L. major ORF-fusion 8264; f–j, of L. major ORF-fusion 4486; k–o, of L. major ORF-fusion 6593. Lanes a, f, k, sample bound to IgG beads; lanes b, g, l, protein eluted with 3C protease; lanes c, h, m: IgG beads after proteolytic cleavage; lanes d, i, n, second wash of the IgG beads after proteolytic cleavage; lanes e, j, o, IgG beads after second wash. (TIFF 541 kb)

Supplementary Figure 2

Evaluation of soluble protein expression based on affinity purification of L. major ORFs on IgG sepharose. Purification of L. major ORF-fusions 6598, 2393 and 6679 on IgG sepharose. Methods and lanes are identical to Supplementary Figure 1. (TIFF 581 kb)

Supplementary Figure 3

Large scale purification of L. major 4089. A. Purification of L. Major 4089 ORF-fusion from 384 OD-L on IgG sepharose, followed by cleavage with 3C protease, and removal of 3C protease with GSH resin. Lanes are identical to Figure 5. B. Purification of L. Major 4089 by sizing chromatography. Lanes a–m contain 25 µl each of fractions 39 to 49 (1.8 ml per fraction). C. Purified, concentrated L. Major 4089 protein. Protein from fractions 43 to 46 (lanes e–h in B) was concentrated to ~5 ml and centrifuged for 10 min at maximum speed in a micro-centrifuge at 4°C. (TIFF 1,752 kb)

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Quartley, E., Alexandrov, A., Mikucki, M. et al. Heterologous expression of L. major proteins in S. cerevisiae: a test of solubility, purity, and gene recoding. J Struct Funct Genomics 10, 233–247 (2009). https://doi.org/10.1007/s10969-009-9068-9

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