Journal of Structural and Functional Genomics

, Volume 10, Issue 3, pp 233–247

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

  • Erin Quartley
  • Andrei Alexandrov
  • Maryann Mikucki
  • Frederick S. Buckner
  • Wim G. Hol
  • George T. DeTitta
  • Eric M. Phizicky
  • Elizabeth J. Grayhack
Article

DOI: 10.1007/s10969-009-9068-9

Cite this article as:
Quartley, E., Alexandrov, A., Mikucki, M. et al. J Struct Funct Genomics (2009) 10: 233. doi:10.1007/s10969-009-9068-9

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.

Keywords

YeastStructural genomicsHeterologous expressionProtein solubilityS. cerevisiae

Abbreviations

LIC

Ligation independent cloning

PSI

Protein structure initiative

PDB

Protein database

L. major

Leishmania major

Supplementary material

10969_2009_9068_MOESM1_ESM.doc (142 kb)
Supplementary Table 1L major ORF targets: Putative Function and Expression in E. coli (DOC 142 kb)
10969_2009_9068_MOESM2_ESM.tif (240 kb)
Supplementary Figure 1Evaluation 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)
10969_2009_9068_MOESM3_ESM.tif (186 kb)
Supplementary Figure 2Evaluation 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)
10969_2009_9068_MOESM4_ESM.tif (213 kb)
Supplementary Figure 3Large 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)

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Erin Quartley
    • 1
  • Andrei Alexandrov
    • 6
    • 7
  • Maryann Mikucki
    • 1
  • Frederick S. Buckner
    • 2
  • Wim G. Hol
    • 3
  • George T. DeTitta
    • 4
    • 5
  • Eric M. Phizicky
    • 1
    • 6
  • Elizabeth J. Grayhack
    • 1
    • 6
  1. 1.Center for Pediatric Biomedical ResearchUniversity of Rochester Medical SchoolRochesterUSA
  2. 2.Division of Allergy and Infectious Diseases, Department of MedicineUniversity of Washington School of MedicineSeattleUSA
  3. 3.Department of BiochemistryUniversity of Washington School of MedicineSeattleUSA
  4. 4.Hauptman-Woodward Medical Research InstituteBuffaloUSA
  5. 5.Department of Structural BiologySUNY at BuffaloBuffaloUSA
  6. 6.Department of Biochemistry and BiophysicsUniversity of Rochester School of Medicine and DentistryRochesterUSA
  7. 7.Yale University School of Medicine, HHMINew HavenUSA