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


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.


Yeast Structural genomics Heterologous expression Protein solubility S. cerevisiae 



Ligation independent cloning


Protein structure initiative


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)


  1. 1.
    Alexandrov A, Vignali M, LaCount DJ, Quartley E, de Vries C, De Rosa D, Babulski J, Mitchell SF, Schoenfeld LW, Fields S, Hol WG, Dumont ME, Phizicky EM, Grayhack EJ (2004) A facile method for high-throughput co-expression of protein pairs. Mol Cell Proteomics 3:934–938PubMedCrossRefGoogle Scholar
  2. 2.
    Aslanidis C, de Jong PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18:6069–6074PubMedCrossRefGoogle Scholar
  3. 3.
    Braun P, LaBaer J (2003) High throughput protein production for functional proteomics. Trends Biotechnol 21:383–388PubMedCrossRefGoogle Scholar
  4. 4.
    Braun P, Hu Y, Shen B, Halleck A, Koundinya M, Harlow E, LaBaer J (2002) Proteome-scale purification of human proteins from bacteria. Proc Natl Acad Sci USA 99:2654–2659PubMedCrossRefGoogle Scholar
  5. 5.
    Burgess-Brown NA, Sharma S, Sobott F, Loenarz C, Oppermann U, Gileadi O (2008) Codon optimization can improve expression of human genes in Escherichia coli: a multi-gene study. Protein Expr Purif 59:94–102PubMedCrossRefGoogle Scholar
  6. 6.
    Burley SK, Joachimiak A, Montelione GT, Wilson IA (2008) Contributions to the NIH-NIGMS protein structure initiative from the PSI production centers. Structure 16:5–11PubMedCrossRefGoogle Scholar
  7. 7.
    Cabantous S, Rogers Y, Terwilliger TC, Waldo GS (2008) New molecular reporters for rapid protein folding assays. PLoS ONE 3:e2387PubMedCrossRefGoogle Scholar
  8. 8.
    Chandonia JM, Brenner SE (2006) The impact of structural genomics: expectations and outcomes. Science 311:347–351PubMedCrossRefGoogle Scholar
  9. 9.
    Chatterjee DK, Esposito D (2006) Enhanced soluble protein expression using two new fusion tags. Protein Expr Purif 46:122–129PubMedCrossRefGoogle Scholar
  10. 10.
    Christendat D, Yee A, Dharamsi A, Kluger Y, Savchenko A, Cort JR, Booth V, Mackereth CD, Saridakis V, Ekiel I, Kozlov G, Maxwell KL, Wu N, McIntosh LP, Gehring K, Kennedy MA, Davidson AR, Pai EF, Gerstein M, Edwards AM, Arrowsmith CH (2000) Structural proteomics of an archaeon. Nat Struct Biol 7:903–909PubMedCrossRefGoogle Scholar
  11. 11.
    Cormack BP, Bertram G, Egerton M, Gow NA, Falkow S, Brown AJ (1997) Yeast-enhanced green fluorescent protein (yEGFP)a reporter of gene expression in Candida albicans. Microbiology 143(Pt 2):303–311PubMedCrossRefGoogle Scholar
  12. 12.
    Esposito D, Chatterjee DK (2006) Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotechnol 17:353–358PubMedCrossRefGoogle Scholar
  13. 13.
    Fan E, Baker D, Fields S, Gelb MH, Buckner FS, Van Voorhis WC, Phizicky E, Dumont M, Mehlin C, Grayhack E, Sullivan M, Verlinde C, Detitta G, Meldrum DR, Merritt EA, Earnest T, Soltis M, Zucker F, Myler PJ, Schoenfeld L, Kim D, Worthey L, Lacount D, Vignali M, Li J, Mondal S, Massey A, Carroll B, Gulde S, Luft J, Desoto L, Holl M, Caruthers J, Bosch J, Robien M, Arakaki T, Holmes M, Le Trong I, Hol WG (2008) Structural genomics of pathogenic protozoa: an overview. Methods Mol Biol 426:497–513PubMedCrossRefGoogle Scholar
  14. 14.
    Flegelova H, Haguenauer-Tsapis R, Sychrova H (2006) Heterologous expression of mammalian Na/H antiporters in Saccharomyces cerevisiae. Biochim Biophys Acta 1760:504–516PubMedGoogle Scholar
  15. 15.
    Froissard M, Belgareh-Touze N, Buisson N, Desimone M, Frommer WB, Haguenauer-Tsapis R (2006) Heterologous expression of a plant uracil transporter in yeast: improvement of plasma membrane targeting in mutants of the Rsp5p ubiquitin protein ligase. Biotechnol J 1:308–320PubMedCrossRefGoogle Scholar
  16. 16.
    Fussenegger M, Hauser H (2007) Protein expression by engineering of yeast, plant and animal cells. Curr Opin Biotechnol 18:385–386PubMedCrossRefGoogle Scholar
  17. 17.
    Gelperin DM, White MA, Wilkinson ML, Kon Y, Kung LA, Wise KJ, Lopez-Hoyo N, Jiang L, Piccirillo S, Yu H, Gerstein M, Dumont ME, Phizicky EM, Snyder M, Grayhack EJ (2005) Biochemical and genetic analysis of the yeast proteome with a movable ORF collection. Genes Dev 19:2816–2826PubMedCrossRefGoogle Scholar
  18. 18.
    Gileadi O, Burgess-Brown NA, Colebrook SM, Berridge G, Savitsky P, Smee CE, Loppnau P, Johansson C, Salah E, Pantic NH (2008) High throughput production of recombinant human proteins for crystallography. Methods Mol Biol 426:221–246PubMedCrossRefGoogle Scholar
  19. 19.
    Graslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, Knapp S, Oppermann U, Arrowsmith C, Hui R, Ming J, dhe-Paganon S, Park HW, Savchenko A, Yee A, Edwards A, Vincentelli R, Cambillau C, Kim R, Kim SH, Rao Z, Shi Y, Terwilliger TC, Kim CY, Hung LW, Waldo GS, Peleg Y, Albeck S, Unger T, Dym O, Prilusky J, Sussman JL, Stevens RC, Lesley SA, Wilson IA, Joachimiak A, Collart F, Dementieva I, Donnelly MI, Eschenfeldt WH, Kim Y, Stols L, Wu R, Zhou M, Burley SK, Emtage JS, Sauder JM, Thompson D, Bain K, Luz J, Gheyi T, Zhang F, Atwell S, Almo SC, Bonanno JB, Fiser A, Swaminathan S, Studier FW, Chance MR, Sali A, Acton TB, Xiao R, Zhao L, Ma LC, Hunt JF, Tong L, Cunningham K, Inouye M, Anderson S, Janjua H, Shastry R, Ho CK, Wang D, Wang H, Jiang M, Montelione GT, Stuart DI, Owens RJ, Daenke S, Schutz A, Heinemann U, Yokoyama S, Bussow K, Gunsalus KC (2008) Protein production and purification. Nat Methods 5:135–146PubMedCrossRefGoogle Scholar
  20. 20.
    Hamilton SR, Gerngross TU (2007) Glycosylation engineering in yeast: the advent of fully humanized yeast. Curr Opin Biotechnol 18:387–392PubMedCrossRefGoogle Scholar
  21. 21.
    Hamilton SR, Davidson RC, Sethuraman N, Nett JH, Jiang Y, Rios S, Bobrowicz P, Stadheim TA, Li H, Choi BK, Hopkins D, Wischnewski H, Roser J, Mitchell T, Strawbridge RR, Hoopes J, Wildt S, Gerngross TU (2006) Humanization of yeast to produce complex terminally sialylated glycoproteins. Science 313:1441–1443PubMedCrossRefGoogle Scholar
  22. 22.
    Hammarstrom M, Hellgren N, van Den Berg S, Berglund H, Hard T (2002) Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli. Protein Sci 11:313–321PubMedCrossRefGoogle Scholar
  23. 23.
    Holz C, Prinz B, Bolotina N, Sievert V, Bussow K, Simon B, Stahl U, Lang C (2003) Establishing the yeast Saccharomyces cerevisiae as a system for expression of human proteins on a proteome-scale. J Struct Funct Genomics 4:97–108PubMedCrossRefGoogle Scholar
  24. 24.
    Jidenko M, Nielsen RC, Sorensen TL, Moller JV, le Maire M, Nissen P, Jaxel C (2005) Crystallization of a mammalian membrane protein overexpressed in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 102:11687–11691PubMedCrossRefGoogle Scholar
  25. 25.
    Kashiwagi K, Taneja SK, Liu TY, Tabor CW, Tabor H (1990) Spermidine biosynthesis in Saccharomyces cerevisiae. Biosynthesis and processing of a proenzyme form of S-adenosylmethionine decarboxylase. J Biol Chem 265:22321–22328PubMedGoogle Scholar
  26. 26.
    Keppler-Ross S, Noffz C, Dean N (2008) A new purple fluorescent color marker for genetic studies in Saccharomyces cerevisiae and Candida albicans. Genetics 179:705–710PubMedCrossRefGoogle Scholar
  27. 27.
    Kjeldsen T (2000) Yeast secretory expression of insulin precursors. Appl Microbiol Biotechnol 54:277–286PubMedCrossRefGoogle Scholar
  28. 28.
    Klock HE, Koesema EJ, Knuth MW, Lesley SA (2008) Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts. Proteins 71:982–994PubMedCrossRefGoogle Scholar
  29. 29.
    Levitt M (2007) Growth of novel protein structural data. Proc Natl Acad Sci USA 104:3183–3188PubMedCrossRefGoogle Scholar
  30. 30.
    Luan CH, Qiu S, Finley JB, Carson M, Gray RJ, Huang W, Johnson D, Tsao J, Reboul J, Vaglio P, Hill DE, Vidal M, Delucas LJ, Luo M (2004) High-throughput expression of C. elegans proteins. Genome Res 14:2102–2110PubMedCrossRefGoogle Scholar
  31. 31.
    Macbeth MR, Lingam AT, Bass BL (2004) Evidence for auto-inhibition by the N terminus of hADAR2 and activation by dsRNA binding. RNA 10:1563–1571PubMedCrossRefGoogle Scholar
  32. 32.
    Macbeth MR, Schubert HL, Vandemark AP, Lingam AT, Hill CP, Bass BL (2005) Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing. Science 309:1534–1539PubMedCrossRefGoogle Scholar
  33. 33.
    Malkowski MG, Quartley E, Friedman AE, Babulski J, Kon Y, Wolfley J, Said M, Luft JR, Phizicky EM, DeTitta GT, Grayhack EJ (2007) Blocking S-adenosylmethionine synthesis in yeast allows selenomethionine incorporation and multiwavelength anomalous dispersion phasing. Proc Natl Acad Sci USA 104:6678–6683PubMedCrossRefGoogle Scholar
  34. 34.
    Marblestone JG, Edavettal SC, Lim Y, Lim P, Zuo X, Butt TR (2006) Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO. Protein Sci 15:182–189PubMedCrossRefGoogle Scholar
  35. 35.
    Mehlin C, Boni E, Buckner FS, Engel L, Feist T, Gelb MH, Haji L, Kim D, Liu C, Mueller N, Myler PJ, Reddy JT, Sampson JN, Subramanian E, Van Voorhis WC, Worthey E, Zucker F, Hol WG (2006) Heterologous expression of proteins from Plasmodium falciparum: results from 1000 genes. Mol Biochem Parasitol 148:144–160PubMedCrossRefGoogle Scholar
  36. 36.
    Nair R, Liu J, Soong TT, Acton TB, Everett JK, Kouranov A, Fiser A, Godzik A, Jaroszewski L, Orengo C, Montelione GT, Rost B (2009) Structural genomics is the largest contributor of novel structural leverage. J Struct Funct Genomics 10:181–191PubMedCrossRefGoogle Scholar
  37. 37.
    Niiranen L, Espelid S, Karlsen CR, Mustonen M, Paulsen SM, Heikinheimo P, Willassen NP (2007) Comparative expression study to increase the solubility of cold adapted Vibrio proteins in Escherichia coli. Protein Expr Purif 52:210–218PubMedCrossRefGoogle Scholar
  38. 38.
    Peti W, Page R (2007) Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost. Protein Expr Purif 51:1–10PubMedCrossRefGoogle Scholar
  39. 39.
    Phizicky EM, Grayhack EJ (2006) Proteome-scale analysis of biochemical activity. Crit Rev Biochem Mol Biol 41:315–327PubMedCrossRefGoogle Scholar
  40. 40.
    Punt PJ, van Biezen N, Conesa A, Albers A, Mangnus J, van den Hondel C (2002) Filamentous fungi as cell factories for heterologous protein production. Trends Biotechnol 20:200–206PubMedCrossRefGoogle Scholar
  41. 41.
    Roodveldt C, Tawfik DS (2005) Directed evolution of phosphotriesterase from Pseudomonas diminuta for heterologous expression in Escherichia coli results in stabilization of the metal-free state. Protein Eng Des Sel 18:51–58PubMedCrossRefGoogle Scholar
  42. 42.
    Roodveldt C, Aharoni A, Tawfik DS (2005) Directed evolution of proteins for heterologous expression and stability. Curr Opin Struct Biol 15:50–56PubMedCrossRefGoogle Scholar
  43. 43.
    Service RF (2002) Structural genomics. Tapping DNA for structures produces a trickle. Science 298:948–950PubMedCrossRefGoogle Scholar
  44. 44.
    Sherman F, Fink G, Hicks JB (1986) In: Methods in yeast genetics. Cold Spring Harbor Laboratory Press, New York, pp 145–149Google Scholar
  45. 45.
    Tran T, Buscher P, Vandenbussche G, Wyns L, Messens J, De Greve H (2008) Heterologous expression, purification and characterisation of the extracellular domain of trypanosome invariant surface glycoprotein ISG75. J Biotechnol 135:247–254PubMedCrossRefGoogle Scholar
  46. 46.
    Waldo GS (2003) Genetic screens and directed evolution for protein solubility. Curr Opin Chem Biol 7:33–38PubMedCrossRefGoogle Scholar
  47. 47.
    Walsh G (2006) Biopharmaceutical benchmarks. Nat Biotechnol 24:769–776PubMedCrossRefGoogle Scholar
  48. 48.
    Waugh DS (2005) Making the most of affinity tags. Trends Biotechnol 23:316–320PubMedCrossRefGoogle Scholar

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

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