title: Heterologous expression of L. major proteins in S. cerevisiae: a test of solubility, purity, and gene recoding.
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.
authors.affiliation: Center for Pediatric Biomedical Research, University of Rochester Medical School, Rochester, NY 14642, USA.
authors.forename: Erin
authors.initials: E
authors.lastname: Quartley
authors.forename: Andrei
authors.initials: A
authors.lastname: Alexandrov
authors.forename: Maryann
authors.initials: M
authors.lastname: Mikucki
authors.forename: Frederick S
authors.initials: FS
authors.lastname: Buckner
authors.forename: Wim G
authors.initials: WG
authors.lastname: Hol
authors.forename: George T
authors.initials: GT
authors.lastname: DeTitta
authors.forename: Eric M
authors.initials: EM
authors.lastname: Phizicky
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Grayhack
mesh_terms: 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
chemical_list: D003062:Codon; D015800:Protozoan Proteins
references.citation: Methods Mol Biol. 2008;426:221-46
references.pmid: 18542867
references.citation: Protein Eng Des Sel. 2005 Jan;18(1):51-8
references.pmid: 15790580
references.citation: Proc Natl Acad Sci U S A. 2005 Aug 16;102(33):11687-91
references.pmid: 16087876
references.citation: Protein Sci. 2002 Feb;11(2):313-21
references.pmid: 11790841
references.citation: Methods Mol Biol. 2008;426:497-513
references.pmid: 18542886
references.citation: Structure. 2008 Jan;16(1):5-11
references.pmid: 18184575
references.citation: Trends Biotechnol. 2005 Jun;23(6):316-20
references.pmid: 15922084
references.citation: Genes Dev. 2005 Dec 1;19(23):2816-26
references.pmid: 16322557
references.citation: Mol Biochem Parasitol. 2006 Aug;148(2):144-60
references.pmid: 16644028
references.citation: J Biotechnol. 2008 Jun 30;135(3):247-54
references.pmid: 18538880
references.citation: PLoS One. 2008 Jun 11;3(6):e2387
references.pmid: 18545698
references.citation: Nat Struct Biol. 2000 Oct;7(10):903-9
references.pmid: 11017201
references.citation: Protein Expr Purif. 2007 Mar;52(1):210-8
references.pmid: 17064934
references.citation: Protein Sci. 2006 Jan;15(1):182-9
references.pmid: 16322573
references.citation: Mol Cell Proteomics. 2004 Sep;3(9):934-8
references.pmid: 15240823
references.citation: Proc Natl Acad Sci U S A. 2002 Mar 5;99(5):2654-9
references.pmid: 11880620
references.citation: Curr Opin Biotechnol. 2006 Aug;17(4):353-8
references.pmid: 16781139
references.citation: Genome Res. 2004 Oct;14(10B):2102-10
references.pmid: 15489332
references.citation: J Struct Funct Genomics. 2009 Apr;10(2):181-91
references.pmid: 19194785
references.citation: Curr Opin Biotechnol. 2007 Oct;18(5):387-92
references.pmid: 17951046
references.citation: Biochim Biophys Acta. 2006 Mar;1760(3):504-16
references.pmid: 16503379
references.citation: Proc Natl Acad Sci U S A. 2007 Apr 17;104(16):6678-83
references.pmid: 17426150
references.citation: J Struct Funct Genomics. 2003;4(2-3):97-108
references.pmid: 14649293
references.citation: Science. 2006 Sep 8;313(5792):1441-3
references.pmid: 16960007
references.citation: Nucleic Acids Res. 1990 Oct 25;18(20):6069-74
references.pmid: 2235490
references.citation: Nat Biotechnol. 2006 Jul;24(7):769-76
references.pmid: 16841057
references.citation: Protein Expr Purif. 2008 May;59(1):94-102
references.pmid: 18289875
references.citation: Crit Rev Biochem Mol Biol. 2006 Sep-Oct;41(5):315-27
references.pmid: 16911958
references.citation: Curr Opin Biotechnol. 2007 Oct;18(5):385-6
references.pmid: 18006295
references.citation: Genetics. 2008 May;179(1):705-10
references.pmid: 18493083
references.citation: Microbiology (Reading). 1997 Feb;143 ( Pt 2):303-311
references.pmid: 9043107
references.citation: Proc Natl Acad Sci U S A. 2007 Feb 27;104(9):3183-8
references.pmid: 17360626
references.citation: J Biol Chem. 1990 Dec 25;265(36):22321-8
references.pmid: 2266128
references.citation: Trends Biotechnol. 2002 May;20(5):200-6
references.pmid: 11943375
references.citation: Science. 2002 Nov 1;298(5595):948-50
references.pmid: 12411682
references.citation: Trends Biotechnol. 2003 Sep;21(9):383-8
references.pmid: 12948670
references.citation: Biotechnol J. 2006 Mar;1(3):308-20
references.pmid: 16897711
references.citation: Appl Microbiol Biotechnol. 2000 Sep;54(3):277-86
references.pmid: 11030562
references.citation: Science. 2005 Sep 2;309(5740):1534-9
references.pmid: 16141067
references.citation: Protein Expr Purif. 2006 Mar;46(1):122-9
references.pmid: 16146696
references.citation: Proteins. 2008 May 1;71(2):982-94
references.pmid: 18004753
references.citation: RNA. 2004 Oct;10(10):1563-71
references.pmid: 15383678
references.citation: Science. 2006 Jan 20;311(5759):347-51
references.pmid: 16424331
references.citation: Curr Opin Struct Biol. 2005 Feb;15(1):50-6
references.pmid: 15718133
references.citation: Nat Methods. 2008 Feb;5(2):135-46
references.pmid: 18235434
references.citation: Protein Expr Purif. 2007 Jan;51(1):1-10
references.pmid: 16904906
references.citation: Curr Opin Chem Biol. 2003 Feb;7(1):33-8
references.pmid: 12547424