Expression platforms for producing eukaryotic proteins: a comparison of E. coli cell-based and wheat germ cell-free synthesis, affinity and solubility tags, and cloning strategies

  • David J. Aceti
  • Craig A. Bingman
  • Russell L. Wrobel
  • Ronnie O. Frederick
  • Shin-ichi Makino
  • Karl W. Nichols
  • Sarata C. Sahu
  • Lai F. Bergeman
  • Paul G. Blommel
  • Claudia C. Cornilescu
  • Katarzyna A. Gromek
  • Kory D. Seder
  • Soyoon Hwang
  • John G. Primm
  • Grzegorz Sabat
  • Frank C. Vojtik
  • Brian F. Volkman
  • Zsolt Zolnai
  • George N. PhillipsJr.
  • John L. Markley
  • Brian G. Fox
Article

Abstract

Vectors designed for protein production in Escherichia coli and by wheat germ cell-free translation were tested using 21 well-characterized eukaryotic proteins chosen to serve as controls within the context of a structural genomics pipeline. The controls were carried through cloning, small-scale expression trials, large-scale growth or synthesis, and purification. Successfully purified proteins were also subjected to either crystallization trials or 1H–15N HSQC NMR analyses. Experiments evaluated: (1) the relative efficacy of restriction/ligation and recombinational cloning systems; (2) the value of maltose-binding protein (MBP) as a solubility enhancement tag; (3) the consequences of in vivo proteolysis of the MBP fusion as an alternative to post-purification proteolysis; (4) the effect of the level of LacI repressor on the yields of protein obtained from E. coli using autoinduction; (5) the consequences of removing the His tag from proteins produced by the cell-free system; and (6) the comparative performance of E. coli cells or wheat germ cell-free translation. Optimal promoter/repressor and fusion tag configurations for each expression system are discussed.

Keywords

Structural genomics NIH Protein Structure Initiative Wheat germ cell-free translation Maltose Binding Protein solubility tag Flexi Vector cloning Gateway cloning 

Abbreviations

CESG

Center for Eukaryotic Structural Genomics

Flexi Vector

Promega Flexi Vector cloning system

HSQC

Heteronuclear single quantum correlation

IMAC

Immobilized metal affinity chromatography

IPTG

Isopropyl β-d-1-thiogalactopyranoside

MBP

Maltose Binding Protein

SeMet

Selenomethionine

TEV

Tobacco etch virus

TVMV

Tobacco vein mottling virus

HRV 3C

Human rhinovirus 3C protease

Supplementary material

10969_2015_9198_MOESM1_ESM.doc (114 kb)
Supplementary material 1 (DOC 114 kb)
10969_2015_9198_MOESM2_ESM.doc (548 kb)
Supplementary material 2 (DOC 548 kb)
10969_2015_9198_MOESM3_ESM.doc (6.2 mb)
Supplementary material 3 (DOC 6328 kb)
10969_2015_9198_MOESM4_ESM.xls (79 kb)
Supplementary material 4 (XLS 79 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • David J. Aceti
    • 1
    • 5
    • 6
  • Craig A. Bingman
    • 1
    • 4
    • 5
    • 6
  • Russell L. Wrobel
    • 1
    • 5
    • 6
  • Ronnie O. Frederick
    • 1
    • 5
    • 6
  • Shin-ichi Makino
    • 1
    • 5
    • 6
  • Karl W. Nichols
    • 1
  • Sarata C. Sahu
    • 1
    • 3
  • Lai F. Bergeman
    • 1
  • Paul G. Blommel
    • 1
  • Claudia C. Cornilescu
    • 1
    • 3
  • Katarzyna A. Gromek
    • 1
    • 5
    • 6
  • Kory D. Seder
    • 1
  • Soyoon Hwang
    • 1
  • John G. Primm
    • 1
    • 5
    • 6
  • Grzegorz Sabat
    • 1
  • Frank C. Vojtik
    • 1
  • Brian F. Volkman
    • 2
  • Zsolt Zolnai
    • 1
  • George N. PhillipsJr.
    • 1
    • 4
    • 7
  • John L. Markley
    • 1
    • 3
    • 5
  • Brian G. Fox
    • 1
    • 6
  1. 1.The Center for Eukaryotic Structural Genomics, Department of BiochemistryUniversity of Wisconsin at MadisonMadisonUSA
  2. 2.The Center for Eukaryotic Structural Genomics, Department of BiochemistryMedical College of WisconsinMilwaukeeUSA
  3. 3.Nuclear Magnetic Resonance Facility at Madison, Department of BiochemistryUniversity of Wisconsin at MadisonMadisonUSA
  4. 4.Natural Product Biosynthesis, BioSciences at Rice and Department of ChemistryRice UniversityHoustonUSA
  5. 5.Mitochondrial Protein Partnership, Department of BiochemistryUniversity of Wisconsin at MadisonMadisonUSA
  6. 6.Transmembrane Protein Center, Department of BiochemistryUniversity of Wisconsin at MadisonMadisonUSA
  7. 7.BioSciences at Rice and Department of ChemistryRice UniversityHoustonUSA

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