title: 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.
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 (1)H-(15)N 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.
authors: The Center for Eukaryotic Structural Genomics, Department of Biochemistry, University of Wisconsin at Madison, 433 Babcock Dr., Madison, WI, 53706, USA.
authors: David J
authors: DJ
authors: Aceti
authors: Craig A
authors: CA
authors: Bingman
authors: Russell L
authors: RL
authors: Wrobel
authors: Ronnie O
authors: RO
authors: Frederick
authors: Shin-Ichi
authors: S
authors: Makino
authors: Karl W
authors: KW
authors: Nichols
authors: Sarata C
authors: SC
authors: Sahu
authors: Lai F
authors: LF
authors: Bergeman
authors: Paul G
authors: PG
authors: Blommel
authors: Claudia C
authors: CC
authors: Cornilescu
authors: Katarzyna A
authors: KA
authors: Gromek
authors: Kory D
authors: KD
authors: Seder
authors: Soyoon
authors: S
authors: Hwang
authors: John G
authors: JG
authors: Primm
authors: Grzegorz
authors: G
authors: Sabat
authors: Frank C
authors: FC
authors: Vojtik
authors: Brian F
authors: BF
authors: Volkman
authors: Zsolt
authors: Z
authors: Zolnai
authors: George N
authors: GN
authors: Phillips
authors: John L
authors: JL
authors: Markley
authors: Brian G
authors: BG
authors: Fox
mesh_terms: D002474:Cell-Free System; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D056890:Eukaryota / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D005854:Germ Cells; D014176:Protein Biosynthesis / Q000235:genetics*; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification; D014908:Triticum / Q000235:genetics
chemical_list: D011506:Proteins