Large-scale protein expression for proteome research.
Access to pure and soluble recombinant proteins is essential for numerous applications in proteome research, such as the production of antibodies, structural characterization of proteins, and protein microarrays. Through the German cDNA Consortium we have access to more than 1500 ORFs encoding uncharacterized proteins. Preparing a large number of recombinant proteins calls for the careful refinement and re-evaluation of protein purification tools. The expression and purification strategy should result in mg quantities of protein that can be employed in microarray-based assays. In addition, the experimental set-up should be robust enough to allow both automated protein expression screening and the production of the proteins on a mg scale. These requirements are best fulfilled by a bacterial expression system such as Escherichia coli. To develop an efficient expression strategy, 75 different ORFs were transferred into suitable expression vectors using the Gateway cloning system. Four different fusion tags (E. coli transcription-termination anti-termination factor (NusA), hexahistidine tag (6xHis), maltose binding protein (MBP) and GST) were analyzed for their effect on yield of induced fusion protein and its solubility, as determined at two different induction temperatures. Affinity-purified fusion proteins were confirmed by MALDI-TOF MS.
Korf
Ulrike
U
Molecular Genome Analysis, DKFZ, Heidelberg, Germany. u.korf@dkfz.de
Kohl
Thorsten
T
van der Zandt
Hans
H
Zahn
Regina
R
Schleeger
Simone
S
Ueberle
Barbara
B
Wandschneider
Silke
S
Bechtel
Stephanie
S
Schnölzer
Martina
M
Ottleben
Holger
H
Wiemann
Stefan
S
Poustka
Annemarie
A
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D018076:DNA, Complementary; D029968:Escherichia coli Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D020543:Proteome; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D012106:Research; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D020543:Proteome; D011993:Recombinant Fusion Proteins