Augmentation of protein production by a combination of the T7 RNA polymerase system and ubiquitin fusion: overproduction of the human DNA repair protein, ERCC1, as a ubiquitin fusion protein in Escherichia coli.
This article presents the development of a set of new expression vectors for overproduction of proteins in Escherichia coli. The vectors, pETUBI-ES1, 2 and 3, allow in-frame cloning of any sequence with the ubiquitin gene driven by the strong T7f10 promoter. Combination of the T7 expression system with ubiquitin fusion appears to have a synergistic effect on protein overproduction. Large amounts of stable RNA are produced by T7 RNA polymerase, and fusion of ubiquitin to the N-terminus of target proteins seems to confer more efficient translation, better folding or protection against proteolytic degradation. The ubiquitin part can be utilized for purification of the fusion protein, after which it can be easily removed from the fusion product by ubiquitin-specific proteases. The advantage of combining both systems is demonstrated by the synthesis of large quantities (up to 40-50% of the total protein) of the human ERCC1 protein that hitherto was refractory to overproduction in various other E. coli and yeast expression systems.
Koken
M H
MH
Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands.
Odijk
H H
HH
van Duin
M
M
Fornerod
M
M
Hoeijmakers
J H
JH
D001483:Base Sequence; D003001:Cloning, Molecular; D004260:DNA Repair; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D015183:Restriction Mapping; D013604:T-Phages / Q000201:enzymology / Q000235:genetics; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D004720:Endonucleases