A spontaneous direct repeat deletion in the pGEX fusion vector decreases the expression level of recombinant proteins in Escherichia coli.
pGEX vectors are widely used for GST-fusion protein expression in Escherichia coli under the control of a strong IPTG inducible tac promoter. While using pGEX-4T-2 vector in heterologous protein expression we noticed that the GST or GST-fusion protein were expressed at a very low levels. Interestingly, we found a spontaneous deletion of 701 bp DNA fragment harbouring the tac promoter in both, native and recombinant pGEX-4T-2 vectors. This deletion took place between two direct repeats of 43 bases and led to the loss of a 701 bp DNA fragment. This explained the decrease in GST or GST-fused protein level since the tac promoter, that directs transcription was deleted. The lacZ promoter, located upstream of the deleted fragment, replaced tac promoter but was less efficient. The deleted DNA also specifies part of the lacZ gene coding for the N-terminal end of the beta-galactosidase (the alpha-peptide), which is slightly functional. Consequently, bacterial cells transformed with the original pGEX are of a faint blue colour while those bearing the deleted ones are white, when plated on X-Gal containing medium. The deletion, did not affect neither the sequence nor the molecular weight of GST and fusion protein since it took place just before the GST start codon. It occurred in E. coli TOP10 cells which are deficient in RecA protein, suggesting that the deletion did not require the RecA recombination system.
Borgi
Ines
I
Laboratoire de Génétique Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, Route Sidi Mansour, BP"K"3038, Sfax, Tunisia.
Gargouri
Ali
A
D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D017384:Sequence Deletion; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase