We are trying to express a eukaryotic cDNA in a prokaryotic expression
system. The E. coli strain is M15 [pREP4] from QIAgen. The pQE40 expression
vector consist of 6His-mouseDHFR and our cDNA (220 bp). Between the
mouseDHFR and the cDNA we placed a factor Xa-digestion site. In the
beginning we had poor expression. At one moment, however, we obtained very
good expression. We always used the same glycerolstock which is defreezed
before used. So repeated freezing and defreezing of the bacteria seems to
result in better expression. Does anybody understand this?
After changing the Xa-site (4 aa) into AspPro (2 aa) expression is again
very low. We tried already lower expression temperature and different
incubation times. Repeated freezing and defreezing didn't result in a
better expression. Can someone help us?
Thanks in advance,
Henri M.H. Spronk
Department of Biochemistry
henri.spronk at bioch.unimaas.nl