IPTG expression w/ GST-fusion gene - (Feb/07/2006 )
I tried IPTG expression with GST-fusion gene ((size about 74KD)) in pGEX-4T1 vector using XL10 as hosts. The control worked fine but the fusion gene did show any sign of expression on SDS-PAGE gel... Can anybody think of any reasons?
not all protein are strongly expressed enough to be seen directly on the gel.
Did you try to western-blot?
how are the bacteria? some proteins are toxic for the bacteria and they die. you can try to reduce IPTG concentration, reduce the temperature, time of incubation...
Yes, I have tried Western-Blot and could not see any bands with right size. I will try reducing IPTG concentration and time... etc. Thanks
Besides that, Do you think it is worth while to trying pET system?
I normally use BL21 coli cells as a host for this vector pGEX-4T1 and works excellent. In the past i had expression problems using other type of host and when i changed for BL21 the expression was excellent under same expression conditions. Sometimes strain is significant for expression of some vectors. Try to change the host may work!
I have tried the pGEX-5T2 vector on BL21 host cells. The expression of my protein of interest (about 50kDa) is high after IPTG induction. I can see a big band on stained SDS-PAGE gel.
However, I found two problems of my system. (1) The expression of large protein (<50 kDa)is significantly low than the small protein (>50 kDa). (2) The protein degardation is serious, even proteinase inhibitors were added.
Therefore, is the problem of low expression caused by the protein degardation?
Anyone can give some comments or advice dealing with this two problems.
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