bacterial protease expression - expression (Jan/04/2007 )
hi friends I am trying to clone a intracellular bactreial protease. But it is not expressing. I sequenced the clone , there was no mutation in the clone what could be the reason ? And the clone is growing well in the antibiotic flask, i am frustated, is there any other reason?
how did you check the expression?
addition of IPTG and SDS PAGE. contol and induced remained same
did you solubilize the bacteria pellet directly with laemmli? (so you have all proteins, soluble and in inclusion bodies)
sometime the protein is toxic, and not well expressed, so you can't see it by coomassie blue staining.
Have you tried a western-blot?
you could try different concentrations of IPTG from 0.1M to 1M and also optimize time of induction, from 1 to 6 hours.
you could also have a look here : http://www.embl-heidelberg.de/ExternalInfo..._e_coli_ext.htm
thanks I will try it and let you know
I am just couldnt find answer.
I will just explain what I did
1. I have cloned a protease gene in Pet16
2. I tried to express in BL 21 -- in dint work
3. I tried cold expression with addion of alcohol- no expression
4. I analysed codon usage- and I found that three arginine rare codondon are present in tendem-
so I used RIL strain to get rare codon tTNA - this also dint work
5. I have sequenced the clone it is perfectly all right-- including vector region
6. Is there any bacilllus strain with T7 polymerase?
7. Any one can tell me whta I shouod do to solve thils problem