Also do you know a company for site-directed mutagenesis and subcloning for a decent price?
I am wondering it is right strategy to change the expression vector alone (e.g. pQE80L to pET19b). I am also curious whether it helps getting better expression for membrane proteins as well. I think it seems too greedy to solicit some sort of hand-on experiences to the expertise in public. But I know you will kindly share your idea.
Secondly, I am also looking at the companies that can manage the sub-cloning project for swapping vector. Our lab does not have any of equipment or enzymes for even simple cloning (e.g. DNA electrophoresis equipments, ligase, polymerase, restriction enzyme and ect.) because I am working in the environment of chemistry instead of biology or structure or molecular biology. I guess that there are many companies which has a service for simple mutagenesis and cloning. I am trying to introduce two restriction enzyme sites for subcloning to new bacterial expression vector and move to new vector that has different purification tags. Would you help me to find the decent company you are using currently?
Genscript offers DNA synthesis, site-directed mutagenesis, and subcloning at a competitive price. I've used them for a while and they are reliabe. http://www.genscript.com/index.html
Hi Sung, to answer your questions regarding bacterial expression vector the choice of vector can make a HUGE difference. This difference comes about as a result of the promoter choice and the various tags/options available for different vectors.
To put it simply different expression vectors have different options that can aid in generating and purifying a soluble recombinant protein. So whereas your pQE80L vector has a T5 promoter and a N terminal His tag the pET vector has a T7 promoter with a HIS and enterokinase site. If your protein requires disulfide bond formation you may need a thioredoxin vector and the BL21 or Rosetta gammi cell line. If you have problems with producing a soluble protein, export, or if your protein is a membrane bound protein different vectors and tags can add in those applications
As for companies Genscript has always been great for us as well as Biobasic. Biomatik is also a good call but usually much more expensive.
All the best,
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