How to chose vector and E.coli strain for protein expression - (Sep/16/2007 )
I already tried to express 2 eukaryotic proteins (69 kDa in pET22b+, 55 kDa in pET41a+) in BL21 and Rosetta cells without success. Now, I need to repeat the expression assay with different vectors a/o E.coli cells. How do I chose which constructs to create and where to express the proteins in? I thought pET28 and 6His-tags could be used, but am not sure if it is promising, as expression in pET22 and 41 did not work. The pET SUMO system for difficult samples is too expensive for us to get, I think.
Thanks for your ideas,
Dumb question, but...
Do you know that the construct is in the correct orientation, that the sequence is correct etc?It sounds very strange that the proteins don't expressing Rosetta. Have you run the sequnece through a codon bias screen? There's one here: http://gcua.schoedl.de/ .
Yes, the constructs are in right orientation and were sequenced as well. I agree that there are rare codons, that`s why we used Rosetta. At least I don`t see an overexpression on SDS-PAGE. I didn`t add a tag, so I can`t verify a possible expression by WB
I really need to get the expression to work asap...