[ccp4bb] Expression vector with NdeI-ClaI sites

[Mark J. van Raaij]

Dear Bulletin Boarders,

We have a protein cloned NdeI-ClaI in pT7.7, which expresses  
insoluble. Ideally, we would like to do a simple sub-cloning using  
NdeI and ClaI into another vector with an N-terminal His-tag, to  
produce large amounts of insoluble protein which can then be easily  
purified by metal chromatography in denaturing conditions. We are not  
looking to crystallise this particular protein, it is for other  
purposes.

I had a look at various vectors (pET, pQE and others), but did not  
find one which has NdeI (CATATG of which the ATG would code for the  
first Met of our protein) as a 5'/Nterm cloning site and ClaI (ATCGAT)  
at the 3'/Cterm, and an N-terminal His-tag. Does anyone know of a  
commercially or otherwise available expression vector with these  
characteristics? For good order, if it is commercially available, we  
will buy it, we are not looking for a free ride here...

If finally there is no such vector, we will just order primers and do  
PCR-cloning, sequencing etc., but I thought I'd try this first, as the  
vector may be useful for other projects, but the primers won't.

Greetings,

Mark