May be old fashioned, but what about cut with ClaI, fill in, and then you have a blunt end to work with.
-----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Artem Evdokimov Sent: Sunday, July 20, 2008 12:02 PM To: [email protected] Subject: Re: [ccp4bb] Expression vector with NdeI-ClaI sites Previous email was incomplete - baby hit the keyboard :) The last thing I was going to mention is that pET15b has NdeI/ClaI option but you have to include T7 terminator because ClaI site is behind it. Artem -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Mark J. van Raaij Sent: Saturday, July 19, 2008 7:15 AM To: [email protected] Subject: [ccp4bb] Expression vector with NdeI-ClaI sites 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
