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Re: [ccp4bb] TEV nucleotude sequence with restriction site |
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- Protein crystallographyMain steps:- Protein purification- Crystallisation Special:- Programs for crystallography- X-ray detectors Basic tutorials:- Chemistry- Protein - Peptide - Amino Acids Xtal community:- CCP4BB |
CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999Subject: Re: TEV nucleotude sequence with restriction site From: Chun Luo chun {- at -} ACCELAGEN {- dot -} COM Date: 2009-06-05 Try type II restriction enzymes. They cleave outside of their recognition sites. So you can use one vector for different inserts. There may be a type I enzyme site for TEV site, I haven't found a good one yet. It's likely some inserts will have the same site internally, making the vector less useful. BTW, Accelagen has TurboTEV with dual GST- and His-tags. TurboTEV is stabilized through a mechanism different from the common S219 mutations. So it does not infringe the recently issued Yale patent. Cheers, Chun Chun Luo, Ph.D. Accelagen, Inc. 6044 Cornerstone Court West, Suite C San Diego, CA 92121 P: 858-678-8618 ext 111 F: 858-678-8628 chun@accelagen.com www.accelagen.com -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Charlie Bond Sent: Friday, June 05, 2009 4:00 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] TEV nucleotude sequence with restriction site Hi Jacob, If you're including the TEV site in your primer, then you won't need a restriction site after it (unless you're planning to recycle the vector afterwards for other inserts). Having the protease site too close to the protein can often make it difficult to cut. Cheers, Charlie Quotfintg Jacob Keller > I checked out the Sheffield et al paper, and the restriction sites > there are all just after the TEV site, thereby including, as Cynthia > mentioned, at least an extra H beyond the obligatory G from the TEV > site. I was hoping to be able to have only the G. (Since I am cloning > in the TEV site with my PCR primer, I have free choice about what > codons to choose, and therefore think it would be nice to have the > restriction site in the TEV site itself, if possible. Also, this will > keep my primer a little shorter.) > > Jacob > > ******************************************* > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > Dallos Laboratory > F. Searle 1-240 > 2240 Campus Drive > Evanston IL 60208 > lab: 847.491.2438 > cel: 773.608.9185 > email: j-keller2@northwestern.edu > ******************************************* > > ----- Original Message ----- From: "Cynthia Kinsland" > To: "Jacob Keller" > Cc: > Sent: Friday, June 05, 2009 5:19 PM > Subject: Re: [ccp4bb] TEV nucleotude sequence with restriction site > > >> I'm not quite sure what you want, but I have a series of vectors >> encoding various N-terminal tags and fusions, all followed by a TEV >> site. They have an MCS standard to many pET vectors. Therefore, >> they are designed to clone your gene in using the NdeI site at the >> 5' end (which will, after proteolysis, leave you with GH at the >> N-terminus of your protein). Other restriction enzymes in the MCS >> can be used, but more amino acids will be left at your N-terminus. >> >> I've used WatCut (from the U. Waterloo) for the silent mutagenesis >> question: http://watcut.uwaterloo.ca/watcut/watcut/template.php >> >> Best, >> >> Cynthia >> >> On Jun 5, 2009, at 5:41 PM, Jacob Keller wrote: >> >>> Dear Crystallographers, >>> >>> Does anybody have a TEV-protease-site-coding nucleotide sequence >>> with a commonly-used restriction site in it, preferably right at >>> the end? Alternatively, does some somebody know of a program to >>> determine all equivalent codon permutations for a small coding >>> region, filtered for resulting restriction site possibilities? It >>> seems like it would be an easy enough script to write... >>> >>> (I have already done some googling around for such a program, with >>> not much luck.) >>> >>> Jacob >>> >>> ******************************************* >>> Jacob Pearson Keller >>> Northwestern University >>> Medical Scientist Training Program >>> Dallos Laboratory >>> F. Searle 1-240 >>> 2240 Campus Drive >>> Evanston IL 60208 >>> lab: 847.491.2438 >>> cel: 773.608.9185 >>> email: j-keller2@northwestern.edu >>> ******************************************* >> -- Charlie Bond Professorial Fellow University of Western Australia School of Biomedical, Biomolecular and Chemical Sciences M310, 35 Stirling Highway Crawley WA 6009, Australia CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999 |
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