Thanks for the clarification; will look into TEV instead. Particularly if it's easier to homebrew in bacteria! All the best, Cathal
On 09/10/12 07:13, DK wrote: > In article <[email protected]>, Cathal Garvey > <[email protected]> wrote: >> Hi DK, >> Is there any reason not to recommend enterokinase? Recognition site is >> DDDDK (right up your alley, no? ;)). I'm working on a project of my own >> and had been planning to use this enzyme instead. > > I am not quite sure why but enterokinase is not a very popular. > TEV is very popular. My guesses why: > > 1. Less specificity (that Lys is definitely not an absolute requirement) > 2. DDDD is pretty crazy - likely to stick to basic patches. > 3. Much harder to express in E.coli ==> more expensive. (?) > 4. Because it has disulfide in the structure, high [reducing agents] > inhibit enterokinase (?) > >> On 04/10/12 05:27, DK wrote: >>> In article <[email protected]>, [email protected] (DK) >> wrote: >>> &species= >>>> Paper describing the construct and outlining purification: >>>> 17543538 >>> >>> Sorry! That number is Pubmed ID. >>> We skip all the fancy equipment in the paper and just do >>> gravity column with SP-Sepharose and step elution followed >>> by gravity Ni-NTA and also step elution. > > Actually, it's the other way around: first Ni-NTA, elute with > imidazole without salt, dilute and load onto SP. > > DK > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods > -- www.indiebiotech.com twitter.com/onetruecathal joindiaspora.com/u/cathalgarvey PGP Public Key: http://bit.ly/CathalGKey _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
