3 replies to this topic

[ManuelM]

Hi everyone!

I´m a PhD student and I´m trying, after some problems, to express a plant protein. Im going to clone in a vector my gene of interest with His-Tag and TEV cleavage site (somepeople tried this way and it works for other specie). My doubt is the next: I know I have to put an ATG start codon before His-Tag but algo my gene of interest has to star with an ATG start codon.

Thanks.

[protolder]

Hola, I don´t know if you want clone your protein in plants or the plant protein in another system. Anyway look the map of the vector, because  some of them have after the protease sequence cut, a NdeI site that mantains the ATG of the cloned protein. Think the possibility of use NdeI to extract your gene by 5´ and choose a vector wich has NdeI after the sequence of protease cut; study the cloning and the digestion of the expressed protein if it wasn´t possible you have design oligonucleotides to create the neccesary end for cloning by PCR. Buena suerte

[ManuelM]

protolder, on 25 January 2011 - 10:36 PM, said:

Hola, I don´t know if you want clone your protein in plants or the plant protein in another system. Anyway look the map of the vector, because  some of them have after the protease sequence cut, a NdeI site that mantains the ATG of the cloned protein. Think the possibility of use NdeI to extract your gene by 5´ and choose a vector wich has NdeI after the sequence of protease cut; study the cloning and the digestion of the expressed protein if it wasn´t possible you have design oligonucleotides to create the neccesary end for cloning by PCR. Buena suerte

Thanks for your suggestions.

Firstly, I´ll express in E. coli and then we will see...The option that you mention is a good idea. The background of this lab, the clone a similar protein only with the His-Tag and without the methionine aa from the protein sequence but they didnt use Tev-protease. Gracias por la rápida respuesta .

[protolder]

Hola Manuel, Here we use the pET system, but when we want recover the protein without tag or we use pGex6T or a modified pET28 with prescision cut, because the experience with trombin isn´t very good and prescision cuts more effectivelly. Buena suerte again