Clone the sequence of a protein in two different vectors, one is pET21a+ which gives a His sequence at the C-terminus and the other vector is pET41A+ which gives His at the C-terminus as well as at the N-terminus, but in addition this last vector gives a GST at the N-terminus. The cloned sequence in both vectors is already verified by PCR and restriction enzyme digestion. These vectors with the protein sequences were transformed into E.Coli BL21 bacteria (this step is also verified) which were grown to an OD of 0.6 and then induced by IPTG at 20ºC overnight. After induction I centrifuge the bacteria and mix the pellet with a solubilization buffer to perform a sonication of 6 pulses of 20 seconds (the whole process was performed on ice). The problem I have is that when I perform the purification either by Ni-NTA resin or Glutathione resin, the protein is not observed in the SDS-PAGE. I am performing the protocol provided by the supplier which I have already performed with other proteins of the same type and which have been purified correctly. So I really do not know where the problem could be, the protein sequence is correct, the protocol works for other proteins of the same type, however with the protein that I am currently working with I have not been able to purify it. The protocols and buffers I have used to purify are as follows: 1) https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FMAN0011804_HisPurTM_NiNTA_SupfLw_Agarose_UG.pdf 2) https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FMAN0011719_Pierce_Glutathione_Agarose_UG.pdf
Dear Brandon Rosales
each protein as its own history that depends from its specific properties as:
- MW;
- isoelettric point;
- presence of cisteines and or hidrofobic regions;
- presence of rare codons (expecially for protein derived from eucariotic)
so a protocoll the work well with some proteins can not work at all for some others.
Can you provide some more details about your specific protein?
From your message, i do not undestand if the problem is at the level of
- expression; (no overexpression band observed in SDS page total extract)
- solubility (no soluble band are observed in the soluble fraction but a band is observed in the total exctract)
- purification (a band is detectable in both total extract and soluble fraction but you are not able to enrich and purify it with IMAC and/or GST purification.
best regards
Manuele
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