Hi Everyone, can you please help me in a problem of protein purification...
I am trying to purify recombinant protein PmST1 (sequence is Gly-Va-lAsp-Lys-Ala-Gly-Cys-Arg-Tyr-Met-Phe-Gly-Gly-Cys-Ser-Val-Asn-Asp-Asp-Cys-Cys-Pro-Arg-Leu-Gly-Cys-His-Ser-Leu-Phe-Ser-Tyr-Cys-Ala-Trp-Asp-Leu-Thr-Phe-Ser-Asp) using the following condition..
37C/2 hr was grown the secondary culture, OD showed 0.9 and then IPTG was induced with a final concentration of 0.1 mM IPTG// 16C, 20 hrs for protein Induction. 1l culture pellet was allowed to purify as below :
Cell pellet was resuspended in 20 ml lysis buffer per lt of cell pellet with the cell lysis buffer composition : 100 mM Tris HCl (pH 8.0), containing 0.1 % Triton-X-100, NaCl and then sonication. Cell lysate separation then Ni NTA purification in ice condition.
Washed by 6 column volume of binding buffer : 5 mM imidazole, 0.5 M NaCl,20 mM Tris HCl, pH 7.5, followed by washing with 8 column volume of washing buffer (20 mM imidazole, 0.5 M NaCl,20 mM Tris HCl, pH 7.5) and eluted with 8 column volume of elution buffer (200 mM imidazole, 0.5 M NaCl,20 mM Tris HCl, pH 7.5), dialyzed against dialysis buffer (50 mM Tris HCl, pH 7.5 containing10% Glycerol).
Protein shows the correct band but not showing any activity in sialyl transferrase assay and also shows an abrupt band at A230..
I understand the A230 band is from the peptide band, but My question is whether A230 band is abruptly high because of unfolding of the protein, may be in time of purifying.....
I am puzzled by the sequence you mention. This is just the beginning of the sequence, as given in the web site of Sigma-Aldrich. PmST1 is a 46 kDa protein and I suppose thatyou are trying to express the whole gene. I wouldn't worry about the 230 nm absorbance. You have currently no means to know whether it is correlated to the activity of the enzyme.
Would it be possible to assay the specific activity of the enzyme in the crude extract and compare the result with an extract of the host not transformed with the target gene ? Or look at the protocol for production of the recombinant protein as described in the original literature ?
Pierre Béguin Thank you for your answer. I was thinking to check the activity with crude cell lysate, that's a good idea.
I followed the exact protocol from the original literature and it did not produce much active protein. though the SDS Page band is very good but it shows 30% assay activity.. That's why I am worried about .. and that's why I am worried whether the 230 nm peak indicating my purified protein is not folded
Interestingly, BLAST shows that the amino acid sequence is for Omega-theraphotoxin-Hg1a (UniProtKB - P56854 (TX482_HYSGI)), not PmST1...
Additionally, you mentioned that you use Ni NTA for protein purification. Do you have HisTag in your sequence (it is not in the sequence provided above in your question)?
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