Dear researchers,
Recently, I have been facing an issue in purifying my recombinant protein because my protein is not binding to the Ni-NTA resin. His-tagged CYP121 protein was expressed in E. coli K12 BL21 and purified using a single affinity chromatography step. The protein cell pellet was resuspended in 100 mL of binding buffer containing (50 mM Tris-HCl, 300 mM NaCl, 10% glycerol, 1% triton-X-100, pH=7.4). Then, the cell lysate was applied to the Ni-NTA affinity column, washed with binding buffer (50 mM sodium phosphate, 300 mM NaCl, (0-20 mM imidazole), 10% glycerol, 1% triton-X-100), and eluted with a one-step gradient in (200-500 mM imidazole) range. Initially, my protein was successfully purified using 0 mM of imidazole (binding buffer) for washing and 200 mM imidazole (elution buffer) for elution. However, recently, I was unable to purify my protein anymore. There is no protein binding to the Ni-NTA resin. When the plasmid was sent for gene sequencing, the sequence was matched, and the His-tags were also available in the gene sequence. However, there is no protein binding. And I need suggestions to fix this issue. thanks.
#protein expression#protein purification#enzyme#recombinant protein#CYP121
The tag could be hidden. If the adsorption is weak, 20 mM could be enough to desorb it. You could corroborate weak or no binding if you completely denature your protein with urea. Are impurities being adsorbed?
Omar Gonzalez-Ortega yes, at the elution fractions there are some impurity bands. Impurities bind to the resin and elute in the elution fraction.
Hi Priya!
I cant really imagine that your Ni-NTA column is the problem. Neither your plasmid, since you checked it. I would suspect the bacteria.
Have you checked that your target protein is expressed by the bacteria? For example via western blot of the crude cell lysate?
How do you prepare the bacteria? Are you doing fresh transformation + selection for each culture?
I previously have run into problems when my glycerol stocks were contaminated and my cultures were overgrown by bacteria that did not express my target protein. Sometimes, just doing everything fresh is really the best way to fix problems.
Hope this helps. Good luck!
Cheers,
Jan
Hi Jan Philipp Dobert
Thanks for your suggestion. I performed a Western blot to check whether my His-tags were still present in the protein. But I see no signal in Western blot, meaning there are no His-tags in my protein and that could be the reason for the purification difficulty. So, currently, I'm cloning my target gene with a new plasmid and transforming it into a new strain (DH5a). Hope it works this time!!
Which strain did you use previously for the expression?
Previously, you were able to express and purify your protein, right? Unless the bacteria mutated your plasmid, there must be some difference in the production of the protein (either expression or purification) yielding either no protein or digested protein without His-tag.
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