It seems that your Histag is not accessible for affinity resin. Put it without MBP and/or put it on C termini. In our case the codon optimization helped for better expression and solubilisation of the protein.

Hi Ketul

The binding of protein with Ni-NTA beads is dependent on PI of your target protein.Ideally the pH of your binding buffer should be +-2 pH of your PI.Try to ionise the protein,it will greatly enhance the binding affinity with your protein.

hello Shashi,

pI of the protien is 6.70 and buffer i'm using is of pH 8.0 tris

even it didnot binds to MBP resin.

can codon optimization help me if i am looking for well stabilized protein?

can i get stable, soluble, active protein after codon optimization?

Probably, codon optimisation will not help for binding of your protein with NI-NTA. This will improve expression.

so may be you can try with changing the tag on C-terminal.

It might help to expose your tag to get bind with NI-NTA properly.

Good Luck !!

Hi Ketul

I think you just calculated the PI of your protein.Actually you have to calculate the entire sequence which is translating including tags.And then use appropriate buffer to bind with Ni-NTA beads.

Indeed the entire pI of protein is 6.70

With fusion protein (MBP) and his tag size of whole construct is 85 kda

In pellet and Sample flow I can clearly see a fatty band at 85Kda

But this band is missing in elution and in wash it is coming with impurities(70%impurity and 30%my protein of interest).

When I dissolved supernatant in 8M urea, thing were same.

It seems that your Histag is not accessible for affinity resin. Put it without MBP and/or put it on C termini. In our case the codon optimization helped for better expression and solubilisation of the protein.

Dear Ketul,

First of all, this is necessary to confirm the sequence of your construct, which should be completely intact, so there is no mutation or deletion that could affect His-tag. This probably you have already done. If the amino acid sequence is correct then, codon optimization would not help your purification problem, since by codon optimization you will get the same protein sequence via different mRNA, which helps the translation phase to become more efficient. So, it can increase your expression, and, in some cases, it can increase the efficiency of purification due to the higher binding affinity of your his-tagged protein compare to untargeted proteins. However, it would not solve your problem with binding.

Regarding the lack of binding to Ni-column, I would recommend you to check your buffers to be sure that your sample is not contaminated with high salt, which can reduce the binding affinity to the column. Also, you need to check if the column working well by easily asking other colleagues who worked with that column recently. If all technical aspects are fine, then as “Jana Schmitzova” mentioned, it could be a possibility that His-tag is buried in the structure in a way that it is not in an exposed position, which finally leads to binding deficiency. Then you need to design your construct again for example by adding a linker behind the His-tag, or using different method of purification.

Dear Amir,

Thank you very much for clearing my confusion about codon optimization.

Yes I got the sequencing result of plasmid and there is no mutation and no frameshift.

I agree with hisTAG masking.

Best

Ketul saharan