Strategy 1)
I have prepared N- Nus and C- His(6-His) tagged fusion construct of my protein of interest in pET43.1a by using SpeI and XhoI restriction enzyme and Prescission protease recognition sequence in between of Nus and protein of interest.
Actual construct in pET43.1a is= N terminal-Nus-SpeI-Prescission protease recognition sequence-Protein of interest-XhoI-6His tag- C terminal.
Protein of interest is cytosolic protein.
I am having following difficulty to purify this protein.
1) Binding issue with Ni -NTA column as >50% protein is going in flow through as C- His tag is not properly available to bind with column.
2) After gradient as well as step Ni NTA purification of Interest of protein, nonspecific band are also coming during elution.
3) Ni NTA purified fusion protein was digested with prescission protease and after cleavage again purified by Ni NTA column, In this Nus tag is also coming during elution along with interest of protein and with non specific bands.
How to troubleshoot these kindly suggest ?
Strategy 2)
Now I wish to re-clone my interest of protein in pET28a (Image attached) as this vector has N- His tag, cloning will be done by using Nde I and XhoI sites , but problem is that, there are some extra amino acid residues after His tag so fusion protein will be = N terminal -His tag -some extra amino acids of vector- Nde I -Nus tag-Prescission protease recognition sequence-Protein of interest-XhoI-6His tag- C terminal.
I wanna ask that, extra residues which will be in between of N- His and Nus tag can have any negative effect in protein expression and purification?.
This new construct will have His tag on both N & C terminal so is that ok to have His tag on both terminals ?
In last I wish to ask is prescission protease ok with this construct?
Image attached for pET 43 and 28 a.vector.
Kindly suggest.
1) Binding issue with Ni -NTA column as >50% protein is going in flow through as C- His tag is not properly available to bind with column.
Are you taking sufficient resin, open or prepacked column and allowing ample time for protein binding. Does your binding buffer contain histidine? that needs to be optimized to concentration which allows binding but removes contaminants. The C-terminal His6 tag in this construct is too small and may be buried, contrary to that in pET28a there is a linker present between the protein and the tag which is the later part of your question.
2) After gradient as well as step Ni NTA purification of Interest of protein, nonspecific band are also coming during elution.
That usually happens and can be minimized by adding imidazole in the binding buffer as recommended by the resin manufacturer and giving proper washing. contaminant will still be there depends the SDS PAGE. Tricine shows the purest of the Ni-NTA purified protein with contaminant. So IMAC is only a first step purification and if needed a second step like SEC is included.
3) Ni NTA purified fusion protein was digested with prescission protease and after cleavage again purified by Ni NTA column, In this Nus tag is also coming during elution along with interest of protein and with non specific bands.
Is the NusTag still attached (optimize protease to protein ratio or incubation time) or the tag is the contaminant (include a second purfication step like SEC or use centrifugal filter).
I wanna ask that, extra residues which will be in between of N- His and Nus tag can have any negative effect in protein expression and purification?.
Most probably no, these extra residues or longer his tag has not affected the proteins cloned in our lab, which are active and purified by IMAC.
This new construct will have His tag on both N & C terminal so is that ok to have His tag on both terminals ?
There will probably be two peaks (due N and C-terminal tag) in protein elution as both tags will be having different affinities to Ni.
In last I wish to ask is prescission protease ok with this construct?
You are only adding extra amino acids on N-terminus of the previous construct, so there will no problem, it will work here as well.
I agree with the comments and answers by Singh. You need to optimize the conditions of your binding buffer, elution buffer, and cleavage conditions. You can also try other proteases for specific cleavage. TEVp works good in this regard.
It is also good to try other tag proteins instead of Nus tag.
Hello mangal
Thanks for you reply.
I asked these questions after optimization. Before asking question, I have performed all experiments as you explained in point 1and 2 still did not get results as expected.
If there is any other possibilities to troubleshoot so please suggest.
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