Here is a weird tale about 6xHis tag. Probable explanations?

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Sun Apr 1 22:27:50 EST 2001

I had a protein tagged with [long linker]+[myc]+[6his]
at C terminus. I needed an ability to remove 6his,
so I modified vector so that linker+myc is replaced
by TEV protease cleavage site. All worked fine, I 
can see the protein expressed and active, his tag
is detectable by anti-6his on western, and the 
protein purifyable on Ni-NTA. The weird thing 
starts now:

I wanted to find and express "minimal" protein, so 
I made 5 truncation mutants using wild type TEV-his 
construct above: -95 and - 108 aa from N terminus, 
and -54, -102 and -130 from C-terminus. All was 
easy and went well and sequencing showed correct 
constructs made in all cases. I then expressed all 
five in Cos-1 cells to test the mutants for activity.
Before bothering with purification, I ran western:
- all mutants run at predicted MW, 
- all are recongized by anti wild type polyclonals,
- none of C-terminal mutants is recognized by
anti-6his Ab! (N-terminal mutants are). 

Since I know the constructs are OK (sequences of
all three + correct sizes on the SDS gel), I have
only two, both extremely unlikely explanations: 
1. limited C-terminal proteolysis of any C-terminal
truncation mutant, but not WT, or
2. 6his is not accesible for anti 6His Ab EVEN after
denaturing gel and random membrane binding (again,
in any C-terminal truncation mutant, but not WT). 

Because these are three pretty different mutants,
made with the idea in mind that "at least one will
work", and because I've never heard about anything
like this, I am having hard time with such 
explanations. OTOH, it's also hard to believe that
all three magically have acquired frame shift 
near C-terminus that magically was not detected
in 6 sequencing reactions (two for each). 

Any other ideas? 

Do antibodies recognizing TEV cleavage site exist? 

    - Dima

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