Re: [ccp4bb] His tag on membrane protein

[Anastassis Perrakis]

In one case, for example, cleaving the N-terminal His-tag improved  
the solubility of the protein dramatically. Interestingly,  
expression levels of the protein without the His-tag were much  
lower than with it!


That is also quite common ;-) Look at the second and third codons:  
optimizing these (as they are optimized in His-tag vectors) can  
dramatically enhance expression levels.


A.

Re: [ccp4bb] His tag on membrane protein

[Savvas Savvides]

Hi Deliang,

My own experience with two membrane proteins suggests that His-tag  
issues pertaining to soluble proteins also seem to apply to membrane  
proteins.


In one case, for example, cleaving the N-terminal His-tag improved the  
solubility of the protein dramatically. Interestingly, expression  
levels of the protein without the His-tag were much lower than with it!


In the other case, we have a construct with a His-tag at the  
N-terminus and another at the C-term. Both behave well when it comes  
to solubility and stability but we thus far only have diffracting  
crystals from the construct with the C-term His-tag. However, we have  
not yet tried to remove the tags from those two constructs to see  
whether that improves crystallizability.


My feeling is that if you do not have solubility/stability/aggregation  
problems with your His-tagged membrane protein, then there is no  
reason to expect lower success rates in crystallization trials. Screen  
with it and hope for the best!


By the way, there is a recent paper on the effects of His-tags on  
crystal structures of soluble proteins. It's worth a look.


Carson M et al. His-tag impact on structure.
Acta Crystallogr D Biol Crystallogr. 2007 Mar;63(Pt 3):295-301.


Best wishes
Savvas


Savvas N. Savvides
L-ProBE, Unit for Structural Biology
Ghent University
K.L. Ledeganckstraat 35
9000 Ghent, BELGIUM
Phone: +32-(0)9-264.51.24 ; +32-(0)472-92.85.19
http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html






Quoting deliang [EMAIL PROTECTED]:


Hi there,

I purified a membrane protein with traditional His-tag on the C   
terminal. Before crystallization, I wonder how this tag may affect   
the result. Does anyone have  experience that the removel of this   
tag may improve the result or not? or can provide some references   
which may give some statistic, like how many membrane proteins have   
been crystallized with or without His-tag?


Thanks so much.

Deliang

[ccp4bb] His tag on membrane protein

[deliang]

Hi there,

I purified a membrane protein with traditional His-tag on the C terminal. 
Before crystallization, I wonder how this tag may affect the result. Does 
anyone have  experience that the removel of this tag may improve the result or 
not? or can provide some references which may give some statistic, like how 
many membrane proteins have been crystallized with or without His-tag?

Thanks so much.

Deliang

Re: [ccp4bb] His tag on membrane protein

[Daniel Picot]

Hi Deliang,
We have crystallized the complex cytochrome b6f with an extension of 
His6 (no linker) at the c-terminal of cytochrome f (Stroebel, 2003). The 
His extension takes part to the crystal contacts. The electron density 
is visible but not very well defined. We have not tried to remove the 
His6. Many other structure of membrane protein have used His-tag, but 
still not enough to  make sound statistics.

Daniel

deliang a écrit :

Hi there,
 
I purified a membrane protein with traditional His-tag on the C 
terminal. Before crystallization, I wonder how this tag may affect the 
result. Does anyone have  experience that the removel of this tag may 
improve the result or not? or can provide some references which may give 
some statistic, like how many membrane proteins have been crystallized 
with or without His-tag?
 
Thanks so much.
 
Deliang