Been there, done that, got the T-shirt.
I do not believe there is a protease like 3C in E. coli.
That said - do you have any rare codons in your protein - that might
cause the stall/termination in expression - leaving you a large amount
of tag. Have you followed through with purifictaion to see if you have
low levels of full length expression? If rare codons are a problem -
multiple vendors have cell lines [such as BL21(DE3) RILP, codonplus, etc.)
With regards to His tag causing inclusion bodies - there have been
publications regarding solubility issues w/ His tags (I think Helena
Berglund has a paper on this).
Have you considered (after taking rare codons into account), expression
with no tag at all? pET3, pET11, etc.
Ezra
Israel Sanchez wrote:
Hello crystallographers in general and E.coli-protein producers in
particular,
I would like to share with all of you a strange behavior of two of my
expression constructs, looking for some advice or just know if anybody
has experienced something similar:
The scenario is the following one, I am trying to produce a NTPase
domain of around 20KDa of a human protein in E.coli. Initial cloning
in a T7-based vector with a N-terminal-hexa histidine tags produced
big quantities of an unfolded protein, in inclusion bodies. I tried
all normal approaches to try to make the construct soluble: lowing
expression temperature, lowing the concentration of IPTG, different
growing mediums, different E.coli strains, ... no success.
Then I decided to try some fusion-protein strategies, I cloned the
same construct as a fusion protein with GST and MBP. Then, I could see
a good soluble expression BUT only of the carrier protein (GST or
MBP). Lowing expression temperature or lowering IPTG concentration
does not produce any improvement. Both fusion-protein construct
contain between the fusion partner and my protein a 3C site for
cleavage with this protease.
So, the question is, does E.coli may posses protease similar to 3C
that may explain the self-cleavage? why my ribosomes are not reaching
until the end of the construct?
Thank you so much for your attention, any comment and/or suggestion
would be highly apreciated¡¡¡¡
--
PhD. Israel Sanchez Fernandez
EM-lab
Departamento de Ciencia de Proteinas
CIB-CSIC Madrid España
