[ccp4bb] Weird expression behavior

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: Weird expression behavior
From: Israel Sanchez israelsanfer {- at -} GMAIL {- dot -} COM
Date: 2009-09-02

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

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Off Topic: Stereo Microscope Recommendations
From: kaiser {- at -} CALTECH {- dot -} EDU kaiser {- at -} CALTECH {- dot -} EDU
Date: 2009-09-02