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Re: [ccp4bb] Weird expression behavior |
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CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999Subject: Re: Weird expression behavior From: Israel Sanchez israelsanfer {- at -} GMAIL {- dot -} COM Date: 2009-09-02 I do have rare codons for E.coli in my construct, indeed I have two stretches of 3 and 4 rare codons (arginines mainly), thus, I tried the expression in BL21(DE3)-codon plus and Rosseta. But the result was the same (GST or MBP alone). Thanks again 2009/9/2 Ezra Peisach > 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 >> >> > -- PhD. Israel Sanchez Fernandez EM-lab Departamento de Ciencia de Proteinas CIB-CSIC Madrid España CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999 |
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