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refolding problem

refolding problem - Protein Forum

refolding problem - Protein Forum

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Old 03-21-2008, 04:51 AM
S. D. Yogesha
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Default refolding problem

Hi everyone,
My protein has c terminal His tag. When I express it more than half remains in IBs. Even after using more culture volume is not helping to get enough protein for work. [What ever little I get is aggregating almost near to 100%. I want to address both problem but one after the other. I know this aggregation is because I mutated 5 glutamine and 1 lysine residue as aa patch (EEE and EKE).} Trying different temperature, strains and induction method did not help me increasing solubilization. I have not tried cheperons. I tried to solubilize the IBs with 8M urea as in standard protocol, was surprised to find that protein is not binding to both Nickle and Talon column. I asked a colleague and he said His6 and His7 had never given him problem. My tag is His8 and I did not get any reason why my protein is not binding in denatured condition when I can purify same in native condition. Can any one educate me if I am missing something in understanding this.
I followed J Struct Funct Genomics. 2005;6(2-3):177-82, and instead of beta cyclodextrin I used 2 hydroxyl beta cyclodextrin. Entire protein was in flowthrough and first wash even after overnight binding.


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