Hi，everyone，
I am purifying a protein containing the contrust: N terminal-10×His+MBP+Tev site+protein-C terminal. The binding buffer is 50mM Tris-HCl, pH7.5, 500mM NaCl,10mM imidazole, 5%glycerin, 0.5mM TCEP, while Elution Buffer is 50mM Tris-HCl, pH7.5, 500mM NaCl,300mM imidazole, 5%glycerin, 0.5mM TCEP. After Ni column elution and dilution 3 times using binding buffer, the full-length protein were cleaved by the Tev protease at 4 ℃, and then Instantly (about 15 min) plenty of white precipitaions occured. Moreover, I further diluted the cleavage products using a buffer(20mM Tris-HCl, pH7.5, 100mM NaCl, 2% glycerin, 0.5mM TCEP) in order to reduce NaCl to 200mM so that the His-MBP tag would be removed subsequently via MBP Trap column.During the processes of dilution I found a lot of precipitaion displayed again. How can I reduce the precipitaion ? I need your help, thanks very much.
A typical reason for making an MBP fusion protein is to help an insoluble protein to remain soluble. This may be the case with your construct. BY removing the MBOP by TEV protease cleavage, you removed the means to keep the protein in solution, at least under those particular conditions. It might be worth trying to include a nonionic detergent with the protein to keep it in solution when the MBP is cleaved.
Adam B Shapiro Thank you very much. When I add final concentration 0.1% TritonX-100 followed by Tev protease, the full length protein did not aggregated and precipitated throughout cleavage and the nonionic detergent did not affact the Tev cleavage activity. Now, 1) I wonder if the addition of the nonionic detergent would influence on the activity of cleaved mature protein (a kind of CRISPR/Cas proteins); 2）how can I improve the binding efficiency of heparin column, by pH change, NaCl concentration or other methods ？ Thanks
1. 0.1% Triton X-100 is less than 10-fold above the critical micellar concentration (CMC). If you will be diluting the protein at least 10-fold into whatever assay you use, the detergent will be below its CMC. It is unlikely to interfere with the activity of the protein unless that protein has a particular affinity for the detergent. A bonus of having 0.01% Triton X-100 is that it helps prevent the protein from sticking to plastic surfaces. However, you should be sure to use only the highest quality Triton X-100, since it can accumulate reactive oxygen species over time if it is not stored under inert gas.
2. The interaction with heparin is largely electrostatic (it is highly sulfated), so lowering the salt concentration or lowering the pH may be effective to increase binding affinity.
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