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
