How can I get protein in soluble fration - (Sep/11/2010 )
I have cloned tow fragments ( 30 and 24 KDa)from the same protein (human)into 3 different vetors ( pGEX, pMAL and pHUE) with 3 different tags ( GST, MBP and HIS6-Ubiquitin tags). I need to purify them in native condition. I did a trail expression in E. coli ( BL21 and Rosseta) at 37C and 20 degree with 0.4 mM IPTG. In all cases, all the six construts were in an insoluble fraction and almost nothing is in the supernatant.
Could you please help me to make these proteins soluble .
I am not sure if this would help but I know that some proteins, in particular certain enzymes, can be solubilised using high concentrations of Urea (i.e. 4-6 M Urea). In this way you can get them out of tissues in their native form. Still, you will need to purify your protein afterwards using dialysis.
Hola, I have answered before a similar question. The fact is that if any of both strains isn´t the subtype lacIq probably you have a quasi constitutive expresion in LB or other mediums without glucose.Have you analyze t=0 of induction ?. In order to facilitate a light expression in my lab grow the cultures at 25 ēC. Good luck