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Dear Elias do you see degradation of your fused protein? My simple mind would think of two reasons for your seeing MBP-alone. 1- multiple vectors in your cells, some with and others without the fused construct. This might always happen, to some level though. One way to fix it would be to transform your cells again and get a better yield of MBP-fused vectors. Some other possibility here could be that the problem comes from the vector itself, which is not pure when you attempted the initial transformation. 2- unstable fused protein. This could cause cleavage of the fused protein and further degradation. You could here either play with stronger protease inhibitor cocktails, or work out your construct and linker size. Either way: cannot you separate cleaved from fused samples afterwards? Cheers, leo > On 15 Feb 2016, at 00:17, Fernandez, Elias J <[log in to unmask]> wrote: > > > Dear Colleagues, > We are attempting to express our target protein fused to MBP in BL21(DE3) and BL21(RIPL). Unfortunately, we get a mixture of MBP-target protein and MBP only, which severely depletes our overall yield of MBP-target protein. We've tried enriched media, such as 2xYT & TB and also optimal high-density induction in minimal media at temperatures ranging from 20-37deg and IPTG from 0.05-0.5microM. > Any suggestions to address this competitively high yield of MBP-only protein? > Cheers, > Elias - Leonard Chavas - Synchrotron SOLEIL Proxima-I L'Orme des Merisiers Saint-Aubin - BP 48 91192 Gif-sur-Yvette Cedex France - Phone: +33 169 359 746 Mobile: +33 644 321 614 E-mail: [log in to unmask] -