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Protein expression in E. coli mutant samples will not work in T7 LysS promoter i

protein expression Biochemistry E.coli

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#1 Doc

Doc

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Posted 22 October 2014 - 04:44 PM

I have made 4 mutants of an enzyme through PCR and expressed cells in NEB5Alpha cells. Sequencing was confirmed. I have conducted protein expression and purification on 4 mutants. Only 2 were successfully expressed, as their OD measurement was in 0.06 to 0.08 range. The other two were a little low on the OD scale but sufficient to move on in expression. These mutants were then induced with IPTG and purified. My point is 2 of these mutant's proteins were successfully expressed and confirmed through SDS-Page electrophoresis and through an enzyme activity assay ( as they showed up pink ). I can't figure out why, since they went through all the same processes as the other two. These were expressed under a T7LysS promoter in BL21(DE3) cells. E.coli mutant stand was used. Please Help! 







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