Hello all, I want to do domain mapping of my protein of intrest for testing its interaction with a few predicted interactors. I have generated N-termius end and C-terminus end of my protein. I have cloned approx150 bp both the terminii in pET28a vector between Nde1 and EcoRI sites(both the constructs). I tried inducing the protein in BL-21DE3 cells using but no success. The full length protein expresses normally without any problem in a different vector backbone(infact gives a big blob of pure protein in induced cells) . I am wondering what went wrong. I have sent my plasmid samples for sequencing and awaiting for results. The constructs were PCR amplified using Pfu polymerase(very less chances of errors in PCR product). Since I have done directional cloning chances of frameshift is also less. I tried for presence of inserts using both colony PCR and plasmid as template both methods show presence of template of correct size. I was wondering if anybody has expressed protein of similar size. Is it getting degraded. Please help me in this regard.
-- Rao Venkatramanan G. Research Scholar UM-DAE-CBS Mumbai University campus, Kalina, Santacruz Mumbai-400098 _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
