Cloning and Expression of a Fused Gene Encoding TNFRI Death Domain and Chloramphenicol Acetyltransferase.

By designing four primers for an overlapping PCR, we created a fusion gene Ddl cat encoding human TNF receptor I death domain and chloramphenicol acetyltransferase(CAT). By DNA sequencing, the whole sequence of the fusion gene is confirmed to be correct. Two hours after induction with IPTG, we could see an 39 kD extra protein band on SDS PAGE pattern. We proved that this 39 kD protein band is DdLcat protein by Western blotting. Then we purified this protein to the purity of 95% through Q Sepharose chromatography.