Expression Of Tumor Necrosis Factor (tnf-alpha) And Mutants

1, An expression plasmid of TNF cDNA designeted pHT-1 was constructed by Xiao in this lab. and was expressed in E.coli JM105 with activity of TNF of 8×10~7u/L culture medium. Structural modifieaion of pHT-1 increaced the efficacy of TNF production to 5-8×10~8u/L culture medium. This modified expression plasmid, pHT-2 was also expressed in JM105. The rTNF was purified by FPLC on columns MonoQ and MonoP. 1×10~8 u of rTNF was isolated from one liter culture medium. The rTNF purified by FPLC showed a single band on SDS-PAGE. Endotoxin in 80μg of the rTNF was assayed by agglutination test using King Crab agent and the result is negative. The reaction of pyrogens of 50ug rTNF on rabbit is also fell normal range. The rTNF as an anti-tumor agent is therefor ready for used in clinical trials.2, A gene fusion plasmid, pAFT-1 was constructed by inserting the TNF cDNA into gene fusion vector pRIT5. The pAFT-1 was expressed in E.coli HB101. The expression product is a fusion protein. It contains protein A' (IgG binding domains of protein A) and TNF protein. Its molecular weight is about 50kD. About 30% of the fusion product was detected in the crude extract of pAFT-l/HB101. The protein was purified by affinity chromoto-graphy throgh IgG sepharose 6FF. The activity of the protein was 4×10~7 u/mg of protein. Mediated by IgG, the protein could bind to the lymphocytes with Fc receptors, so it may be a potential agent in treatment of chronic lymphocytic leukemia.3, In order to produce interested characteristics, TNF cDNA was amplified by PCR in the imbalanced ratio of dNTPs. The PCR products...