The Fusion Expression Of Lymphotoxin Deletion In E.coli And Its Biological Activity

Lymphotoxin(LT) is a kind of pleiotropic lymphocyte-secreted cytokine which mediates a large variety of inflammatory, immunostimulatory, and antiviral responses. In order to increase the antitumor activity of lymphotoxin and reduce its side effects, the recombinant plasmid pET36b-LT 27 was constructed to express soluble fusion protein CBD-LT 27. The active form of LT 27 could be collected directly with several simple steps by three kind of components on the expressed fusion protein.A human lymphotoxin deletion gene fragment which lacking N-terminal 27 amino acid residues of the protein was PCR amplified using a pair of designed primers and cloned into expression vector pET36b(+) to construct a fusion protein with CBD tag, resulting a recombinant plasmid pET36b-LT 27. The recombinant plasmid was transformed into host E.coli BL21 (DE3) pLysS. According to the results of SDS-PAGE analysis and Western blot analysis, the fusion protein CBD-LT 27 with a molecular weight 37kDa was expressed after induced by 1PTG. The expressed fusion protein occupied more than 20% of total bacterial protein. The fusion protein induced mainly existed in the insoluble inclusion body of the cell, only little parts of fusion protein expressed in the cytoplasm, excreted into periplasm and secreted into the cultural medium as soluble protein.Through ultrasonic treatment at low temperature, soluble expressed fusion protein could be obtained from the supernatant of lysed cell. The soluble fusion proteins CBD-LT 27 in cytoplasm and periplasm of E.coli were purified with CBIND 100 Resin, the purity of fusion protein was more than 80% in eluted solution. The CBD tag of the fusion protein was cut by site-specific protease enterokinase at starting Met of the target protein LT27. it was released from the CBD fusion tag efficiently. With Ni2+-lDA-Sepharose, the CBD tag was removed. According to the results of SDS-PAGE and Western blot analysis, the product of outflow was the target protein LT A 27. The purity of LT A 27 was about 95%.The assay system of the biological activity of lymphotoxin was established using L929 cell as the sensitive target, LT international standard as the positive control and crystal violet staining method to detect viable cell after treated with LT. The best relationship between dosage and effect could be got if the cell seeding density in cell plate was 1.6 0.1 104 the dosage of AMD was lug/ml, and the starting concentration of dilution in the plate of LT standard was 10 IU/ml with two fold dilution. The credibility of the established system was detected with rhTNFp developed by R&D. The results were consistent with its label. The biological activity of purified LT 27 was tested with the assay system, and its biological activity was 2-3 107 Iu/mg.pro.The cytotoxicity of purified LT 27 was in the same level with rhTNFp and LT international standard. It shows that LT deletion could keep its high cytotoxicity towards tumour cell L929 in vitro after 27 amino acids deleted from its N-terminal. Researches showed that purified LTA27 had the same antitumor activity with LT but far more low toxicity in vivo. Since the production and purification of LT A 27 are simple, LT A 27 is prospected to be developed to a kind of high efficiency, low toxicity antitumor drug.