The cDNA encoding human TRAIL extracellular region(amino acids 41—281) was amplified by reverse transcription(RT) PCR from total RNA derived from human acute promyelocytic leukemia cell line HL 60. After sequencing, ...The cDNA encoding human TRAIL extracellular region(amino acids 41—281) was amplified by reverse transcription(RT) PCR from total RNA derived from human acute promyelocytic leukemia cell line HL 60. After sequencing, the cDNA was cloned into the vector pQE 80L and transformed into E.coli DH5 α . By IPTG induction, the soluble TRAIL 41—281 (sTRAIL 41—281 ) protein was expressed with the 40% of total bacteria protein. Inclusion bodies were dissolved into 8 mol/L urea, purified by Ni NTA chromatography column, the product with over 90% purity was obtained. After refolding by dialysis, the active trimer form of sTRAIL 41～281 was derived from the renatured proteins by gel filtration chromatography. The MTT assay, flow cytometry and DNA fragmentation assay showed that the refolded sTRAIL 41～281 could potently inhibit the growth of Jurkat cells and induce apoptosis, confirmed the apoptosis inducing activity of sTRAIL 41～281 on tumor cells, it will benefit the further research of TRAIL.展开更多
文摘The cDNA encoding human TRAIL extracellular region(amino acids 41—281) was amplified by reverse transcription(RT) PCR from total RNA derived from human acute promyelocytic leukemia cell line HL 60. After sequencing, the cDNA was cloned into the vector pQE 80L and transformed into E.coli DH5 α . By IPTG induction, the soluble TRAIL 41—281 (sTRAIL 41—281 ) protein was expressed with the 40% of total bacteria protein. Inclusion bodies were dissolved into 8 mol/L urea, purified by Ni NTA chromatography column, the product with over 90% purity was obtained. After refolding by dialysis, the active trimer form of sTRAIL 41～281 was derived from the renatured proteins by gel filtration chromatography. The MTT assay, flow cytometry and DNA fragmentation assay showed that the refolded sTRAIL 41～281 could potently inhibit the growth of Jurkat cells and induce apoptosis, confirmed the apoptosis inducing activity of sTRAIL 41～281 on tumor cells, it will benefit the further research of TRAIL.
