Construction, Expression And Bioactivity Assay Of Self-splicing Prokaryotic Expression Vector PTWIN1/TRAIL_111-281

【Background and object】TRAIL(TNF-related apoptosis-inducing Ligand) was an new members of TNF family. It was an apoptosis molecular and was named Apo2L (Apo2 Ligand). Its gene was located on chromosome 3q26, and The full length of TRAIL cDNA is 1042 base pairs. Its molecular weight is 32. 5kD, PI is 7.63. TRAIL is a member of typeâ…¡transmembrane protein family. Human TRAIL contains 281 aa, of which 14 aa in N-termini consists of its cytoplasm region, following 26 aa makes its transmembrane region and 241 aa as extracellular region. The region from 114 to 281 aa contributes its function.TRAIL induces apoptosis through the interaction of TRAIL and its receptors. So far, five receptors of TRAIL have been found: DR4(TRAILR1), DR5(TRAILR2), DcR1(TRAILR3), DcR2(TRAILR4) and OPG. DR4 and DR5 are death receptor having completely cytoplasm death domain and can induce apoptosis when TRAIL binds to them. But DcR1 and DcR2 are decay receptor which can not induce apoptosis because they have not death domain in cytoplasm and can not transform the signal of apoptosis. DR4 and DR5 are expressed broadly in many tissues, such as spleen, liver, lung, thymus,PBC,activated T cells and some tumor cells. DcR1 is expressed only in normal tissues, not in tumor tissues, while DcR2 is expressed only in embryonic liver tissue and adult testis tissue. The binding of TRAIL to DR4 and DR5 can induce apoptosis while to DcR1 and DcR2 can protect the cells from apoptosis. Because many tumor cells only express DR4 and DR5 and don't express DcRl and DcR2, TRAIL can specifically kill tumor cells while have no toxicity to normal tissues. An another receptor is OPG(osteoprotegerin), which can not induce apoptosis because they have not death domain in cytoplasm and can not transform the signal of apoptosis. It was confirmed that TRAIL can kill selectively various tumor cells and transformed cells while have no toxicity to normal tissues. So TRAIL and its receptor are considered as a potential drug in tumor therapy.To further investigate the mechanism of how TRAIL inducing apoptosis and the prospect of TRAIL as a clinic drug in cancer therapy, we made 513bp. cDNA fragment of the soluble region of TRAIL by gene chemical semi-synthesized and PCR. After DNA sequencing in pMD-18T/TRA1L_111-281, FRAIL_111-281 cDNA was cloned into self-splicing expression vector pTWIN1, then transformed into E. coli ER2566. Soluble TRAIL_111-281 fusion protein was expressed after 14-16 hours induction at 15℃. TRAIL_111-281 fusion protein was purified by self-splicing on chitin column at 4℃, pH7.0, after 48-72 hours, high purity TRAIL_111-281 protein was gained, its bioactivity was analysised with human breast tumor MDA-MB-435 cell line in vitro. 【methods】According to the high-usage codons in Escherichia coli and the multiple cloning site of expression vector pTWINI of a self-splicing prokaryotic expression system, the extracellular region of TRIL_111-281 gene was designed and synthesized, which was cloned into pMD18-T vector. After pMD-18T/TRAIL_111-281 and pTWIN1 were digested by Nruâ… and EcoRâ… , the target fragment purified was linked to the self-splicing expression vector pTWIN1, which was transferred into the competent cell ER2566, and positive recombination was screened and identified with restrictive endonuclease digesting and sequencing. After positive recombination vector pTWIN1/TRAIL_111-281 identified, the expression was induced by different concentration of IPTG at different temperature and culture time. The expression products were analyzed by 15%SDS-PAGE. The fusion protein was identified by the special anti-body of CBD. The TRAIL_111-281 protein in supernatant of bacterial lysate was purified with chitin column. With SRB assay, its bioactivity was analysised with human breast tumor MDA-MB-435 cell line in vitro.【Results】The extracellular region of TRAIL_111-281 gene was obtained successfully by gene chemical synthesis and PCR, which was 550bp in lenghth. After TRAIL_111-281 gene fragment was identifiedwith restrictive endonuclease digesting and sequencing, it was confirmirmed to be constructed successfully into self-splicing prokaryotic expression vector prWIN1/TRAIL_111-281. With 0.3mmol/L IPTG at 15℃for 14 to 16 hours, the CBD-Ssp-Intein / TRAIL_111-281 fusion protein was expressed efficiently and it come to the half of total bacterial proteins. The molecular weight of the soluble fusion protein was estimated as approximately 4.5×10⁴Da by western-blot. With SRB assay the TRAIL_111-281 was showed initially inhibit the growthof human breast tumor MDA-MB-435 cell line in vitro with a dose-dependent mode, and its IC₅₀ is 0.005μg/ml.【Conclusions】TRAIL_111-281 cDNA wasmade successfully and selfsplicing pTWIN1/TRAIL_111-281 was constructed. High-expression level of the extracellular region of TRAIL_111-281 fusion protein was made in E. coli ER2566, The fusion protein was probed with the special anti-body of CBD. and the soluble TRAIL_111-281 protein without any additional amino acid was successfully purified by simple treatment. With SRB assay TRAIL_111-281 was showed potently inhibiting the growth of human breast tumor MDA-MB-435 cell line in vitro.