TRAIL的基因克隆及其表达条件优化研究
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摘要
肿瘤坏死因子相关凋亡诱导配体(TRAIL)能够选择性地诱导多种肿瘤细胞凋亡,而对正常组织细胞基本没有毒性,因此成为肿瘤治疗中的研究热点。TRAIL一旦正式投入使用后,必然需要进行大规模生产。目前已报道的TRAIL主要以包涵体形式表达。然而包涵体表达的蛋白需复性后才能得到功能蛋白,且包涵体的复性通常效率低、过程复杂,这在一定程度上影响了TRAIL的大规模生产。因此,减少包涵体的形成和提高重组蛋白的可溶性表达,将会推动TRAIL的产业化发展。
本实验通过PCR技术扩增出人可溶性TRAIL114-281氨基酸残基的cDNA序列,扩增产物克隆到大肠杆菌表达载体pET-28a,并在大肠杆菌BL21(DE3)中实现高表达。将表达的目的蛋白经Western Blot实验显示具有明显抗原性。
然后采用响应面法对TRAIL的可溶性表达条件进行优化,优化后,TRAIL的可溶性表达量从146.84 mg/L提高到454.85 mg/L,提高了3.1倍。在该基础上进行了高密度发酵实验,进一步提高TRAIL的可溶性表达量至3.66 g/L。
将发酵得到的菌液直接取破壁上清进行纯化,通过亲和层析和离子交换层析两步纯化得到目的蛋白。目的蛋白纯化后最终产量为1.04 g/L。最后对纯化后的可溶性TRAIL蛋白进行了性质鉴定,结果表明纯化后的目的蛋白纯度高,分子量与理论值相符,活性高。
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in numerous tumor cell lines while sparing normal cells. Once it officially put into use, large quantities of biologically active TRAIL are needed. As has been reported, TRAIL mainly expressed in the form of inclusion body. Whereas, the low efficiency and complicated process of renaturation, which is inevitable for the inclusion body to get functional, obstructs its mass production. Therefore, reducing the inclusion body and increacing the soluble protein will promote the industrial development of TRAIL.
In this study, the cDNA sequence of TRAIL (encoding residues 114-281) was amplified by PCR. Then, the genes were cloned into plasmid pET-28a and expressed in E.coli BL21(DE3). Western blot analysis showed specific reaction of the expressed recombinant protein.
The response surface methodology (RSM) was adopted to optimize the culture conditions of TRAIL. The production of soluble TRAIL was improved from 146.84 mg/L to 454.85mg/L after optimization. Furthermore, high-density fermentation was performed. Production of soluble TRAIL concentration reached to 3.66 g/L under the fermentation conditions.
By sonication and centrifugation, the supernatant was collected and loaded onto chromatography for purification. TRAIL were purified by Ni-affinity chromatography and ion-exchange chromatography combinational. 1.04g/L of purified TRAIL was obtained after purification. Finally, the purified soluble TRAIL was identified, and the results showed that the recombinant protein was in the condition of high purity, high activity and the molecular weight matched well with the theoretical value.
本实验通过PCR技术扩增出人可溶性TRAIL114-281氨基酸残基的cDNA序列,扩增产物克隆到大肠杆菌表达载体pET-28a,并在大肠杆菌BL21(DE3)中实现高表达。将表达的目的蛋白经Western Blot实验显示具有明显抗原性。
然后采用响应面法对TRAIL的可溶性表达条件进行优化,优化后,TRAIL的可溶性表达量从146.84 mg/L提高到454.85 mg/L,提高了3.1倍。在该基础上进行了高密度发酵实验,进一步提高TRAIL的可溶性表达量至3.66 g/L。
将发酵得到的菌液直接取破壁上清进行纯化,通过亲和层析和离子交换层析两步纯化得到目的蛋白。目的蛋白纯化后最终产量为1.04 g/L。最后对纯化后的可溶性TRAIL蛋白进行了性质鉴定,结果表明纯化后的目的蛋白纯度高,分子量与理论值相符,活性高。
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in numerous tumor cell lines while sparing normal cells. Once it officially put into use, large quantities of biologically active TRAIL are needed. As has been reported, TRAIL mainly expressed in the form of inclusion body. Whereas, the low efficiency and complicated process of renaturation, which is inevitable for the inclusion body to get functional, obstructs its mass production. Therefore, reducing the inclusion body and increacing the soluble protein will promote the industrial development of TRAIL.
In this study, the cDNA sequence of TRAIL (encoding residues 114-281) was amplified by PCR. Then, the genes were cloned into plasmid pET-28a and expressed in E.coli BL21(DE3). Western blot analysis showed specific reaction of the expressed recombinant protein.
The response surface methodology (RSM) was adopted to optimize the culture conditions of TRAIL. The production of soluble TRAIL was improved from 146.84 mg/L to 454.85mg/L after optimization. Furthermore, high-density fermentation was performed. Production of soluble TRAIL concentration reached to 3.66 g/L under the fermentation conditions.
By sonication and centrifugation, the supernatant was collected and loaded onto chromatography for purification. TRAIL were purified by Ni-affinity chromatography and ion-exchange chromatography combinational. 1.04g/L of purified TRAIL was obtained after purification. Finally, the purified soluble TRAIL was identified, and the results showed that the recombinant protein was in the condition of high purity, high activity and the molecular weight matched well with the theoretical value.
引文
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[2]Daniel PT,Wieder T,Sturm I,et al.The kiss of death:promises and failures of death receptors and ligands in cancer therapy[J].Leukemia,2001,15(7):1022-32.
[3]Nagata S.Apoptosis by death factor[J].Cell,1997,88(3):355-365.
[4]Kruyt FA.TRAIL and cancer therapy[J].Cancer Lett,2008,263(1):14-25.
[5]Wang YH,Feng T,Zhang Q,et al.Optimization of fermentation condition for antibiotic production by Xenorhabdus nematophila with response surface methodology[J].J Appl Microbiol,2008,104(3):735-744.
[6]Xiong ZQ,Tu XR,Tu GQ.Optimization of medium composition for actinomycin X2production by Streptomyces spp JAU4234 using response surface methodology[J].J Ind Microbiol Biotechnol,2008,[Epub ahead of print].
[7]Anisha GS,Sukumaran RK,Prema P.Evaluation of alpha-galactosidase biosynthesis by Streptomyces griseoloalbus in solid-state fermentation using response surface methodology[J].Lett Appl Microbiol,2008,46(3):338-343.
[8]Wang Q,Hou Y,Xu Z,et al.Optimization of cold-active protease production by the psychrophilic bacterium Colwellia sp.NJ341 with response surface methodology[J].Bioresour Technol,2008,99(6):1926-31.
[9]Wang X,Niu D J,Yang XS,et al.Optimization of methane fermentation from effluent of bio-hydrogen fermentation process using response surface methodology.Bioresour Technol[J],2008,99(10):4292-99.
[10]李涛,岳田利,袁亚宏.Plackett-burman设计法筛选超声波提取苹果多酚上艺的主要影响因子[J].农产品加工,2007,3:18-21.
[11]张锋华.产PCA基因工程菌M18G发酵条件优化[D].上海:上海交通大学,2007年1月.
[12]Nam GH,Choi YK.Association of human tumor necrosis factor-related apoptosis inducing ligand with membrane upon acidification[J]. Eur J Biochem, 2002, 269(21): 5280-87.
[13] Ashkenazi A, Pai RC, Fong S, et al. Safety and antitumor activity of recombinant soluble Apo2 Hgand[J]. J Clin Invest, 1999, 104(2): 155-162.
[14] Cha SS, Shin HC, Choi KY, et al. Expression, purification and crystallization of recombinant human TRAIL[J]. Acta Crystallogr D Biol Crystallogr, 1999, 55(5): 1101-04.
[15] Kim SH, Kim K, Kwagh JG, et al. Death induction by recombinant native TRAIL and its prevention by a caspase 9 inhibitor in primary human esophageal epithelial cells[J]. J Biol Chem, 2004, 279(38): 40044-52.
[16] Walczak H, Miller RE, Ariail K,et al. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo[J]. Nat Med, 1999, 5(2): 157-163.
[17] Shen YL, Zhang Y, Sun Y, et al. High-level production of soluble tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) in high-density cultivation of recombinant Escherichia coli using a combined feeding strategy[J]. Biotechnol Lett, 2004, 26(12): 981-984.
[18] Lin Z, Lei H, Cao P. Expression, purification, and in vitro refolding of soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) [J]. Protein Expr Purif, 2007, 51(2): 276-282.
[19] Gasparian ME, Ostapchenko VG, Yagolovich AV, et al. Overexpression and refolding of thioredoxin/TRAIL fusion from inclusion bodies and further purification of TRAIL after cleavage by enteropeptidase[J]. Biotechnol Lett, 2007, 29(10): 1567-73.
[20] Luo Q, Shen YL, Wei DZ, et al. Optimization of culture on the overproduction of TRAIL in high-cell-density culture by recombinant Escherichia coli[J]. Appl Microbiol Biotechnol, 2006, 71(2): 184-191.
[21] Sun AY, Shen YL, Yin JC, et al. Improvement of expression level and bioactivity of soluble tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) by a novel zinc ion feeding strategy[J]. Biotechnol Lett, 2006, 28(15): 1215-19.
[22] Chekhonin VP, Pavlov KA, Shkoporov AN, et al. Cloning and expression of rat GFAP cDNA in Escherichia coli[J]. Bull Exp Biol Med, 2007, 143(1): 68-71.
[23] He HJ, Yuan QS, Yang GZ, et al. High-level expression of human extracellular superoxide dismutase in Escherichia coli and insect cells[J].Protein Expr Purif,2002,24(1):13-17.
[24]Shen YL,Xia XX,Zhang Y,et al.Refolding and purification of Apo21/TRAIL produced as inclusion bodies in high-cell-density cultures of recombinant Escherichia coli[J].Biotechnol Lett,2003,25(24):2097-101.
[25]Maia MM,Heasley A,Camargo MM,et al.Effect of culture conditions on lipase production by Fusarium solani in batch fermentation[J].Bioresour Technol,2001,76(1):23-27.
[26]Paul S,Chaudhuri TK.Chaperone mediated solubilization of 69-kDa recombinant maltodextrin glucosidase in Escherichia coli[J].J Appl Microbiol,2008,104(1):35-41.
[27]Johnston W,Cord-Ruwisch R,Cooney MJ.Industrial control of recombinant E.coli fed-batch culture:new perspectives on traditional control variables[J].Bioprocess Biosyst Eng,2002,25(2):111-120.
[28]Korz D L,Rinas U,et al.Simple fed-bbatch technique for high cell density cultivation of Escherichia coli[J].Journery Biotechnol,1995,39(1):59-65.
[29]Baetu TM,Kwon H,Sharma S,et al.Disruption of NF-kappaB signaling reveals a novel role for NF-kappaB in the regulation of TNF-related apoptosis-inducing ligand expression[J].J Immunol,2001,167(6):3164-73.
[30]Kavurma MM,Bennett MR.Expression,regulation and function of trail in atherosclerosis[J].Biochem Pharmacol,2008,75(7):1441-50.
[31]El-Deiry WS.Insights into cancer therapeutic design based on p53 and TRAIL receptor signaling[J].Cell Death Differ,2001,8(11):1066-75.
[32]郦萍,吴雪昌.肿瘤细胞抗TRAIL凋亡诱导的分子机制[J].细胞生物学杂志,2006,28:153-159.
[33]Chen X,Kandasamy K,Srivastava RK.Differential roles of RelA(p65)and c-Rel subunits of nuclear factor kappa B in tumor necrosis factor-related apoptosis-inducing ligand signaling[J].Cancer Res,2003,63(5):1059-66.
[34]Pitti RM,Marsters SA,Ruppert S,et al.Induction of apoptosis by Apo-2 ligand,a new member of the tumor necrosis factor cytokine family[J].J Biol Chem,1996,271:12687-90.
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