大肠杆菌表达的新一代重组人内皮抑素复性条件的优化
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摘要
目的：优化重组人血管内皮细胞生长抑制素(rhED)的复性方案,以提高其复性率,并初步检测复性后rhED抗血管生成的生物活性,为rhED的进一步研究与利用提供大量的有活性的rhED蛋白。方法：用带有rhED cDNA的pQE3表达载体转染大肠杆菌E.ColiM15,在IPTG的诱导下发酵产生rhED包涵体并提取rhED包涵体。复性时,先用6mol/L盐酸胍溶解提取后rhED包涵体,在适宜的尿素浓度下稀释蛋白液并搅拌足够长的时间,结合逐级透析法进行复性,找出复性的主要影响因子,并优化最适复性条件。然后,利用标准曲线法测量rhED的浓度,SDS-PAGE验证并计算复性得率。复性结束后阳离子交换层析纯化rhED并超滤浓缩至适宜浓度。最后,用rhED特异性单克隆抗体和鸡胚绒毛尿囊膜抑制血管生长实验检测复性后蛋白的活性。结果：1L的rhED菌种发酵液能产生15g rhED包涵体。通过优化复性条件,rhED的复性率可达到46%。其中有三大重要的复性影响因素——复性时蛋白液的浓度、尿素浓度及pH值。复性时蛋白的浓度为0.3mg/ml、尿素浓度为3.5mol/L及pH为8时是适宜的复性条件。而阳离子交换层析能很好的纯化复性后的rhED。通过复性、纯化后的rhED能与rhED特异性单抗反应,并在鸡胚绒毛尿囊膜抑制血管生长实验中显示出抑制血管生长的活性。结论：至此提高rhED复性率的复性条件已经确立,本复性方案将极大地促进rhED的临床前研究及临床研究。
Objectives:To optimize the conditions for enhancing the refolding of rh-endostatin(rhED) from E. coli and test the biological activities of refolded rhED and for the futher research and use of rhED.
Methods:The rhED inclusion body was obtained by fermenting E.coli (strain M15) containing human endostatin cDNA which was ligated to plasmid pQE3 when it was induced by IPTG. The partial purified inclusion bodies of rhED were dissolved with 6 mol/L Guanidine-HCL following by combination of dilution in a diluent containing appropriate concentration of urea for an enough time and dialysis in series buffers of the dissolved endostatin.The rhED concentration after refolded was determined by a standard curve assay and SDS-PAGE, and calculated the refolding yield. The refolded rhED was then purified by cation-exchange method and concentrated by ultrafiltration. The biological activities of purified rhED were assessed by using rhED-specific monoclonal antibody and chick embryo chorioallantoic membrane assay.
Results:Fifteen grams per liter of rhED inclusion body was achieved by fermentation of E.coli with recombinant endostatin fragment in 20L bio-reactor. And the 46%(w/w) refolding yield was achieved after optimizing the conditions of refolding method after a numbers of comparison studies. Three conditions were critical to obtain high yield of refolded rhED:the starting refolding protein concentration, urea concentration, and pH in dialysis buffers during the refolding process. Our studies indicated that the best optimizing conditions of refolded rhED was achieved with 0.3mg/ml rhED in the starting refolding solution,3.5mol/L urea, and pH 8.0 in s series dialysis buffers. The high yield of refolded protein was observed by SDS-PAGE and protein concentration assay.The cation-exchange method was very suitable for purification the refolded rhED. The purified rhED was reacted with rhED-specific monoclonal antibody and significantly reduced the formation of blood vessels in rhED group compared with saline group in chick embryo chorioallantoic membrane assay, indicating that refolded rhED significantly inhibited the process of angiogenesis.
Conclusion:The method of highest refolding yield of human endostatin so far was developed in this study. This optimized method will greatly increase the application of our human endostatin to preclinical and clinical studies.
Objectives:To optimize the conditions for enhancing the refolding of rh-endostatin(rhED) from E. coli and test the biological activities of refolded rhED and for the futher research and use of rhED.
Methods:The rhED inclusion body was obtained by fermenting E.coli (strain M15) containing human endostatin cDNA which was ligated to plasmid pQE3 when it was induced by IPTG. The partial purified inclusion bodies of rhED were dissolved with 6 mol/L Guanidine-HCL following by combination of dilution in a diluent containing appropriate concentration of urea for an enough time and dialysis in series buffers of the dissolved endostatin.The rhED concentration after refolded was determined by a standard curve assay and SDS-PAGE, and calculated the refolding yield. The refolded rhED was then purified by cation-exchange method and concentrated by ultrafiltration. The biological activities of purified rhED were assessed by using rhED-specific monoclonal antibody and chick embryo chorioallantoic membrane assay.
Results:Fifteen grams per liter of rhED inclusion body was achieved by fermentation of E.coli with recombinant endostatin fragment in 20L bio-reactor. And the 46%(w/w) refolding yield was achieved after optimizing the conditions of refolding method after a numbers of comparison studies. Three conditions were critical to obtain high yield of refolded rhED:the starting refolding protein concentration, urea concentration, and pH in dialysis buffers during the refolding process. Our studies indicated that the best optimizing conditions of refolded rhED was achieved with 0.3mg/ml rhED in the starting refolding solution,3.5mol/L urea, and pH 8.0 in s series dialysis buffers. The high yield of refolded protein was observed by SDS-PAGE and protein concentration assay.The cation-exchange method was very suitable for purification the refolded rhED. The purified rhED was reacted with rhED-specific monoclonal antibody and significantly reduced the formation of blood vessels in rhED group compared with saline group in chick embryo chorioallantoic membrane assay, indicating that refolded rhED significantly inhibited the process of angiogenesis.
Conclusion:The method of highest refolding yield of human endostatin so far was developed in this study. This optimized method will greatly increase the application of our human endostatin to preclinical and clinical studies.
引文
[1]翟中和,王喜忠,丁明孝.细胞生物学第三版[M].北京：高等教育出版社,2007：480-482.
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[17]Xie D, Grulich P, Erickson J. Method for refolding recombinant endostatin[P]. WO Patent:wo/2001/060839,2001-08-23
[18]罗永章,周兵.生产内皮抑制素的方法[P].中国专利：00107569.1,2001-12-05
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[21]Hurrell J. Monoclonal hybridoma antibodies:techniques and applications[M]. Florida:CRC Press,1982,49-51.
[22]Jiang YP, Pannell R, Liu JN, et al. Evidence for a novel binding protein to urokinase-type plasminogen activator in platelet membranes [J]. Blood,1996,87(7): 2775-2781.
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[24]王克夷.疏水作用和蛋白质[J].生命的化学,1999,19(5):233-235.
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[26]Seckler R, Jaenice R. Protein folding and protein refolding [J]. FASEB J,1992,6(8): 2545-2552.
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[2]赵立希,孙晶晶,蒋永平.基因工程技术在生物制药领域的应用和发展[J].基础医学与临床,2009,29(9)：996-998.
[3]金惠铭,李先涛.血管新生与疾病[J].中国微循环,2001,5(3)：173-177.
[4]Folkman J. Tumor angiogenesis:therapeutic implications[J]. N Engl J Med,1971, 285(21):1182-1186.
[5]杨健,陈增良.血管新生与肿瘤生长的研究进展[J].浙江大学学报(医学版),2001,30(4)：173-177.
[6]Greenberg S, Rugo HS. Triple-negative breast cancer:role of antiangiogenic agents[J]. Cancer J,2010,16(1):33-38.
[7]林丽彬,林建银.内皮抑素及其抗血管生成作用[J].福建医科大学学,2007,41(2)：189-190.
[8]吕碧文,胡敏,齐义鹏.内皮细胞生长抑制素—抗肿瘤血管生成的细胞因子[J].武汉大学学报(理学版),2001,47(2)：251-256.
[9]O'Reilly MS, Boehm T, Shing Y, et al. Endostatin:An endogenous inhibitor of angiogenesis and tumor growth[J]. Cell,,1997,88(2):277-285.
[10]Lee TY, Robert M, Sjin TT, et al. Linking antibody Fc domain to endostatin significantly improves endostatin half-life and efficacy[J]. Clin Cancer Res,2008, 14(5):1487-1493.
[11]Boehem T, Folkman J, Browder T, et al. Antiangiogentic therapy of experimental cancer does not induce acquired drug resistance[J]. Nature,1997,390:404-407.
[12]Fu Y, Chen Y, Luo X, et al. The Heparin Binding Motif of Endostatin Mediates Its Interaction with Receptor Nucleolin[J]. Biochemistry,2009,48(49):11655-11663.
[13]Urbich C, Reissner A, Chavakis E, et al. Dephosphorylation of endothelial nitric oxide synthase contributes to the anti-angiogenic effects of endostatin[J]. FASEB J, 2002,16(7):706-708.
[14]Sudhakar A, Sugimoto H, Yang C, et al. Human tumstatin and human endostatin exhibit distinct antiangiogenic activities mediated by alpha v beta 3 and alpha 5 beta 1 integrins[J].Proc Natl Acad Sci U S A,2003,100(8):4766-4771.
[15]Dhanabal M, Ramchandran R, Waterman MJ, Lu H, Knebelmann B, Segal M, et al. Endostatin induces endothelial cell apoptosis[J]. Biol Chem,1999,274(17): 11721-11726.
[16]李忠义,汪军远.血管内皮细胞生长抑制剂——Endostatin的研究进展[J].中国公共卫生,2002,18(9)：1135-1136.
[17]Xie D, Grulich P, Erickson J. Method for refolding recombinant endostatin[P]. WO Patent:wo/2001/060839,2001-08-23
[18]罗永章,周兵.生产内皮抑制素的方法[P].中国专利：00107569.1,2001-12-05
[19]孙玉蓓,胡冰,王勇等.重组人血管内皮抑素联合化疗治疗晚期大肠杆癌的临床观察[J].临床肿瘤学杂志,2009,14(9)：837-840.
[20]Robert M, Sjin TT, Satchi-Faninaro R, et al. A 27-amino-acid synthetic peptide corresponding to the NH2-terminal zinc-binding domain of endostatin is responsible for its antitumor activity[J]. Cancer Res,2005,65(9):3656-3663.
[21]Hurrell J. Monoclonal hybridoma antibodies:techniques and applications[M]. Florida:CRC Press,1982,49-51.
[22]Jiang YP, Pannell R, Liu JN, et al. Evidence for a novel binding protein to urokinase-type plasminogen activator in platelet membranes [J]. Blood,1996,87(7): 2775-2781.
[23]Chiou GCY. Ophthalmic toxicology (target organ toxicology series)[M].2nd Edition, Philadephia:Taylor&Francis,1999,189-191.
[24]王克夷.疏水作用和蛋白质[J].生命的化学,1999,19(5):233-235.
[25]周爱儒,查锡良.生物化学第6版[M].北京：人民卫生出版社,2005：12-22.
[26]Seckler R, Jaenice R. Protein folding and protein refolding [J]. FASEB J,1992,6(8): 2545-2552.
[27]Chura-Chambi RM, Genora LA, Affonso R, et al. Refolding of endostatin from inclusion bodies using high hydrostatic pressure[J]. Anal Biochem,2008,379(1): 32-39.
[1]Folkman J. Tumor angiogenesis:therapeutic implications[J]. N Engl J Med,1971, 285(21):1182-1186.
[2]Folkman J. Proceedings:Tumor angiogenesis factor[J]. Cancer Res,1974,34(8): 2109-2113.
[3]O'Reilly MS, Boehm T, Shing Y, et al. Endostatin:An endogenous inhibitor of angiogenesis and tumor growth[J]. Cell,1997,88(2):277-285.
[4]O'Reilly MS, Boehm T, Shing Y, et al. Endostatin:an endogenous inhibitor of angiogenesis and tumor growth[J]. Cell,1997,88(2):277-85.
[5]Zatterstrom UK, Felbor U, Fukai N, et al. Collagen XVIII/endostatin structure and functional role in angiogenesis[J]. Cell Struct Funct,2000,25(2):97-101.
[6]林丽彬,林建银.内皮抑素及其抗血管生成作用[J].福建医科大学学报,2007,41(2)：189-190.
[7]Yamaguchi N, Anand-Apte B, Lee M, et al. Endostatin inhibits VEGF-induced endothelial cell migration and tumor growth independently of zinc binding[J]. The EMBO Journal,1999,18(6):4414-4423.
[8]Hohenester E, Sasaki T, Mann K, et al. Varible zinc coordination in endostatin[J]. Journal of molecular biology,2000,297(1):1-6.
[9]Robert M, Sjin TT, Satchi-Faninaro R, et al. A 27-amino-acid synthetic peptide corresponding to the NH2-terminal zinc-binding domain of endostatin is responsible for its antitumor activity[J]. Cancer Res,2005,65(9):3656-3663
[10]吕碧文,胡敏,齐义鹏.内皮细胞生长抑制素—抗肿瘤血管生成的细胞因子[J].武汉大学学报(理学版),2001,47(2)：251-256.
[11]Dhanabal M,Ram chandran R,Waterman MJ,et al. Endostatin induces endothelial cell apoptosis[J]. The journal of biological chemistry,1999,274(17):11721-11726
[12]Kim YM, Jang JW, Lee OH,et al. Endostatin inhibits endothelial and tumor cellular invasion by blocking the activation and catalytic activity of matrix metalloproteinase 2[J]. Cancer Research,2000,60(19):5410-5413
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