rhBMP-6的表达、纯化与鉴定

【作者】 闫继东；

【导师】 朱天慧；

【作者基本信息】 南开大学 ， 生物化学与分子生物学， 2009， 博士

【副题名】BMP-6抗纤维化及抗氧化应激作用与机制初探

【摘要】 第一部分rhBMP-6的表达、纯化与鉴定骨形态发生蛋白（Bone morphogentic protein,BMP）属于TGF-β蛋白超家族（BMP-1缺乏碳段保守结构而除外）。BMP-6为BMP家族成员之一,与其它成员相比,BMP-6有较强的骨诱导作用。BMP-6在骨折愈合过程中表达上调,并在不同的骨缺损模型中,能加快缺损骨的修复。另外,系统给予BMP-6能有效治疗实验性骨质疏松引起的骨量减少和骨微观结构的改变,提示BMP-6对骨缺损和骨质疏松有潜在的治疗作用。本研究分别采用大肠杆菌和培养哺乳动物细胞为表达系统,通过基因工程途径获取具有生物活性的BMP-6蛋白。1.BMP-6在大肠杆菌的表达、复性与纯化以非融合形式分别在大肠杆菌胞质中表达不同截短模式,同时带有克隆位点引入氮端延伸序列（MA或MG）的BMP-6单体蛋白或BMP-6突变体(Ala⁵⁶-His)。表达菌株与表达条件筛选试验发现序列MA-BMP-6（1323）的表达量较高。因此选用MA-BMP-6（1323）在大肠杆菌BL-21（DE3）中表达BMP-6成熟肽单体蛋白。诱导表达后,BMP-6约占菌体总蛋白的20%,以包涵体形式存在。包涵体分离纯化后,获得组成均一的BMP-6单体蛋白。溶解试验发现高浓度变性剂（6MGdn-HCl）合并还原剂（0.1M DTT）能有效溶解包涵体蛋白。经溶解、折叠、添加剂试验逐步筛选、优化复性条件后,约10%蛋白形成同源二聚体。BMP-6突变体对二聚体的形成没有显著影响。复性所得二聚体蛋白能够通过反相色谱纯化。可能由于缺乏糖基化缘故,纯化的二聚体蛋白碱性磷酸酶诱导活性的量效关系不显著。2.BMP-6在CHO细胞中的表达、纯化与鉴定将含BMP-6信号肽、前肽和成熟肽以及含BMP-2信号肽、前肽与BMP-6成熟肽融合的基因序列分别克隆至哺乳动物细胞表达载体。利用脂质体瞬时转染分析发现BMP2/6融合基因能有效分泌表达BMP-6蛋白。然后采用二氢叶酸还原酶（Dihydroflolate reductase,DHFR）缺失的中华仓鼠卵巢细胞（Chinese hamster ovary cell,CHO）细胞为宿主,通过混合克隆、单克律秆∫约澳康幕蚶┰龊?获得表达水平约20 ng/10⁶ cells/24 h的稳定表达细胞株。对稳定表达细胞株的比生长速率,BMP-6的比生成速率与分泌特性,以及BMP-6在培养环境与条件培养基中的稳定性进行了初步研究。条件培养基中的BMP-6通过肝素亲和色谱与反相色谱分离纯化,对纯化产物的纯度和生物活性进行了鉴定。最后,纯化产物剂量和时间依赖地诱导碱性磷酸酶活性。与对照相比,BMP-6在浓度为25 ng/ml时活性显著,在400 ng/ml时达到最大反应剂量,而半数有效量介于100-150 ng/ml。用50 ng/ml的BMP-6处理细胞三天,碱性磷酸酶活性与对照相比差异显著。结论:本研究采用大肠杆菌表达系统,以非融合包涵体形式表达BMP-6单体蛋白,通过优化表达、体外氧化复性和纯化获得成熟肽二聚体;同时采用CHO细胞分泌表达BMP-6,通过载体优化、细胞株筛选、纯化流程建立等途径获取具有显著生物活性的BMP-6蛋白。第二部分BMP-6抗纤维化和氧化应激的作用与机制除骨诱导作用外,BMP-6还参与机体发育和成体组织器官重塑过程的调节。研究发现慢性肾损伤时BMP-6表达下调,而慢性肾损伤直接抑制骨形成,提示抑制肾损伤可能能够促进骨形成。因此本文对BMP-6的抗肾纤维化以及抗氧化应激作用与机制进行研究。1.BMP-6抑制TGF-β1诱导的PTEC转化肾间质纤维化是慢性肾损伤的主要特征而TGF-β1在其中发挥重要作用。TGF-β1能诱导人近曲小管上皮细胞（Pmximal tubular epithelial cell,PTEC）HK-2细胞增殖抑制、上皮间质转化和黏附性增加,能诱导Fibtonectin和TIMP-2（Tissueinhibitors of metalloproteinase-2）表达,而抑制MMP-2（Matrix metalloproteinase 2）的表达和活性。同时,TGF-β1能激活Smad3和JNK激酶信号途径。BMP-6单独处理时,对HK-2细胞的增殖、形态表型、胞外基质蛋白的合成以及黏附性无明显影响。但是BMP-6能抑制TGF-β1诱导HK-2细胞增殖抑制,形态、表型转化,胞外基质蛋白的堆积以及黏附性增加,同时能抑制TGF-β1诱导的Smad3和JNK激酶的激活。本文研究证实BMP-6能抑制TGF-β1诱导的纤维化作用,这种作用可能是通过抑制JNK激酶和Smad3信号途径的活化而实现。2.BMP-6通过Smad5途径诱导HO-1表达而抗PTEC氧化应激损伤研究发现氧自由基参与TGF-β1诱导的PTEC转化而血红素氧合酶1（hemeoxygenase 1,HO-1）是机体抗氧化应激的重要机制。BMP-6能剂量和时间依赖地抑制双氧水诱导的HK-2细胞氧化应激损伤,以及诱导HO-1表达。HO-1诱导、过表达以及HO-1产物胆红素或一氧化碳能减轻HK-2细胞的氧化应激损伤,而新基因合成抑制、HO-1活性抑制、HO-1基因干扰或一氧化碳捕获等能减弱BMP-6的抗氧化应激作用。BMP-6能激活Smad5途径,而Smad5基因干扰则抑制HO-1的诱导表达。HO-1启动子上Smad5结合位点缺失或突变能抑制BMP-6诱导的HO-1启动子活性增加。最终证实BMP-6通过Smad5途径诱导HO-1表达,通过HO-1的产物胆红素和一氧化碳来发挥抗氧化应激损伤作用。结论:本研究在培养的肾近曲小管上皮细胞中发现,BMP-6能抑制TGF-β1诱导的肾近曲小管上皮细胞纤维化转变,并且能通过激活Smad5信号途径诱导HO-1表达而减轻肾近曲小管上皮细胞氧化应激损伤。这一结果为进一步深入研究BMP-6的骨生成和肾保护作用、机制与应用奠定了基础。更多还原

【Abstract】 PartⅠExpression,purification,and characterization of rhBMP-6Bone morphogenetic proteins（BMPs） belong to the transformation growth factorsβsuperfamily（TGF-β） except BMP-1,which lack the C-terminal conserve domain of TGF-β.BMP-6 is one member of BMPs family and is one of the most potent osteogenesis factors as compared with the other members of BMP family.BMP-6 is induced in different stages of bone fracture.BMP-6 promotes bone regeneration in different bone deficiency model.In addition,BMP-6 administered systematically, restores bone volume and micro-architecture in osteoporotic rat and mouse.These results indicate BMP-6 as a therapeutic potential candidate for treating bone deficiency and osteoporosis.In the present study,we recombinant produced BMP-6 in Escherichia coli and cultivated mammalian cells.1.Expression,refolding,and purification of BMP-6 in E.ColiThe variants of BMP-6 and BMP-6 mutant(Ala⁵⁶-His) plus an N-terminal extension（MA or MG duo to the clone site） were expressed in E.Coli as non-fusion protein,respectively.The expression level of the mature peptide with MA extension （MA-BMP-6（1323）） was significantly higher than the other’s by screening of host cells and expression constructs under different growth conditions.This construct was transformed into an E.coli BL21（DE3） for the expression of BMP-6 mature peptide.BMP-6 was expressed as IBs after induction,contributing up to 20%of the total bacterial protein.Protein was near homogenous after a simplified procedure of IBs isolation.The IBs were dissolved in 6M Gdn-HCl and 0.1M DTT containing buffer. The refolded dimeric protein was obtained by dilution the dissolved IBs protein in an optimized refolding buffer and about 10%of BMP-6 formed disulphide-linked homodimers.This buffer system was optimized step by step,including solubility, refolding,and refolding adding agent determined.BMP-6 mutant could not improve efficiency of refolding.The refolded dimeric BMP-6 was purified by one step reverse phase liquid chromatography.Howerver,the dose-dependent alkaline phosphatase （ALP） induction activity of the purified dimeric protein was not observed,probably due to lack of N-glycosylation produced in E.Coli.2.Expression,purification,and characterization of BMP-6 in CHO cellsThe prepromature peptide of BMP-6 and prepro-peptide of BMP-2 fused with mature peptide of BMP6（BMP2/6） was subcloned into the mammalian expression vector,respectively.The BMP2/6 construction could efficiently pruduce BMP-6 protein after transient transfection in Cos 7 via liposame reagent.BMP-6 was stable epressed in dihydroflolate reductase deficiency Chinese hamster ovary（CHO-dhfr-） cells.BMP-6 expression levels of the most productive subclones was about 20 ng/10⁶ cells/24 h after individual and cell pool based selection followed by target gene amplification.The specific growth rate,BMP-6 production, and stability of BMP-6 in culture condition and conditioned medium were characterized.BMP-6 was purified by combination of heparin affinity and reverse phase chromatography.The purified product was indentified by SDS-PAGE and western blot.Finally,the purified BMP-6 induced alkaline phophatase（ALP） activity in a time-course and dose-dependent manner.The ALP activity was significantly induced at concentration of 25ng/ml.EC50 was about 100-150ng/ml and the maximal effective dose was about 400ng/ml.The ALP activity was significantly induced after 3 days incubation at concentration of 50ng/ml.In summary,the non-fusion human BMP-6 was expressed in E.coli as IBs with high level.After the optimized procedure for IBs isolation and refolding,the refolded dimeric protein was purified by one step reverse phase liquid chromatography. Furthermore,the BMP-6 was expressed in CHO cells.After vector construction, stable cell line screening,and purification scheme optimazation,the bioactive BMP-6 protein was produced.PartⅡEffect and mechanism of BMP-6 on fibrosis and oxidant stressBMP-6 plays a role during embryonal development and tissue morphogenesis postnatal as well as bone formation.Downregulation of BMP-6 may predispose to renal deficiency and chronic renal deficiency directly reduced bone formation, indicating that suppression of chronic renal deficiency my improve bone regeneration. In the present study,the effect and mechamism of BMP-6 on renal fibrosis and oxidant injury were investigated.1.BMP-6 reverses TGF-β1-initiated changes of PTECTransforming growth factor-beta 1（TGF-β1） plays a crucial role in the inception and progression of renal tubulointerstitial fibrosis which lead to renal deficiency. Treatment with TGF-β1 reduced cell proliferation,induced epithelial-to-mesenchymal transition（EMT）,deceased expression and activity of matrix metalloproteinases 2（MMP-2）,increased expression of fibronectin and tissue inhibitors of matrix metalloproteinases 2（TIMP-2）,and increased cell adhesion in renal proximal tubular epithelial cell（PTEC） HK-2 cells.All these effects were inhibited when TGF-β1 was combined with BMP-6,whereas BMP-6 alone had no such effects.In addition,BMP-6 abrogated the JNK and Smad3 signaling activated by TGF-β1.BMP6 ameliorated TGF-β1 induced fibrosis changes in HK-2 cells and suppression of TGF-β1-mediated JNK and Smad3 activation maybe implicated.2.BMP-6 attenuates oxidant injury in PTEC via Smad-dependent HO-1 inductionReactive oxygen species are involved in TGF-β1-induced transition and heme oxygenase 1（HO-1） induction is a protective response to oxidative stress.BMP-6 effectively protected renal proximal tubule cells（HK-2） against hydrogen peroxide （H₂O₂）-induced cell injury.BMP-6 also increased HO-1 gene expression.Inhibition of de novo gene expression,HO-1 inhibitor,HO-1 knockdown,or carbon monoxide （CO） scavenger attenuated the cytoprotective effect of BMP-6,whereas HO-1 inducer, HO-1 constitutive expression,or HO-1 products bilirubin and CO ameliorated H₂O₂-induced cell injury.Stimulation of HK-2 cells with BMP-6 activated Smad5 signaling and BMP6-mediated induction of HO-1 expression was inhibited by Smad5 knockdown.Furthermore,deletion or mutation of the Smad-binding element in HO-1 promoter inhibited the BMP-6-induced HO-1 promoter activity.These findings suggest that induction of HO-1 through a Smad-dependent manner is responsible for the cytoprotective effect of BMP-6 in H₂O₂-mediated renal cell injury.In summery,the antifibrotic and antioxidant effect of BMP-6 were determined in renal proximal tubular epithelial cells,useful for further therapeutic investigations of BMP-6 in the treatment of renal interstitial fibrosis and bone deficiency.更多还原