CN112553231A - 一种重组人热休克蛋白HSP90-His及其表达和纯化方法 - Google Patents
一种重组人热休克蛋白HSP90-His及其表达和纯化方法 Download PDFInfo
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Abstract
本发明公开了一种重组人热休克蛋白HSP90‑His及其表达和纯化方法。本发明通过构建重组人热休克蛋白HSP90的原核表达载体,使重组人热休克蛋白HSP90在大肠杆菌中高效表达,表达的重组蛋白以融合蛋白形式存在,经纯化、鉴定获得重组人热休克蛋白HSP90‑His。本发明的重组人热休克蛋白HSP90‑His通过SPR技术检测与糖皮质激素受体GRα结合,显示重组人热休克蛋白HSP90‑His具有较强的亲和力,其亲和力为1.44×10‑8M,有利于构建GRα‑HSP90蛋白复合体介导的分子开关型糖皮质激素检测方法,为糖皮质激素检测研究提供新的思路和依据。
Description
技术领域
本发明涉及生物技术和基因工程领域,具体涉及一种重组人热休克蛋白HSP90及其表达和纯化方法。
背景技术
热休克蛋白90(Heat shock protein 90,HSP90)是热休克蛋白家族中的重要成员之一,广泛存在于原核和真核生物中,多以分子伴侣的形式参与相关蛋白的折叠、装配、细胞内运输及蛋白质降解等过程。在正常细胞内有较高的组成性表达,约占细胞内蛋白含量的1~2%,它通过与类固醇激素受体及蛋白激酶的结合,在细胞信号传导中起着重要作用。
通常HSP90与其靶蛋白结合使其维持稳态,从而抑制其活性,如在介导糖皮质激素受体GRα的核转位时,HSP90与糖皮质激素受体结合形成复合体,当其配体(糖皮质激素)进入细胞后与受体结合,使HSP90从复合物中解离出来,配体-受体复合物才能进入核内发挥作用。研究表明,HSP90与GRα的结合能够改变其立体构象,使GRα的激素结合区像一个口袋一样打开,从而能够与糖皮质激素结合。当激素浓度增加,HSP90从多种蛋白组成的GRα复合体解离,导致GRα的DNA结合区及受体二聚化位点暴露,从而GRα二聚化,并具备结合DNA的能力,在细胞核内GRα可以上调或者下调目标基因的转录从而发挥生理效应。鉴于HSP90蛋白可与糖皮质激素受体GRα结合并呈现出典型的分子开关信号转导机制,与糖皮质激素特异性识别,有利于构建GRα-HSP90蛋白复合体介导的分子开关型糖皮质激素检测方法。
目前有关于人热休克蛋白HSP90基因表达载体的构建和蛋白表达虽已有文献报道,但这些方法都没有对表达的HSP90蛋白进行纯化,也没有检测对糖皮质激素受体的亲和力,其蛋白活性是未知的。本发明拟通过重组表达、纯化人HSP90蛋白并检测其与糖皮质激素受体结合的亲和力,为构建糖皮质激素检测方法提供新的思路和依据。
发明内容
本发明的目的是提供一种重组人热休克蛋白HSP90及其表达和纯化方法,通过构建重组人热休克蛋白HSP90的原核表达载体,实现重组人热休克蛋白HSP90在大肠杆菌中的高效表达。
本发明的目的是提供一种重组人热休克蛋白HSP90-His的表达和纯化方法。
本发明的另一目的提供所述HSP90-His蛋白的表达和纯化方法所制备的HSP90-His蛋白。
本发明是通过以下技术方案予以实现的:
一种重组人热休克蛋白HSP90-His的表达和纯化方法,其特征在于,包括以下步骤:
S1.将含有人热休克蛋白HSP90基因的重组表达载体质粒转化到宿主菌,获得重组菌株;
S2.对重组菌株进行诱导,表达HSP90-His蛋白;
S3.采用镍离子亲和层析柱对步骤S2表达的蛋白进行纯化,用平衡缓冲液平衡层析柱,用洗脱缓冲液进行洗脱,收集洗脱液,得HSP90-His蛋白溶液;
S4.将步骤S3所得的蛋白溶液加入到透析缓冲液中进行透析,即得纯化的HSP90-His蛋白。
优选地,步骤S1所述表达载体为原核表达载体pET-28a(+)。
优选地,步骤S1所述宿主菌为大肠杆菌。
优选地,步骤S1的具体步骤为:将人热休克蛋白HSP90基因构建到原核表达载体pET-28a(+)中得到重组原核表达载体pET-28a-HSP90质粒,再将重组表达载体质粒转化至大肠杆菌E.coli BL21(DE3)感受态细胞中。
优选地,步骤S2所述诱导的方法为采用诱导剂IPTG进行诱导。
更优选地,所述诱导的方法为:在含氨苄青霉素的LB培养基中37℃培养至OD600nm为0.4~0.6,加入终浓度为0.2~0.6mM的IPTG进行诱导。
更优选地,所述IPTG的终浓度为0.2mM。
更优选地,所述诱导的条件为:在18℃条件下诱导表达24h。
优选地,步骤S3所述平衡缓冲液含有浓度为15~20mmol/L的Tris-HCl缓冲液、250~300mmol/L的NaCl,pH为7.8~8.0。
更优选地,步骤S3所述平衡缓冲液含有浓度为20mmol/L的Tris-HCl缓冲液、300mmol/L的NaCl,pH为8.0。
优选地,步骤S3所述洗脱缓冲液含有浓度为15~20mmol/L的Tris-HCl缓冲液、250~300mmol/L的NaCl、200~250mmol/L的咪唑,pH为7.8~8.0。
更优选地,步骤S3所述洗脱缓冲液含有浓度为20mmol/L的Tris-HCl缓冲液、300mmol/L的NaCl、250mmol/L的咪唑,pH为8.0。
优选地,步骤S4所述透析缓冲液含有浓度为15~20mmol/L的Tris-HCl缓冲液、250~300mmol/L的NaCl、0.5~1mmol/L的二硫苏糖醇、体积浓度为8~10%的丙三醇,pH为8.0~8.5。
更优选地,步骤S4所述透析缓冲液含有浓度为20mmol/L的Tris-HCl缓冲液、300mmol/L的NaCl、1mmol/L的二硫苏糖醇、体积浓度为10%的丙三醇,pH为8.5。
因此,本发明还要求保护以上任一所述HSP90-His蛋白的表达和纯化方法所制备的HSP90-His蛋白。
与现有技术相比,本发明具有如下有益效果:
本发明通过构建重组人热休克蛋白HSP90的原核表达载体,并通过合理调整、优化蛋白表达和纯化的实验条件,使重组人热休克蛋白HSP90在大肠杆菌中高效表达,经纯化、鉴定获得重组人热休克蛋白HSP90。本发明的重组人HSP90蛋白可与糖皮质激素受体GRα结合,具有较强的结合亲和力,为构建糖皮质激素检测方法提供新的思路。本发明通过成本较低的表达系统体外获得大量的HSP90蛋白,并成功检测到与糖皮质激素受体相结合,可用于构建GRα-HSP90蛋白复合体介导的分子开关型糖皮质激素检测方法,为开展糖皮质激素检测的研究提供基础。
附图说明
图1为HSP90基因PCR扩增产物电泳分析结果;其中,M:1kb DNA Marker。
图2为重组表达载体pET-28a-GRα酶切鉴定结果;其中,M1:1kb DNA Marker,M2:DNA Marker DL2000,1-2:HSP90-His的质粒与酶切后产物。
图3为重组人HSP90蛋白的诱导表达条件结果,M:marker;1:0.6mM IPTG诱导24h沉淀,2:0.4mM IPTG诱导24h沉淀,3:0.2mM IPTG诱导24h沉淀,4:0.6mM IPTG诱导24h上清,5:0.4mM IPTG诱导24h上清,6:0.2mM IPTG诱导24h上清。
图4为重组人HSP90-His蛋白的镍离子亲和层析柱纯化结果;其中,1:不含咪唑的缓冲液,2:含10mmol/L咪唑的缓冲液,3:含25mmol/L咪唑的缓冲液,4:含250mmol/L咪唑的缓冲液。
图5为重组人HSP90-His蛋白的Western blotting检测结果;其中,1-2:重组HSP90-His蛋白。
图6为重组人HSP90-His蛋白与糖皮质激素受体GRα的浓度梯度结合曲线。
具体实施方式
下面结合说明书附图和具体实施例对本发明作出进一步地详细阐述,所述实施例只用于解释本发明,并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明,为可从商业途径得到的试剂和材料。
本发明中的原核表达载体pET-28a(+)、大肠杆菌E.coli BL21(DE3)感受态细胞购自生工生物工程(上海)股份有限公司;限制性内切酶BamHI和XhoI、质粒提取试剂盒购自宝日医生物技术(北京)有限公司;IPTG、SDS-PAGE凝胶制备试剂盒购自北京索莱宝科技有限公司;His标签Ni-NTA纯化柱购自亚科因(武汉)生物技术有限公司;抗His标签鼠单克隆抗体、辣根过氧化物酶标记山羊抗鼠二抗购自艾博抗(上海)贸易有限公司。
实施例1重组人热休克蛋白HSP90表达载体pET-28a-HSP90及表达菌株的构建
1、通过NCBI GeneBank数据库查找人HSP90基因序列(登录号为AJ890083.1,序列如SEQ ID NO:1所示),采用全基因合成方法获取HSP90基因的质粒,以质粒为模板,加入上游引物和下游引物进行PCR扩增(扩增产物结果如图1所示),上、下游引物如下所示:
上游引物HSP90-F(SEQ ID NO:2):
5'-cgcggatccATGCCTGAGGAAACCCAGACCCAAGAC-3';
下游引物HSP90-R(SEQ ID NO:3):
5'-ccgctcgagGTCTACTTCTTCCATGCGTGATGTGTC-3';
将扩增产物经1%琼脂糖凝胶电泳后,切胶回收纯化PCR扩增产物;将回收纯化的产物和pET-28a(+)表达载体用BamHI和XhoI进行双酶切鉴定,将酶切鉴定正确的阳性质粒送铂尚生物技术(上海)有限公司测序,测序结果与目的序列一致(如图2所示),成功构建重组原核表达载体pET-28a-HSP90。
2、将重组原核表达载体pET-28a-HSP90转化至大肠杆菌E.coli BL21(DE3)感受态细胞中:取出保存于-80℃超低温冰箱中的BL21(DE3)感受态细胞,置于冰上解冻,将5~10μL的连接产物加入至50μL的BL21(DE3)感受态细胞中,轻轻吹打混匀,冰浴30min,42℃热激90s,立即放置在冰中,冷却10min;加入预冷的750μL LB液体培养基中,37℃条件下200rpm振荡培养2h;将培养好的菌液涂布于含氨苄青霉素(100μg/mL)的LB平板上,37℃过夜培养。
3、筛选阳性菌落:在上述平板上,随机挑选多个白色菌落,用30μL无菌水稀释后用作PCR扩增验证模板,经PCR扩增后送铂尚生物技术(上海)有限公司测序,最终获得重组人HSP90基因序列的大肠杆菌工程菌。
实施例2重组人HSP90-His蛋白的表达、纯化及鉴定
(1)重组人HSP90-His蛋白的表达
挑取实施例1转化重组的阳性质粒的单菌落接种于1mL含氨苄青霉素(100μg/mL)LB液体培养基中,37℃过夜培养;将培养物以1:100转入LB液体培养基中扩大培养,37℃培养至OD600nm为0.6时,加入终浓度0.2mmol/L的异丙基-β-D-硫代半乳糖苷(IPTG),在18℃条件下诱导表达24h。同时设置空载体和pET28a-HSP90未诱导为对照。
诱导完后离心,将收集的菌体悬浮于100mL预冷PBS(含PMSF 1mmol/mL)中,4℃进行超声破碎至澄清,15000rpm,离心15min,分离上清和沉淀,进行SDS-PAGE分析;用考马斯亮蓝染色液染色20~30min后,置于脱色液中脱色数小时,直至蓝色背景消失。图3中SDS-PAGE电泳检测结果的泳道3和6显示,采用0.2mmol/L IPTG诱导使得目的蛋白在大肠杆菌中获得了大量表达。
(2)重组人HSP90-His蛋白的纯化
根据pET-28a(+)表达载体上带有的6×His标签,采用镍离子亲和层析柱进行蛋白纯化,具体步骤为:用平衡缓冲液平衡柱子,用柱子的5~10倍体积含咪唑的洗脱缓冲液洗脱目的蛋白,收集洗脱液;将收集的洗脱液放入透析缓冲液中,透析缓冲液为含NaCl、DTT、丙三醇的Tris-HCl缓冲液,4℃条件下透析8~12h,即获得纯化的重组人HSP90-His蛋白。
其中,平衡缓冲液为含NaCl的Tris-HCl缓冲液,pH为8.0,Tris-HCl缓冲液的浓度为20mmol/L,NaCl浓度为300mmol/L;
洗脱缓冲液为含NaCl、咪唑的Tris-HCl缓冲液,pH为8.0,Tris-HCl缓冲液的浓度为20mmol/L,NaCl浓度为300mmol/L,咪唑浓度为250mmol/L;
透析缓冲液为含NaCl、DTT、丙三醇的Tris-HCl缓冲液,pH为8.5,Tris-HCl缓冲液的浓度为20mmol/L,NaCl浓度为300mmol/L,DTT浓度为1mmol/L,丙三醇体积浓度为10%。
将纯化得到的重组人HSP90-His蛋白进行SDS-PAGE电泳,结果如图4的泳道4所示,当使用含有浓度为250mmol/L咪唑的洗脱缓冲液时,SDS-PAGE电泳结果显示具有清晰的单一条带,说明所得到的重组人HSP90-His蛋白纯化效果较好。
(3)重组人HSP90-His蛋白的鉴定
将以上得到的SDS-PAGE胶转至0.45μm PVDF膜,加入5%脱脂牛奶室温封闭1-2h,TBST洗膜后加入一抗(抗His标签鼠单克隆抗体,稀释比例为1:1000),4℃孵育过夜,TBST清洗3次,再加入二抗(辣根过氧化物酶标记山羊抗鼠,稀释比例为1:5000),室温孵育1-2h,TBST洗膜,采用ECL发光液(A液和B液等量混匀)显影目的条带,进行成像拍照。
Western blotting检测结果如图5所示,在84KD处具有明显的蛋白条带,证明已获得重组人HSP90-His蛋白,其氨基酸序列如SEQ ID NO:4所示。
实施例3重组人HSP90-His蛋白与糖皮质激素受体结合检测
1、实验步骤
采用表面等离子共振(SPR)检测方法检测重组人HSP90-His蛋白与糖皮质激素受体的结合,具体操作步骤如下:
(1)将羧基修饰的芯片作为SPR传感芯片安装在Open SPR仪器上;
(2)先以最大流速150μL/min,注入缓冲液PBS进行流通通道校正;
(3)仪器达到信号基线后,上样200μL 80%的异丙醇,运行10s后排气泡,达到基线后,再用PBS冲洗样本环,并用空气排空;
(4)上样200μL的EDC/NHS混合溶液,EDC浓度为400mM,NHS浓度为100mM,比例为1:1,流速为20μL/min,运行10min,从而活化SPR芯片表面的羧基基团;
(5)将实施例1得到的热休克蛋白HSP90-His用PBS稀释,经PBS稀释后浓度分别为0、12.5、25、50、100、200nM的HSP90-His稀释液;
(6)在所得的活化表面羧基后的SPR芯片上流入200μL热休克蛋白HSP90稀释液进行芯片偶联;流速为20μL/min,运行10min,用PBS冲洗样本环,并用空气排空;
(7)在HSP90-His偶联后的SPR芯片上注入200μL乙醇胺溶液,流速为20μL/min,运行10min,用以封闭未反应的活化过的羧基位点,用PBS冲洗样本环,并用空气排空;
(8)用PBS将糖皮质激素受体GRα稀释成浓度为0、12.5、25、50、100、200nM的GRα溶液,以20μL/min上样,HSP90与糖皮质激素受体GRα结合反应温度为25℃,结合时间为240s,自然解离180s;
(9)采用TraceDrawer分析软件中的1:1结合模型计算HSP90-His与糖皮质激素受体GRα的结合动力学和亲和力。
2、实验结果
结果如图6和表1所示,显示HSP90-His与GRα的结合亲和力为1.44×10-8M(表1),证明本发明制备得到的重组人HSP90-His蛋白亲和力较强,具有很好的活性。
表1 HSP90与糖皮质激素受体GRα的动力学及亲和力参数
对比例1不同浓度咪唑的洗脱缓冲液对重组人HSP90-His蛋白纯化效果的影响
重组人HSP90-His蛋白的表达和纯化方法步骤同实施例2,只是在重组人HSP90-His蛋白的纯化过程中,洗脱缓冲液分别为不含咪唑、含浓度为10mmol/L、25mmol/L咪唑。
实验结果如图4的SDS-PAGE电泳检测结果所示,其中,泳道1~4分别为不含咪唑、含浓度为10mmol/L、25mmol/L、250mmol/L咪唑的洗脱缓冲液;可以看出,只有当使用含有浓度为250mmol/L咪唑的洗脱缓冲液时,SDS-PAGE电泳结果显示具有较少的杂带,且表达效果较好,说明所得到的重组人HSP90-His蛋白纯化效果较好。
最后应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,对于本领域的普通技术人员来说,在上述说明及思路的基础上还可以做出其它不同形式的变化或变动,这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明权利要求的保护范围之内。
序列表
<110> 华南农业大学
<120> 一种重组人热休克蛋白HSP90-His及其表达和纯化方法
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2199
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
atgcctgagg aaacccagac ccaagaccaa ccgatggagg aggaggaggt tgagacgttc 60
gcctttcagg cagaaattgc ccagttgatg tcattgatca tcaatacttt ctactcgaac 120
aaagagatct ttctgagaga gctcatttca aattcatcag atgcattgga caaaatccgg 180
tatgaaagct tgacagatcc cagtaaatta gactctggga aagagctgca tattaacctt 240
ataccgaaca aacaagatcg aactctcact attgtggata ctggaattgg aatgaccaag 300
gctgacttga tcaataacct tggtactatc gccaagtctg ggaccaaagc gttcatggaa 360
gctttgcagg ctggtgcaga tatctctatg attggccagt tcggtgttgg tttttattct 420
gcttatttgg ttgctgagaa agtaactgtg atcaccaaac ataacgatga tgagcagtac 480
gcttgggagt cctcagcagg gggatcattc acagtgagga cagacacagg tgaacctatg 540
ggtcgtggaa caaaagttat cctacacctg aaagaagacc aaactgagta cttggaggaa 600
cgaagaataa aggagattgt gaagaaacat tctcagttta ttggatatcc cattactctt 660
tttgtggaga aggaacgtga taaagaagta agcgatgatg aggctgaaga aaaggaagac 720
aaagaagaag aaaaagaaaa agaagagaaa gagtcggaag acaaacctga aattgaagat 780
gttggttctg atgaggaaga agaaaagaag gatggtgaca agaagaagaa gaagaagatt 840
aaggaaaagt acatcgatca agaagagctc aacaaaacaa agcccatctg gaccagaaat 900
cccgacgata ttactaatga ggagtacgga gaattctata agagcttgac caatgactgg 960
gaagatcact tggcagtgaa gcatttttca gttgaaggac agttggaatt cagagccctt 1020
ctatttgtcc cacgacgtgc tccttttgat ctgtttgaaa acagaaagaa aaagaacaac 1080
atcaaattgt atgtacgcag agttttcatc atggataact gtgaggagct aatccctgaa 1140
tatctgaact tcattagagg ggtggtagac tcggaggatc tccctctaaa catatcccgt 1200
gagatgttgc aacaaagcaa aattttgaaa gttatcagga agaatttggt caaaaaatgc 1260
ttagaactct ttactgaact ggcggaagat aaagagaact acaagaaatt ctatgagcag 1320
ttctctaaaa acataaagct tggaatacac gaagactctc aaaatcggaa gaagctttca 1380
gagctgttaa ggtactacac atctgcctct ggtgatgaga tggtttctct caaggactac 1440
tgcaccagaa tgaaggagaa ccagaaacat atctattata tcacaggtga gaccaaggac 1500
caggtagcta actcagcctt tgtggaacgt cttcggaaac atggcttaga agtgatctat 1560
atgattgagc ccattgatga gtactgtgtc caacagctga aggaatttga ggggaagact 1620
ttagtgtcag tcaccaaaga aggcctggaa cttccagagg atgaagaaga gaaaaagaag 1680
caggaagaga aaaaaacaaa gtttgagaac ctctgcaaaa tcatgaaaga catattggag 1740
aaaaaagttg aaaaggtggt tgtgtcaaac cgattggtga catctccatg ctgtattgtc 1800
acaagcacat atggctggac agcaaacatg gagagaatca tgaaagctca agccctaaga 1860
gacaactcaa caatgggtta catggcagca aagaaacacc tggagataaa ccctgaccat 1920
tccattattg agaccttaag gcaaaaggca gaggctgata agaacgacaa gtctgtgaag 1980
gatctggtca tcttgcttta tgaaactgcg ctcctgtctt ctggcttcag tctggaagat 2040
ccccagacac atgctaacag gatctacagg atgatcaaac ttggtctggg tattgatgaa 2100
gatgacccta ctgctgatga taccagtgct gctgtaactg aagaaatgcc accccttgaa 2160
ggagatgacg acacatcacg catggaagaa gtagactaa 2199
<210> 2
<211> 36
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
cgcggatcca tgcctgagga aacccagacc caagac 36
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<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 3
ccgctcgagg tctacttctt ccatgcgtga tgtgtc 36
<210> 4
<211> 740
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 4
Met Pro Glu Glu Thr Gln Thr Gln Asp Gln Pro Met Glu Glu Glu Glu
1 5 10 15
Val Glu Thr Phe Ala Phe Gln Ala Glu Ile Ala Gln Leu Met Ser Leu
20 25 30
Ile Ile Asn Thr Phe Tyr Ser Asn Lys Glu Ile Phe Leu Arg Glu Leu
35 40 45
Ile Ser Asn Ser Ser Asp Ala Leu Asp Lys Ile Arg Tyr Glu Ser Leu
50 55 60
Thr Asp Pro Ser Lys Leu Asp Ser Gly Lys Glu Leu His Ile Asn Leu
65 70 75 80
Ile Pro Asn Lys Gln Asp Arg Thr Leu Thr Ile Val Asp Thr Gly Ile
85 90 95
Gly Met Thr Lys Ala Asp Leu Ile Asn Asn Leu Gly Thr Ile Ala Lys
100 105 110
Ser Gly Thr Lys Ala Phe Met Glu Ala Leu Gln Ala Gly Ala Asp Ile
115 120 125
Ser Met Ile Gly Gln Phe Gly Val Gly Phe Tyr Ser Ala Tyr Leu Val
130 135 140
Ala Glu Lys Val Thr Val Ile Thr Lys His Asn Asp Asp Glu Gln Tyr
145 150 155 160
Ala Trp Glu Ser Ser Ala Gly Gly Ser Phe Thr Val Arg Thr Asp Thr
165 170 175
Gly Glu Pro Met Gly Arg Gly Thr Lys Val Ile Leu His Leu Lys Glu
180 185 190
Asp Gln Thr Glu Tyr Leu Glu Glu Arg Arg Ile Lys Glu Ile Val Lys
195 200 205
Lys His Ser Gln Phe Ile Gly Tyr Pro Ile Thr Leu Phe Val Glu Lys
210 215 220
Glu Arg Asp Lys Glu Val Ser Asp Asp Glu Ala Glu Glu Lys Glu Asp
225 230 235 240
Lys Glu Glu Glu Lys Glu Lys Glu Glu Lys Glu Ser Glu Asp Lys Pro
245 250 255
Glu Ile Glu Asp Val Gly Ser Asp Glu Glu Glu Glu Lys Lys Asp Gly
260 265 270
Asp Lys Lys Lys Lys Lys Lys Ile Lys Glu Lys Tyr Ile Asp Gln Glu
275 280 285
Glu Leu Asn Lys Thr Lys Pro Ile Trp Thr Arg Asn Pro Asp Asp Ile
290 295 300
Thr Asn Glu Glu Tyr Gly Glu Phe Tyr Lys Ser Leu Thr Asn Asp Trp
305 310 315 320
Glu Asp His Leu Ala Val Lys His Phe Ser Val Glu Gly Gln Leu Glu
325 330 335
Phe Arg Ala Leu Leu Phe Val Pro Arg Arg Ala Pro Phe Asp Leu Phe
340 345 350
Glu Asn Arg Lys Lys Lys Asn Asn Ile Lys Leu Tyr Val Arg Arg Val
355 360 365
Phe Ile Met Asp Asn Cys Glu Glu Leu Ile Pro Glu Tyr Leu Asn Phe
370 375 380
Ile Arg Gly Val Val Asp Ser Glu Asp Leu Pro Leu Asn Ile Ser Arg
385 390 395 400
Glu Met Leu Gln Gln Ser Lys Ile Leu Lys Val Ile Arg Lys Asn Leu
405 410 415
Val Lys Lys Cys Leu Glu Leu Phe Thr Glu Leu Ala Glu Asp Lys Glu
420 425 430
Asn Tyr Lys Lys Phe Tyr Glu Gln Phe Ser Lys Asn Ile Lys Leu Gly
435 440 445
Ile His Glu Asp Ser Gln Asn Arg Lys Lys Leu Ser Glu Leu Leu Arg
450 455 460
Tyr Tyr Thr Ser Ala Ser Gly Asp Glu Met Val Ser Leu Lys Asp Tyr
465 470 475 480
Cys Thr Arg Met Lys Glu Asn Gln Lys His Ile Tyr Tyr Ile Thr Gly
485 490 495
Glu Thr Lys Asp Gln Val Ala Asn Ser Ala Phe Val Glu Arg Leu Arg
500 505 510
Lys His Gly Leu Glu Val Ile Tyr Met Ile Glu Pro Ile Asp Glu Tyr
515 520 525
Cys Val Gln Gln Leu Lys Glu Phe Glu Gly Lys Thr Leu Val Ser Val
530 535 540
Thr Lys Glu Gly Leu Glu Leu Pro Glu Asp Glu Glu Glu Lys Lys Lys
545 550 555 560
Gln Glu Glu Lys Lys Thr Lys Phe Glu Asn Leu Cys Lys Ile Met Lys
565 570 575
Asp Ile Leu Glu Lys Lys Val Glu Lys Val Val Val Ser Asn Arg Leu
580 585 590
Val Thr Ser Pro Cys Cys Ile Val Thr Ser Thr Tyr Gly Trp Thr Ala
595 600 605
Asn Met Glu Arg Ile Met Lys Ala Gln Ala Leu Arg Asp Asn Ser Thr
610 615 620
Met Gly Tyr Met Ala Ala Lys Lys His Leu Glu Ile Asn Pro Asp His
625 630 635 640
Ser Ile Ile Glu Thr Leu Arg Gln Lys Ala Glu Ala Asp Lys Asn Asp
645 650 655
Lys Ser Val Lys Asp Leu Val Ile Leu Leu Tyr Glu Thr Ala Leu Leu
660 665 670
Ser Ser Gly Phe Ser Leu Glu Asp Pro Gln Thr His Ala Asn Arg Ile
675 680 685
Tyr Arg Met Ile Lys Leu Gly Leu Gly Ile Asp Glu Asp Asp Pro Thr
690 695 700
Ala Asp Asp Thr Ser Ala Ala Val Thr Glu Glu Met Pro Pro Leu Glu
705 710 715 720
Gly Asp Asp Asp Thr Ser Arg Met Glu Glu Val Asp Gly Ser His His
725 730 735
His His His His
740
Claims (9)
1.一种重组人热休克蛋白HSP90-His的表达和纯化方法,其特征在于,包括以下步骤:
S1.将含有人热休克蛋白HSP90基因的重组表达载体质粒转化到宿主菌,获得重组菌株;
S2.对重组菌株进行诱导,表达HSP90-His蛋白;
S3.采用镍离子亲和层析柱对步骤S2表达的蛋白进行纯化,用平衡缓冲液平衡层析柱,用洗脱缓冲液进行洗脱,收集洗脱液,得HSP90-His蛋白溶液;
S4.将步骤S3所得的蛋白溶液加入到透析缓冲液中进行透析,即得纯化的HSP90-His蛋白。
2.根据权利要求1所述HSP90-His蛋白的表达和纯化方法,其特征在于,步骤S2所述诱导的方法为采用诱导剂IPTG进行诱导。
3.根据权利要求2所述HSP90-His蛋白的表达和纯化方法,其特征在于,所述诱导的方法为:在含氨苄青霉素的LB培养基中37℃培养至OD600nm为0.4~0.6,加入终浓度为0.2~0.6mM的IPTG进行诱导。
4.根据权利要求3所述HSP90-His蛋白的表达和纯化方法,其特征在于,所述IPTG的终浓度为0.2mM。
5.根据权利要求3所述HSP90-His蛋白的表达和纯化方法,其特征在于,所述诱导的条件为:在18℃条件下诱导表达24h。
6.根据权利要求1所述HSP90-His蛋白的表达和纯化方法,其特征在于,步骤S3所述平衡缓冲液含有浓度为15~20mmol/L的Tris-HCl缓冲液、250~300mmol/L的NaCl,pH为7.8~8.0。
7.根据权利要求1所述HSP90-His的表达和纯化方法,其特征在于,步骤S3所述洗脱缓冲液含有浓度为15~20mmol/L的Tris-HCl缓冲液、250~300mmol/L的NaCl、200~250mmol/L的咪唑,pH为7.8~8.0。
8.根据权利要求1所述HSP90-His蛋白的表达和纯化方法,其特征在于,步骤S4所述透析缓冲液含有浓度为15~20mmol/L的Tris-HCl缓冲液、250~300mmol/L的NaCl、0.5~1mmol/L的二硫苏糖醇、体积浓度为8~10%的丙三醇,pH为8.0~8.5。
9.权利要求1~8任一所述HSP90-His蛋白的表达和纯化方法所制备的HSP90-His蛋白。
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