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1.
为探讨诱导温度对于HIV-1 Gag在大肠杆菌中表达产物状态以及尿素浓度对蛋白纯化效果的影响, 将30oC和37oC诱导表达的包涵体分别溶于不同浓度的尿素, 比较溶解性的差异, 并比较复性的不同。将30oC诱导的目的蛋白分别用2 mol/L和8 mol/L尿素溶解后做层析分离, 比较两者的分离效果。结果发现, 与37oC相比, 30oC诱导表达的蛋白能有效溶于低浓度尿素, 并且更容易复性。与8 mol/L尿素溶解相比, 30oC诱导的包涵体用2 mol/L尿素溶解后通过凝胶过滤和离子交换层析纯化能得到更好的分离效果。这提示低温诱导的Gag包涵体中可能含有更多类似天然态构象的蛋白, 而低浓度尿素有利于保持包涵体中蛋白的天然态构象。从而为包涵体蛋白的诱导表达和分离纯化提供了参考。  相似文献   

2.
[目的]探索不同条件对包涵体复性的影响,优化重组融合蛋白包涵体中释放大环肽的工艺条件。[方法]对纯化后的包涵体进行不同变性液、透析方式及是否超声破碎处理的比较;并在此基础上探讨不同透析温度、谷胱甘肽浓度与L-Arg浓度对大环肽产量的影响。采用Tanon凝胶分析系统与紫外吸收法对大环肽的产量进行分析。[结果]使用8 mol/L的尿素溶解包涵体后,获得的上清液在25℃条件下,在含有1 mmol/L GSH、0.1 mmol/L GSSG、0.8mmol/L L-Arg的透析液中进行梯度透析,其得率为每克包涵体获得约1.4 mg大环肽。[结论]成功从包涵体中获得大环肽并优化工艺条件。  相似文献   

3.
将表达人重组IL-3的工程菌热诱导表达后超声裂菌,洗涤处理沉淀物,得到纯化包涵体.8 mol/L尿素将其变性溶解后,直接利用制备性等电聚焦仪Rotofor一步纯化,获得纯度90%以上的人重组IL-3,回收率约60%,每次回收15—20mg蛋白.纯化的人重组蛋白保持了IL-3生物活性。  相似文献   

4.
目的将前期在大肠埃希杆菌中获得表达的A型人呼吸道合胞病毒兰州分离株截短的F1重组蛋白进行纯化和复性,为后期动物免疫制备抗原。方法 37℃诱导重组菌体p ET-42b-F1J/Rossata,诱导完毕后离心收集菌体,高压破碎菌体并收集包涵体后用不同浓度的Triton X-100(细胞裂解液)洗涤包涵体3次。洗涤的包涵体用8 mol/L尿素进行溶解并用镍离子亲和层析方法进行初步纯化,用阳离子交换层析方法对初步纯化蛋白进行最终的纯化。亲和层析纯化蛋白用3种不同的复性液进行了稀释复性。结果 37℃诱导5 000 m L重组菌p ET-42b-F1J/Rossata共收获37 g湿菌体,经过不同浓度Triton X-100洗涤包涵体后纯度可达75%。包涵体用8 mol/L尿素溶解后经镍离子亲和层析纯化纯度约为40%,再用阳离子交换层析介质SP HP进一步纯化样品后纯度可达90%。纯化蛋白以3种不同的复性液都能得到复性,其中复性液3的复性效果相对较好。结论实验中探索了人呼吸道合胞病毒截短F1重组蛋白包涵体的纯化方法及步骤,为后期的蛋白制备及动物免疫奠定了基础。  相似文献   

5.
本文继大肠杆菌表达含凝血酶切点的人重组IL-3融合蛋白成功的基础上进一步探讨了天然型rhIL-3的纯化。超声破碎细菌细胞得包涵体,经洗涤处理可使包涵体纯度达90%,用8mol/L尿素变性溶解包涵体沉淀后直接稀释复性,再超滤浓缩、凝血酶消化,释放天然型rhIL-3。经DEAE Sepharose Fast Flow和Sepharcyl-100 HR层析得到天然型IL-3,纯度达96%,回收率20%以上,具有刺激正常人骨髓细胞形成集落的活性。本实验为大批量重组IL-3的生产创造了条件。  相似文献   

6.
利用大肠杆菌表达重组人转化生长因子β3(humantransformgrowthfactor,hTGFβ3),目的蛋白以包涵体形式表达。用8mol/L尿素溶解的包涵体蛋白,经CMSepharoselFastFlow柱和SphacrylS100分子筛可获得纯度达90%以上的rhTGF-β3单体。将单体蛋白加到复性缓冲液(1mol/LNaCl,0.5mol/LLArg,0.5mmol/LGSSG,30mmol/LCHAPS,20%(v/v)DMSO)进行复性后,再经DEAESepharoselFastFlow柱和SphacrylS100分子筛可分离得到纯度达94%的二聚体rhTGFβ3,纯化后的产量为720mg/L,纯化总回收率为47%。MTT法测活表明,该重组蛋白具刺激成纤维细胞Balb/c3T3生长的作用。  相似文献   

7.
旨在建立并优化融合蛋白Trx-IFN-CSP的复性工艺。对重组融合Trx-IFN-CSP进行体外复性研究,考查p H、温度、蛋白浓度、氧化还原体系及辅助复性小分子等复性条件对融合蛋白重折叠的影响。结果显示,适合Trx-IFN-CSP复性的方法为,反复冻融联合超声破菌获得包涵体;用含有1%Triton X-100、2 mol/L尿素、2%DOC洗涤液初步纯化包涵体;再用6 mol/L盐酸胍溶解液变性包涵体;脉冲加样稀释变性液后4℃条件下梯度透析复性,使用L-Arg辅助复性。经肠激酶切去Trx标签后,每升发酵液最终获得110-130 mg肝靶向干扰素,每批蛋白纯度都在95%以上,比活性在1.9-2.4×108 U/mg之间,制备工艺稳定。  相似文献   

8.
利用8 mol/L尿素溶液对表达在大肠杆菌包涵体中的GST-TRAF6融合蛋白进行变性,通过逐级稀释复性的方法对尿素溶解后的GST-TRAF6融合蛋白进行复性,将复性后的GST-TRAF6融合蛋白进一步利用谷胱甘肽琼脂糖树脂亲和层析的方法进行分离纯化,将分离纯化后的蛋白通过Western blot方法进行验证,最后利用体外泛素化反应检测经包涵体变性、复性和纯化后的GST-TRAF6融合蛋白的生物学活性。经过包涵体变性、梯度稀释复性和谷胱甘肽琼脂糖树脂亲和层析3个步骤后纯化得到纯度达90%以上、浓度为396 ng/μL的蛋白质溶液。利用GST蛋白作为对照,经Western blot验证表明,纯化得到的蛋白确为GSTTRAF6融合蛋白。进一步利用体外泛素化反应分析其泛素连接酶活性发现,17 ng/μL浓度的GST-TRAF6融合蛋白能够以泛素分子作为底物在5 min内快速催化自由泛素链的生成。结果表明,表达在大肠杆菌包涵体中的GST-TRAF6融合蛋白经尿素变性溶解后能够成功复性并分离纯化,在溶解性改变的同时恢复了其泛素连接酶活性。为从大肠杆菌包涵体中大规模分离纯化蛋白质提供了一种新的复性方法。  相似文献   

9.
碱性条件下溶解包涵体提高凝乳酶原复性率的机制   总被引:1,自引:0,他引:1  
重组凝乳酶原的复性效率不仅依赖于复性条件,而且与包涵体的溶解条件(即变性条件)有关.含有凝乳酶原的包涵体在pH11,8mol/L脲中溶解后,其复性率较在pH8,8mol/L脲中溶解的可提高1倍,由20%左右提高到40%左右.其机制是碱性条件有利于破坏分子间交联的二硫键从而增加凝乳酶原的溶解度.更为重要的是碱性条件使凝乳酶原分子处于一种还原性更强更为伸展的状态,这种状态容易进行正确再折叠.  相似文献   

10.
由E.coli表达的重组白细胞介素2(rIL—2)以包涵体形式存在于工程菌胞浆中。用变性剂将包涵体溶解后得到的还原态rIL—2没有生物活性,必须对其进行复性才能最终得到有用的产品。本实验研究了以盐酸胍(GUHCl)为变性剂条件下,变性剂浓度、复性剂、pH值以及温度和时间等对rIL—2复性的影响。  相似文献   

11.
The renaturation efficiency of recombinant prochymosin depends on not only the renaturation condi-tions but also the solubilization (denaturation) conditions. Compared with pH 8, solubilization of prochymosin-contain-ing inclusion bodies at pH 11 (8 mol/L urea) results in onefold increase of renaturation efficiency ( ~ 40% vs. ~ 20 % ). Alkaline pH facilitates the solubilization of inclusion bodies via the breakage of intermolecular disulfide bonds. Moreover, alkaline pH renders prochymosin molecules to be in a more reduced and more unfolded state which undergoes refolding readily.  相似文献   

12.
利用内蛋白子剪切功能一步纯化重组人神经营养因子-3   总被引:3,自引:0,他引:3  
将人神经营养因子 - 3(h NT3)基因插入含内蛋白子 -几丁质结合区 (Intein- CBD)片段的质粒p TXB1的多克隆位点 ,构建成重组子 p TXB- h NT3,随后转化入 E.coli 2 566并进行融合表达 .表达产物包涵体经 8mol/ L脲变性 ,并在 GSH,GSSG存在下复性 .复性后的融合蛋白经几丁质珠亲和柱吸附 .待洗涤杂蛋白后 ,加入 50 mmol/ L DTT在 4℃或 2 5℃进行剪切反应 48h,再用缓冲液洗脱 ,即得 h NT3.SDS- PAGE分析表明 ,h NT3达电泳纯 .其分子量约为 1 4 k D  相似文献   

13.
利用 PCR扩增出人血管生成素 (h ANG)成熟肽的基因片段 .与绿脓杆菌外毒素缺失突变体PE40的基因连接后 ,克隆入 p UC1 9载体中 .测序后克隆入表达载体 p RSETB,构建成 h ANG-PE40融合基因的表达载体 .IPTG诱导 ,表达出分子量约为 58k D的 His6- ANG- PE40融合蛋白 ,占菌体总蛋白的 8% .Ni2 +- NTA树脂纯化表达蛋白 ,SDS- PAGE结果显示纯化重组蛋白为单一条带 .鸡胚绒毛尿囊膜鉴定表明重组蛋白体外能够有效地抑制血管的形成 .  相似文献   

14.
Recombinant human growth hormone (r-hGH) was expressed in Escherichia coli as inclusion bodies. In 10 h of fed-batch fermentation, 1.6 g/L of r-hGH was produced at a cell concentration of 25 g dry cell weight/L. Inclusion bodies from the cells were isolated and purified to homogeneity. Various buffers with and without reducing agents were used to solubilize r-hGH from the inclusion bodies and the extent of solubility was compared with that of 8 M urea as well as 6 M Gdn-HCl. Hydrophobic interactions as well as ionic interactions were found to be the dominant forces responsible for the formation of r-hGH inclusion bodies during its high-level expression in E. coli. Complete solubilization of r-hGH inclusion bodies was observed in 100 mM Tris buffer at pH 12.5 containing 2 M urea. Solubilization of r-hGH inclusion bodies in the presence of low concentrations of urea helped in retaining the existing native-like secondary structures of r-hGH, thus improving the yield of bioactive protein during refolding. Solubilized r-hGH in Tris buffer containing 2 M urea was found to be less susceptible to aggregation during buffer exchange and thus was refolded by simple dilution. The r-hGH was purified by use of DEAE-Sepharose ion-exchange chromatography and the pure monomeric r-hGH was finally obtained by using size-exclusion chromatography. The overall yield of the purified monomeric r-hGH was approximately 50% of the initial inclusion body proteins and was found to be biologically active in promoting growth of rat Nb2 lymphoma cell lines.  相似文献   

15.
大肠杆菌表达的人IL-6复性条件研究   总被引:1,自引:0,他引:1  
采用稀释法复性,筛选重组人白细胞介素-6(rhIL-6)包涵体蛋白的复性条件。结果显示,复性液的浓度、pH值、复性时间和复性蛋白浓度,对重组人白细胞介素-6复性效果有很大影响。在复性液为2mol/L尿素、pH8.5、复性时间为24h和复性蛋白浓度50μg/ml条件下,重组人白细胞介素-6包涵体蛋白的复性效果最佳。  相似文献   

16.
Purification of the recombinant human renin receptor (rhRnR) is a major aspect of its biological or biophysical analysis, as well as structural research. A simple and efficient method for the refolding and purification of rhRnR expressed in Escherichia coli with weak anion‐exchange chromatography (WAX) was presented in this work. The solution containing denatured rhRnR in 8.0 mol/L urea extracted from the inclusion bodies was directly injected into the WAX column. The aggregation was prevented and the soluble form of renatured rhRnR in aqueous solution was obtained after desorption from the column. Effects of the extracting solutions, the pH values and urea concentrations in the mobile phase, as well as the sample size on the refolding and purification of rhRnR were investigated, indicating that the above mentioned factors had remarkable influences on the efficiency of refolding, purification and mass recovery of rhRnR. Under the optimal conditions, rhRnR was successfully refolded and purified simultaneously by WAX in one step within only 30 min. The result was satisfactory with mass recovery of 71.8% and purity of 94.8%, which was further tested by western blotting. The specific binding of the purified rhRnR to recombinant human renin was also determined using surface plasmon resonance (SPR). The association constant of rhRnR to recombinant human renin was calculated to be 3.25 × 108 L/mol, which demonstrated that rhRnR was already renatured and simultaneously purified in one step using WAX. All of the above demonstrate that protein folding liquid chromatography (PFLC) should be a powerful tool for the purification and renaturation of rhRnR. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:864–871, 2014  相似文献   

17.
姚燕  周开亚  宋大祥 《动物学报》2006,52(1):209-214
蜕皮抑制激素(Moltinhibitinghormone,MIH)属于甲壳动物高血糖激素家族神经肽,对甲壳类的蜕皮起抑制作用。本研究用DNA重组技术将中华绒螯蟹(Eriocheirjaponicasinensis)的蜕皮抑制激素1(ErsMIH1)成熟肽的cDNA序列亚克隆至原核表达载体pET28a( )中,并在大肠杆菌BL21(DE3)中进行高效表达。SDSPAGE检测结果显示,融合蛋白pET-MIH1的Mr约为12kD,与理论值相符。融合蛋白的表达量约占菌体总蛋白的15%,表达产物以包涵体形式存在。对包涵体进行变性、复性及纯化处理,并以8mol/L尿素溶解的包涵体作为免疫原免疫BALB/c小鼠制备多克隆抗体。ELISA和Westernblot的结果表明制备的抗体效价高、特异性强  相似文献   

18.
The puromycin-sensitive aminopeptidase was found to be resistant to proteolysis by trypsin, chymotrypsin, and protease V8 but was cleaved into an N-terminal 60-kDa fragment and a C-terminal 33-kDa fragment by proteinase K. The two proteinase K fragments remain associated and retained enzymatic activity. Attempts to express the 60-kDa N-terminal fragment in Escherichia coli produced inclusion bodies. A hexa-histidine fusion protein of the 60-kDa N-terminal fragment was solubilized from inclusion bodies with urea and refolded by removal of the urea through dialysis. The refolded protein was devoid of aminopeptidase activity as assayed with arginine-beta-naphthylamide. However, the refolded protein bound the substrate dynorphin A(1-9) with a stoichiometry of 0.5 mol/mol and a K(0.5) value of 50 microM. Dynorphin A(1-9) binding was competitively inhibited by the substrate dynorphin B(1-9), but not by des-Tyr(1)-leucine-enkephalin, a poor substrate for the enzyme.  相似文献   

19.
Protein folding liquid chromatography (PFLC) is a powerful tool for simultaneous refolding and purification of recombinant proteins in inclusion bodies. Urea gradient size exclusion chromatography (SEC) is a recently developed protein refolding method based on the SEC refolding principle. In the presented work, recombinant human granulocyte colony-stimulating factor (rhG-CSF) expressed in Escheriachia coli (E. coli) in the form of inclusion bodies was refolded with high yields by this method. Denatured/reduced rhG-CSF in 8.0 mol.L(-1) urea was directly injected into a Superdex 75 column, and with the running of the linear urea concentration program, urea concentration in the mobile phase and around the denatured rhG-CSF molecules was decreased linearly, and the denatured rhG-CSF was gradually refolded into its native state. Aggregates were greatly suppressed and rhG-CSF was also partially purified during the refolding process. Effects of the length and the final urea concentration of the urea gradient on the refolding yield of rhG-CSF by using urea gradient SEC were investigated respectively. Compared with dilution refolding and normal SEC with a fixed urea concentration in the mobile phase, urea gradient SEC was more efficient for rhG-CSF refolding--in terms of specific bioactivity and mass recovery, the denatured rhG-CSF could be refolded at a larger loading volume, and the aggregates could be suppressed more efficiently. When 500 microL of solubilized and denatured rhG-CSF in 8.0 mol.L(-1) urea solution with a total protein concentration of 2.3 mg.mL(-1) was loaded onto the SEC column, rhG-CSF with a specific bioactivity of 1.0 x 10(8) IU.mg(-1) was obtained, and the mass recovery was 46.1%.  相似文献   

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