【作者】 丁兆阳；

【导师】 曹学君；

【作者基本信息】 华东理工大学 ， 生物化工， 2013， 博士

【摘要】 蛋白质的大规模分离纯化一直是一项非常有挑战性的工作,制约很多蛋白质、酶制剂的大规模应用的关键环节就是分离纯化。亲和沉淀技术是目前分离纯化蛋白质的研究热点之一,其有着环境友好,分离效率高等优势,适合大规模分离应用。亲和沉淀研究的重点之一是溶解可逆聚合物的合成,本文分别合成了pH响应溶解可逆聚合物和热响应溶解可逆聚合物。其中使用甲基丙烯酸、甲基丙烯酸二甲氨基乙酯、甲基丙烯酸丁酯和N-羟甲基丙烯酰胺合成了pH响应溶解可逆聚合物PMMDN,通过红外技术分析其结构,通过测量Zeta电位得知该聚合物等电点为4.51,通过凝胶色谱技术测得其分子量为6.78×104,多次回收率均高于96.0%以上。使用N-异丙基丙烯酰胺、丙烯酸丁酯和N-羟甲基丙烯酰胺为反应单体无规聚合制备热响应聚合物PNBN。通过分光光度计法测得该聚合物的最低临界温度为30.8℃,测得其分子量为3.17×104,在添加0.5mol/LNaCl的情况下,其回收率高达99.8%,基本达到完全回收。其次分别使用两种聚合物连接不同亲和配基,使用PMMDNn连接Cibacron Blue F3GA配基(PMMDNn-CB)、金属离子配基(PMMDN-IDA-Cu2+)和L-甲状腺素配基(PMMDN-T)；使用PNBN连接L-甲状腺素配基(PNBN-T)。对pH响应聚合物及连接配基的亲和聚合物测量了等电点,分别为4.45、4.35和4.65,各自多次回收率均高于96.0%以上。PNBN-T的最低临界温度为32.8℃,在有NaCl存在的情况下,五次回收率约99.0%。随后将得到的四种不同亲和沉淀载体分别应用到纤维素酶、内切葡聚糖酶、溶菌酶、人血清白蛋白和谷氨酰胺转氨酶的纯化中。同时使用分子模拟技术、红外光谱、圆二色谱、生物大分子相互作用仪(Octet)、SDS-PAGE电泳等来进行研究。使用PMMDN-CB分离纯化纤维素酶,发现在pH7.2,添加NaCl浓度为1.0mol/L的条件下,在30℃下吸附两个小时达到平衡。通过酶活以及蛋白含量的测量确定了含有20.0%乙醇的pH7.1的Tris-HCl缓冲液作为洗脱液可以得到最合适的洗脱效率,总活力洗脱率约84.5%,内切酶活洗脱率高达99.0%,通过SDS-PAGE鉴定也达到电泳纯。首先使用分子模拟手段,使用DOCK软件计算辅助确定了金属螫合剂的种类和金属离子配基的种类,亚氨基二乙酸作为金属蝥合剂在稳定性和活性上都最合适,金属铜离子在结合蛋白质上最稳定,实验也验证了模拟结果。使用PMMDN-IDA-Cu2+亲和沉淀内切葡聚糖酶,在pH5.0,添加NaCl浓度为1.0mol/L的条件下,在30℃下混合吸附两个小时达到平衡；含有0.5mol/L咪唑与1.0mol/L盐酸胍作为洗脱液可以得到最佳的洗脱效率,蛋白检测和内切酶酶活洗脱率分别高达98.5%和99.3%。使用PMMDN-T作为亲和沉淀剂从咸鸭蛋清中提取溶菌酶。在pH5.5,添加NaCl以增加离子强度,配基密度为60.0μmol/g的条件下,在25℃下振荡吸附两个小时,通过吸附等温线得到该聚合物对溶菌酶的最大吸附容量和解离常数分别22.8mg/g和0.085mg/g,使用的Octet进行分析也印证了这一结果的正确性,圆二色谱证明了L-甲状腺素对溶菌酶有亲和作用力,影响了其二级结构的变化,得出pH7.0磷酸盐缓冲液复合0.5mol/LUrea可以作为洗脱液得到较高效率的溶菌酶,蛋白和酶活洗脱率分别高达85.0%和95.4%。对于热响应聚合物,使用PNBN-T从人血清中亲和沉淀提取人血清白蛋白,在pH7.0,不添加NaCl以增加离子强度,配基密度为60.0μmol/g的条件下,在25℃下振荡吸附两个小时,吸附达到平衡,最大吸附容量和解离常数分别14.87mg/g和0.11lmg/g,与Octet的实验结果吻合。圆二色谱证明了人血清白蛋白因L-甲状腺素的影响而二级结构发生了变化；1.0mol/L的NaSCN可以作为洗脱液,洗脱率高达93.8%,对亲和沉淀的结果使用SDS-PAGE分析,证明PNBN-T可以分离出电泳纯的HSA。使用PNBN-T通过亲和沉淀技术从TG发酵液中提取谷氨酰胺转氨酶(TG),最佳吸附条件为pH5.0,配基密度为59.50μmol/g,在15℃下振荡吸附两个小时达到最大吸附效率。通过吸附等温线得到该聚合物对TG的最大吸附容量和解离常数分别169.4mg/g和1.35mg/g,Octet实验基本验证了这一结果的正确性。使用圆二色谱证明L-甲状腺素使TG二级结构中螺旋和折叠发生变化,证明了其对TG的亲和作用。实验得到的最佳洗脱条件为pH10.0Gly-NaOH,蛋白洗脱率和酶活洗脱率分别高达99.01%和95.85%,使用SDS-PAGE分析最终结果,得到电泳纯的TG。更多还原

【Abstract】 Protein purification on a large scale is still a challenge nowadays.Affinity precipitation was reported as a potential technique for the purification of proteins at early stage of downstream processing. The technique could be achieved by using reversibly soluble-insoluble polymers coupled with ligand as an affinity carrier to purify proteins from large volume of dilute solution material. The key of affinity precipitation is the syntheisi of the response polymers. In this study, pH-response and thermo-response polymers were synthesized. Methyl acrylate, methacrylate, dimethylaminoethyl methacrylate, butyl methacrylate and N-methylol acrylamide were used to synthesize the pH-response polymer PMMDN, IR was used to analyze its structure. The pI of PMMDN was4.51obtained by measuring the Zeta potential. The molecular weight was6.78×104, whick was tested by gel chromatography technique. The multiple recoveries were higher than96.0%. N-isopropyl acrylamide, butyl acrylate and N-methylol acrylamide were used as monomers to synthize the thermo-response polymer PNBN by random polymerization. The lowest critical temperature of the polymer was30.8℃measured by the spectrophotometer. Its molecular weight was3.17×104, and the recovery of PNBN was99.8%with adding0.5mol/LNaCl.There are four different ligands were connect to two polymers. PMMDN was connected Cibacron Blue F3GA(PMMDN-CB), metal ions(PMMDN-IDA-Cu2+) and L-thyroxine ligand (PMMDN-T); PNBN was connected to the L-thyroxine(PNBN-T).The pI of these different ligand to the pH-response polymer were4.45,4.35and4.65, respectively. The recoveries of these polymers were all higher than96.0%. The LCST of PNBN-T was32.8℃,five times recoveries of PNBN-T were99.0%in the presence of NaCl.Subsequently, the obtained four different affinity precipitation polymers were applied to purify cellulase, endoglucanase. lysozyme, human serum albumin and glutamine transaminase. Molecular simulation technology, infrared spectroscopy, circular dichroism, biological macromolecules interaction instrument (Octet), SDS-PAGE electrophoresis were all used in this study. Cellulase was purified by PMMDN-CB. Optimal adsorptions could be achieved at pH7.2and1.0mol/1NaCl. FTIR confirmed that ligand CB was immobilized on the polymer and cellulase was adsorbed on PMMDN-CB. The optimal eluant was0.1mol/1pH7.1Tris-HCl with20.0%glycol. The elution recovery of total cellulase activity and endo-glucanase activity was almost84.4%and99.8%, respectively. The SDS-PAGE showed the main bands with a molecular weight corresponding to that of the native cellulase. Molecular docking by DOCK software was used to calculate and help to determine the species of the metal chelating agent and a metal ion ligand. Iminodiacetic acid was chose as metal chelating agent for its stability and activity, copper ions was decided because it could be combined with protein stably.All experiments datas tesefy the simulation results. Use PMMDN-IDA-Cu2+to separation endoglucanase in the conditon of pH5.0,1.0mol/L NaCl concentration and30℃in two hours to reach equilibrium; the eluant was0.5mol/L imidazole with l.Omol/L guanidine hydrochloride, and the elution ratio of protein and endonuclease activity were up to98.5%and99.3%, respectively. Use PMMDN-T as affinity polymer to get lysozyme from salted duck eggs. The optimal adsorption condition was pH5.5with some NaCl to increase ionic strength, ligand density was60.0μmol/g. The maximum adsorption capacity of lysozyme and the dissociation constant of the polymer obtained by adsorption isotherm22.8mg/g and0.085mg/g, the Octet analysis also confirms the correctness of the results. Circular dichroism proved that L-thyroxine had affinity force to lysozyme. pH7.0PB with0.5mol/LUrea was used as the eluent to obtain higher efficiency, the elution ratio of protein and enzyme activity up to85.0%and95.4%, respectively. L-thyroxin which was first used as affinity ligand was immobilized on the PNBN for affinity precipitation of HSA. The optimal adsorption condition was0.02M Tris-HCl buffer (pH7.0) and the adsorption capacity of HSA on the polymer was14.9mg/g polymer in affinity precipitation process. Circular Dichroism spectra and ForteBio Octet system were used to analyze the interactions between the affinity polymer and HSA during adsorption and desorption. The elution recovery of total HSA was93.6%by1.0mol/L NaSCN. When the affinity polymer was applied in the purification of HSA from human serum, the SDS-PAGE showed that a purified HSA single band was obtained.PNBN-T was used to extract pure glutamine transaminase (TG) from the fermentation broth, the optimum adsorption conditions was pH5.0, ligand density for59.5.0μmol/g, shaken at15°C for two hours to achieve maximum efficiency. The maximum adsorption capacity and dissociation constant were169.4mg/g and1.35mg/g, respectively. The Octet experimental verified the correctness of the results. Circular dichroism proved that L-thyroxine make the secondary structure of TG change by the affinity force between. The optimum elution condition was pH10.0Gly-NaOH, and the elution ratio of protein amount and activity were up to99.01%and95.85%, respectively. The SDS-PAGE was used to analyze the final results, and the electrophoretically pure TG was obtaind.更多还原