快速纯化在大肠杆菌中表达的增强型绿色荧光蛋白

As an excellent reporter molecule, enhanced green fluorescent protein （eGFP） was widely used for gene expression and regulation and was generally expressed in Escherichia coli strain. A rapid procedure consisting of ammonium sulfate precipitation, size exclusion chromatography, and anion exchange chromatography was developed for the purification of eGFP. Based on the proposed procedure, recombinant eGFP with an electrophoretic purity was achieved in combination with an overall yield of 66% and a purification factor of 17.9. The fluorescent spectrometry of purified eGFP and lysate from E. coli strain expressing eGFP exhibited the same wavelength of excitation and emission maxima, indicating that the purification procedure did not influence the construct and fluorescent characteristics of desired protein. The procedure mentioned was easy to scale up for the purification of large quantities of eGFP.     

Purification and characterization of recombinant human erythropoietin from milk of transgenic pigs

BACKGROUND: Human erythropoietin (hEPO), a hydrophobic acidic glycoprotein responsible for the regulation of red blood cell production in mammals, is used for the treatment of anemia. In general, the purification of transgenic animal‐derived therapeutic proteins is not easy due to their low titer concentrations and abundant contaminant proteins. For the first time, here the purification and characterization of rhEPO from the milk of transgenic pigs are described. RESULTS: The rhEPO was purified by heparin chromatography, reverse‐phase chromatography, and gel filtration chromatography, resulting in a 16.5% yield and > 98% purity. The rhEPO purified from the milk of transgenic pigs contained less acidic isoforms and was underglycosylated in contrast to CHO‐derived rhEPO. Cell proliferation of the F‐36/EPO‐dependent cell line was proportional to the dose of transgenic pig‐derived rhEPO. CONCLUSION: Transgenic pig‐derived rhEPO with high purity was achieved after three‐step chromatography following two‐step precipitation. The transgenic pig‐derived rhEPO was demonstrated to have comparable potency with CHO‐derived rhEPO. Transgenic pig‐derived rhEPO may not be therapeutically feasible because of different glycosylation, and thus further studies are required to elucidate the effect of this aberrant glycosylation on the biological activity and stability in vivo. Copyright © 2008 Society of Chemical Industry     

抗氯霉素卵黄抗体的制备及其分离纯化

将合成的氯霉素(CAP)与牛血清白蛋白(BSA)的偶联物(CAP-BSA)作为免疫抗原注射到母鸡体内得到氯霉素卵黄抗体(IgY). 比较了不同的提取方法,用优化的辛酸-硫酸铵沉淀法从卵黄液中初步提取卵黄抗体. 选用3,3￠-二氨丙基亚胺(DADPA)作为亲和凝胶与配基氯霉素之间的"间隔臂",合成了分离纯化抗氯霉素特异性IgY的亲和层析柱. 将IgY粗品经过亲和层析柱,得到的特异性IgY的抗体活性提高了10倍,收率约为3.3%.     

Two-step purification strategy for enhanced recovery of recombinant hepatitis B surface antigen from Pichia pastoris

Univariate screening on factors affecting the purification performance of recombinant hepatitis B surface antigen (HBsAg) on ion exchange chromatography (IEC) and size exclusion chromatography (SEC) and the establishment of a two-step purification strategy were performed. Amongst four IEC adsorbents examined, the use of Q Sepharose XL IEC adsorbent under optimized conditions together with optimized SEC purification was able to efficiently purify HBsAg. An established purification strategy comprising the two techniques further demonstrated adaptability for scale-up operations with a final total purification factor (PF) of 94.82 ± 16.20, HBsAg purity of 95.48% and recovery yield of 78.07%.     

肾综合征出血热原代地鼠肾细胞灭活疫苗纯化方法的研究

目的 建立简便、适合大规模肾综合征出血热灭活疫苗的纯化方法。方法 采用超滤浓缩、醋酸锌沉淀和柱层析（亲和层析、离子交换层析和凝胶过滤层析）等方法，对出血热原代地鼠肾细胞灭活疫苗进行提纯对比试验。结果 用超滤浓缩和Sepharose 4FF凝胶柱层析或用醋酸锌沉淀和凝胶柱层析，纯化的疫苗均可达到质量标准，而用 cellufine sulfate gel亲和层析和离子交换层析均不理想。结论 建立了经济、适合大规模肾综合征出血热灭活疫苗的纯化方法。     

液固层析辅助蛋白质从变性态恢复到活性态

层析是化工中分离纯化一些价格昂贵的精细化工产品的常用手段,而液固层析因其温和、快捷、高效等优点已经成为蛋白质纯化过程中必不可少的工具.近年来层析技术的一个拓展是用于辅助蛋白质从变性态恢复到活性态,即所谓的复性,取得了令人瞩目的进展.液固层析辅助蛋白质复性可在高蛋白浓度下操作,适用于大规模生产,同时还能使目标蛋白得到部分纯化.对近年来发展的利用液固层析辅助蛋白质复性的方法做了归纳,包括凝胶过滤层析、离子交换层析、疏水相互作用层析、亲和层析和固定化的折叠催化剂及人工分子伴侣辅助蛋白质复性的方法,并对各自的优缺点和适用范围进行了比较分析.     

葡萄球菌蛋白A的纯化新工艺

目的建立一种高效、简便、实用的分离纯化葡萄球菌蛋白A(SPA)的工艺。方法采用超声波破菌和IgG-Sepharose4B亲和层析分离纯化SPA;以Lowry法检测蛋白含量;双向琼脂免疫扩散法测定效价和特异性;用还原和非还原SDS-PAGE法检测纯度和相对分子质量。结果此法制得的3批SPA的蛋白含量分别为4·7、4·4和5·4mg/ml,收率分别为2·70、2·64和3·78g/100g湿菌。对人IgG的免疫双扩散效价均为1:64;与正常人、豚鼠、家兔和小鼠的血清反应均出现一条沉淀线,而与鸡和羊不出现沉淀线。经非还原SDS-PAGE检测只呈现一条蛋白带,相对分子质量约为160000～180000;经还原SDS-PAGE检测呈现两条蛋白带,相对分子质量分别约为67000和34000。结论已成功建立了分离纯化SPA的新工艺。     

3种不同Protein A亲和层析填料纯化抗CD52单克隆抗体的比较

目的比较3种Protein A亲和层析填料MabSelect SuRe、Protein A Diamond及UniMab 50纯化抗CD52单克隆抗体的载量及纯化效果。方法将浓度为1.24 mg/mL的抗CD52单克隆抗体分别流经3种Protein A亲和层析填料,确保保留时间一致,检测流穿液的抗体浓度,以流穿液中抗体浓度达10%上样浓度时的上样量确定填料的最大动态载量。3种填料均按80%最大载量上样,纯化3批抗CD52单克隆抗体培养上清样品,比较洗脱体积、抗体回收率及洗脱收集液中抗体浓度、抗体纯度、电荷异质性、宿主蛋白残余量、宿主DNA残余量、填料配基Protein A脱落量。结果填料MabSelect SuRe与Protein A Diamond在载量、宿主蛋白残余量、填料配基Protein A脱落量方面接近;在抗体纯度、电荷异质性、宿主DNA残余量方面,3种填料表现相当;在抗体回收率、洗脱体积及抗体浓度方面,UniMab 50表现最佳。结论 3种填料亲和纯化抗CD52单克隆抗体的效果各有优劣,但均可满足该纯化步骤的要求,可根据实际工艺状况及质控要求进行选择。     

人免疫球蛋白G酶解制备Fab和Fc片段及其分离纯化

为了制备抗体Fab和Fc片段,采用木瓜蛋白酶酶解人免疫球蛋白G(IgG),通过优化酶解条件和色谱法分离过程,得到了纯度较高的Fab片段和Fc片段。考察了酶解pH、酶加入量、添加半胱氨酸和酶解时间等对IgG酶解过程的影响,优化了酶解条件,提高酶解效率,IgG转化率大于98%。酶解产物通过Protein A亲和色谱法和DEAE阴离子交换色谱法进行了纯化,分离得到Fc片段和Fab片段,收率分别为72.6%和40.1%。经SEC-HPLC分析,Fc片段纯度达95.7%,Fab片段纯度达96.6%。     

亲和色谱技术研究进展

亲和色谱具有高选择、高活性回收率和高纯度等特点，已成为生物工程中分离纯化最有效的技术之一，是生物化工研究的重要方向。综述了亲和色谱技术及其发展趋势。     

用盐析与疏水层析相偶合快速分离提纯猪胰激肽释放酶

将疏水层析技术用于从猪胰脏中分离纯化激肽释放酶,建立了一种简便、快速的分离提纯方法：将粗品溶解后经过硫酸铵沉淀处理,然后经过Butyl Sepharose FF疏水层析后得到目标蛋白,分析其纯度大于500U／mg,盐析和疏水两步纯化的收率大于85．0％,同时比较了Phenyl Sepharose FF,Octyl Sepharose FF和Butyl Sepharose FF三种疏水介质分离纯化胰K的效果,本实验工艺与传统工艺相比,具有操作简单、快速、回收率和纯化倍数高等优点,有望成为一种从动物组织中快速分离纯化药用蛋白质的有效技术平台。     

自制填充介质对人血浆凝血因子Ⅸ的层析分离

目的采用自制的DEAE Bio-SepFF和肝素Bio-Sep FF介质,从人血浆中快速分离纯化凝血因子Ⅸ(FⅨ)。方法低温离心去除冷沉淀后的人血浆,在不同淋洗条件下,经过两步弱阴离子交换和一步亲和层析分离纯化FⅨ,用活性检测试剂盒检测各组分凝血因子的活性,Bradford法测定蛋白浓度,聚丙烯酰胺凝胶电泳分析样品的纯度。结果经过三步层析分离,得到的FⅨ比活达到99.40IU/mg,纯化倍数为3823倍,回收率约为30%,纯度较高。结论采用该方法和介质,可从人血浆中纯化FⅨ,且效果较好。     

Application of peptide chromatography for the isolation of antibodies from bovine skim milk,acid whey and colostrum

Protein A mimetic peptide ligands have several benefits over conventional Protein A/G ligands, namely that they are small in size, have low production costs, are stable over a wide range of pH values and can withstand cleaning by harsh sanitization agents such as sodium hydroxide. In this paper, a hexamer peptide (HWRGWV) affinity matrix was used for the isolation of bovine immunoglobulins from various dairy streams (skim milk, acid whey and colostrum). Bound immunoglobulins were recovered in elution buffer (0.2 M sodium acetate buffer, pH 4.0) fractions with a purity of >85% in a single step. The peptide resin has achieved a maximum equilibrium adsorption capacity of 23 ± 0.58 mg mL⁻¹ of resin for bovine IgG and had a dynamic binding capacity of 11.8 ± 0.03 mg mL⁻¹ at residence time of 2 min. These results suggest that the hexamer peptide chromatography could potentially be used for the selective purification of bovine immunoglobulins from dairy streams. This method has promise as an alternative to conventional Protein A/G chromatography for direct capture of immunoglobulins from streams containing relatively high immunoglobulin concentrations such as colostrum, transgenic or hyper-immune milk.     

金属螯合亲和层析介质及应用研究的进展

金属螯合亲和层析具有螯合介质制备简单，吸附容量大，选择性及通用性较好，易于再生，成本低等优点，是分离纯化蛋白质等生物工程产品最有效的技术之一。本文综述了金属螯合亲和层析介质及最新应用方面的进展。     

Directly resistively heated-column gas chromatography for the evaluation of cow milk fat purity

Cow milk fat purity is currently evaluated by triacylglycerol (TAG) analysis. The method is based on the gas chromatographic analysis of TAG according to their total number of carbon atoms, followed by the application of formulae deriving from multiple linear regressions. The original procedure was set up by using a packed column, but different successive works showed its suitability also with the adoption of more modern adjustments. Recently, the use of high-speed gas chromatography has been spreading. In this work, the suitability of the directly resistively heated-column gas chromatography [Ultrafast module (UFM)-GC] for the evaluation of milk fat purity by the Official EU method was tested. Pure milk fat, together with mixtures of milk fat containing two levels of four foreign fats (coconut fat, sunflower oil, lard and beef tallow), were analyzed by both the conventional capillary GC method and UFM-GC. Sheep, goat and buffalo milk fats were also analyzed. The reference material CRM-519 was used for calibration. Repeatability and reproducibility values were always below the limits reported in the Official method, demonstrating the suitability of UFM-GC for the evaluation of milk fat purity. The investigation on sheep, goat and buffalo milk fat confirmed that the S ranges, calculated for cow milk fat, are not applicable to the evaluation of genuineness of non-bovine milk fats.     

PEG沉淀结合层析分离重组乙肝病毒表面抗原

应用聚乙二醇(PEG)沉淀结合层析技术纯化乙肝病毒表面抗原(HBsAg),对PEG浓度、pH值、离子强度等影响PEG沉淀的因素进行了正交实验研究. 结果表明,浓度为0.12 g/mL的PEG6000在4℃, pH 9.0条件下沉淀HBsAg纯化效果比较理想,纯化倍数达3.7,回收率96.8%,有效地去除了生物大分子杂质和牛血清白蛋白(BSA),PEG在后续的凝胶过滤中可以除去,整个工艺的回收率可提高到41%.     

Purification and Characterization of Iso-Ribonucleases from a Novel Thermophilic Fungus

A thermophilic fungus previously isolated from composted horse manure was found to produce extracellular iso-RNases that were purified 127.6-fold using a combination of size exclusion chromatography and a novel affinity membrane purification system. The extent of purification was determined electrophoretically using 4%–15% gradient polyacrylamide gels. RNase activity was dependent on the presence of a metal co-factor with significantly more activity with Zn²⁺ or Mn²⁺ than Mg²⁺. The RNases exhibited maximum activity at both pH 3.0 and pH 7.0 with no activity at pH 2.0 or 10.0. The optimal temperature for the iso-RNase was 70 °C. The molecular weight of the iso-RNase was determined to be 69 kDa using a Sephadex G-75 column.     

沉析法与粉碎法制备高纯度聚苯砜的性能对比

针对聚苯砜(PPSU)树脂生产流程中纯化困难的特点,设计出一种沉析纯化方法,即PPSU在聚合完成后以粉末态析出,再经过纯化过程得到高纯度的PPSU树脂。对沉析纯化法和传统粉碎纯化法制备的两种PPSU树脂进行了灰分含量、特性黏度、粒径、热稳定性等系列对比测试,同时比较了两种PPSU薄膜的透光率、雾度和拉伸性能。结果表明,沉析法大幅提高了PPSU的纯化效果,其制备的PPSU粉体粒径更小,产物灰分含量更低;沉析法有利于保证PPSU树脂分子量和分子链结构的完整,其特性黏度比粉碎法PPSU提高了34.5%;由于PPSU树脂分子结构和组成未变,两种方法制备的PPSU树脂热稳定性基本一致;沉析法制备的PPSU薄膜的透光率更高、雾度更低,拉伸性能更好。     

柱层析法纯化基因工程HBsAg中试工艺的研究

由乙肝病毒S基因转化的哺乳动物细胞培养收液，经过Butyl-S-SepharoseFF层析，DEAESepharoseFF层析和Sepharose4FF层析，可获HBsAg纯品。产品检定结果表明，PAGE和SDS-PAGE电泳纯度均合格，小牛血清残余量、细胞DNA残余量和动物免疫效力也均符合规定标准．HBsAg终收率达40％左右．在此工艺中，疏水作用柱层析的纯化效率比较高，可去除绝大部分杂蛋白和细胞DNA。     

亲合层析法纯化白细胞介素11

白细胞介素 1 1 (IL - 1 1 )是一种具有调节功能的蛋白质。一般采用含有IL - 1 1基因的硫氧还蛋白系统在重组大肠杆菌中来表达制备IL - 1 1融合蛋白 ,产物经金属鏊合层析进行初次分离。本文对融合蛋白IL - 1 1进行亲合纯化工艺研究 ,结果表明 ,IL - 1 1的纯度和回收率均达到较高的水平 ,为后续的纯化奠定了基础