明胶亲和层析介质分离纯化猪血浆纤维结合蛋白

目的：研究猪血浆中纤维结合蛋白的分离纯化方法,得到高纯度、高活性的目标蛋白。方法：以明胶亲和层析介质对猪血浆纤维结合蛋白进行分离纯化,考察了不同洗脱方式对纯化结果的影响,对产品进行了纯度、生物活性的测定。结果：通过对纯化工艺条件的摸索,采用含不同浓度尿素缓冲液进行分步洗脱,可以得到纯度为95％以上的产品,回收率达到50．96％;经BHK细胞培养实验证明其具有与人血浆纤维结合蛋白同样的生物活性。结论：采用了较温和的洗脱方式以明胶亲和层析介质对猪血浆纤维结合蛋白进行分离纯化,提高了动物资源的可利用性。     

Thermal elution chromatography and the resolution of nucleic acids on hydroxylapatite

Thermal elution chromatography of nucleic acids on hydroxylapatite was studied from a technical standpoint. It is shown that current methods for selecting elution buffers are inadequate. The construction of window diagrams for the purpose of determining suitable conditions is demonstrated. The resolving ability of various buffer-hydroxylapatite systems was studied in some detail. The best system for resolving single- from double-stranded nucleic acids was found to be the use of potassium phosphate together with Bio-Rad HTP (non-DNA grade) which has been preheated in phosphate buffer. Sodium phosphate gives the best resolution among various species of double-stranded nucleic acid.     

An ethidium-acrylamide affinity medium for recovery of nucleic acids from free solution and from polyacrylamide and agarose gels

The simple preparation of an ethidium-bromide-based nucleic acid affinity medium is described. The medium is composed of an acrylamide matrix to which ethidium bromide is attached. Its use in preparative purification and fractionation of nucleic acids in solution and in electrophoretic elution of nucleic acids from gels is reported. Nucleic acids can be eluted from this medium with a buffered salt solution and concentrated by ethanol precipitation without persistent contamination with undesirable impurities.     

High Performance DNA Purification using a Novel Ion Exchange Matrix

Ion exchange chromatography has emerged as a reliable alternative to classic CsCl-ethidium bromide gradients for isolating nucleic acids of the highest purity. A plasmid purification method based on a unique anion exchange membrane (IEXM) was developed for the production of superior quality plasmids. This method was simpler and more efficient than conventional bead-based methods. Plasmids were extracted from bacterial cells through alkaline lysis. The crude lysate was clarified by a sequential filtration device that not only removed cell debris but micellar aggregates as well. The clarified lysate was mixed with an extraction solution and loaded into a spin column containing IEXM. Binding, washing, and elution conditions were optimized to achieve efficient isolation of plasmids from the impurities. IEXM had an exceedingly high dynamic binding capacity, excellent selectivity, and a near 100% recovery for plasmids. The binding capacity for pUC19 was 2.93 mg/cm³ of IEXM, which is several times greater than the values for conventional ion exchange beads. The superior selectivity of the method was reflected in the extremely low levels of endotoxin, and thus it is well-suited for critical applications in eukaryotic systems.     

A slow gradient approach for the purification of synthetic polypeptides by reversed phase high performance liquid chromatography

Unquestionably, the purification of polypeptides by chromatographic methods is a considerable bottleneck in their preparation. Peptides synthesised by solid phase synthesis typically contain chromatographically similar impurities that complicate purification by reversed phase high performance liquid chromatography (HPLC) techniques. We report on the application of a slow gradient HPLC protocol that allows, in a single chromatographic step, the purification of hundreds of milligrammes of material. This technique was applied to an extensive collection of synthetic polypeptides some incorporating non‐proteinogenic functionality. In all cases examined, the peptides were not only obtained in high purity peptides but were also recovered in multi‐milligramme amounts. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

A Process for large-scale isolation of beta-galactosidase from E. coli in an aqueous two-phase system

A method suitable for large-scale isolation of beta-galactosidase from a suspension of disintegrated E. coli cells has been developed. In an aqueous two-phase system consiting of PEG 6000 and potassium phosphate, all cell debris and the major part of the proteins and nucleic acids were partitioned to the denser salt phase. Seventy-five percent of the beta-galactosidase was recovered in the lighter PEG phase, giving a purification ratio of about 12.     

Purification of nucleic acids by hybridization to affinity tagged PNA probes

The use of affinity tagged PNA capture probes offers an efficient means for the purification of nucleic acids by hybridization. Two different approaches are described. A sequence specific method and a generic method. The sequence specific method requires sequence information on the target and synthesis of a dedicated PNA. It can be used to selectively purify the nucleic acid containing the target from non-related nucleic acids and other cellular components. The generic method uses a "universal" triplex forming PNA and requires no sequence information on the target. It can be used in the bulk purification of large nucleic acids.     

大孔树脂吸附与反相色谱纯化恩拉霉素

恩拉霉素作为多肽类抗生素,是一种新型、安全的饲料添加剂。本文建立了一条基于大孔树脂初纯和反相色谱精制的分离纯化工艺。该工艺路线首先使用AB-8大孔树脂在0.012 mol/L盐酸溶液-甲醇(50:50,V/V)缓冲液条件下洗脱实现恩拉霉素初步纯化,再使用制备型C18反相色谱柱在0.05 mol/L磷酸二氢钠-乙腈(70:30,V/V)(p H 4.5)缓冲液洗脱下实现恩拉霉素a和b的有效分离,a、b两个组分纯度分别达到98.5%和98.0%,a和b两种有效成分的总收率为29.2%。本研究为恩拉霉素a和b两种纯品的制备以及高纯度恩拉霉素产品的生产提供了参考。     

A new method for the purification of RNA polymerase II (or B) from the lower eukaryote Physarum polycephalum. The presence of subforms

The DNA-dependent RNA polymerase II or B from the lower eukaryote Physarum polycephalum has been purified to apparent homogeneity by a new method employing poly(ethylene imine) precipitation and elution, and heparin-Sepharose affinity chromatography. The method is readily scaled up or down and affords a purification of over 5000-fold with a yield of 35-45%. The procedure is easy to perform and can be carried out in less than three days even on a large scale. Furthermore, it gives enzyme of higher purity and in at least 10-fold greater yield than previously published procedures for its purification from this organism. These improvements have allowed the detection of a series of subforms of the enzyme. The combination of precipitation using poly(ethylene imine) with chromatography on heparin-Sepharose may prove useful in the preparation of other proteins which interact with nucleic acids.     

Composite fluorine polymer-containing sorbents for isolation and purification of biopolymers

Composite fluoropolymer-containing sorbents based on porous silicas were synthesized for the isolation and purification of biopolymers under nondenaturing conditions. Examples of the application of these sorbents in the separation of various mixtures of peptides and proteins and purification of nucleic acids from various sources (plasmid DNA and DNA from nucleated human blood cells) using the cartridge, column, and batch (sorption in a stirred volume) methods are presented. It was shown that the sorbents can be used in laboratory practice because they are selective to nucleic acids (DNA and RNA) and proteins. These materials combine the mechanical properties of the inorganic matrix with the specific sorption properties of the polymer phase and exhibit enhanced stability to alkaline hydrolysis. Alternative methods of preparing sorbents containing polytetrafluoroethylene, polytrifluorostyrene, and polyfluorobutadiene are described. By the example of polyfluorobutadiene-containing sorbents, a completely new method for obtaining fluorinated polymer phases was developed: the polymer phase was preliminarily formed on the surface of porous disperse carriers and was fluorinated with xenon difluoride.     

Single step purification of a series of wheat recombinant proteins with expanded bed absorption chromatography

Expanded bed absorption chromatography (EBA) was used to improve and simplify the purification of several wheat recombinant proteins. Binding and elution conditions were set to allow the purification of the over expressed protein in a single step. In comparison with our previous multi step protocol, same purity was obtained while EBA required less time (one day instead of five) and gave a higher yield (63% instead of 10%). This new procedure was then used for the successful purification of five other wheat ns-LTP. Despite their important polymorphism (identity from 44 to 97 %-pHi from 8 to 10), the EBA protocol allowed their purification in a single step.     

Integrated removal of nucleic acids and recovery of LDH from homogenate of beef heart by affinity precipitation

Lactate dehydrogenase (LDH) was purified from beef heart homogenate by affinity precipitation. The protein purification was integrated with nucleic acid removal and was done by precipitation of nucleic acids by addition of poly(ethylene imine) PEI onto which a ligand, Cibacron blue, had been coupled. The yield of LDH after elution from the precipitate was 63%, the purification factor 6.9 and the nucleic acid content was reduced by 98%. The capacity of the affinity polymer Cibacron blue-PEI is dependent on the nucleic acid concentration in the homogenate. The beef heart homogenate had an unfavourable ratio of nucleic acids to LDH. Precipitation with recirculated Cibacron blue-PEI, already complexed with some nucleic acids, improved the yield of the enzyme to 74%. The loss of Cibacron blue-PEI, when recirculated, was less than 1% after each cycle. This revised version was published online in July 2006 with corrections to the Cover Date.     

Affinity-purification of Transferrin-binding protein B under nondenaturing conditions

The commonly used purification procedures for Transferrin-binding protein B (TbpB) are based on an affinity chromatography step using resins onto which human transferrin had been immobilized. These protocols involve protein elution using denaturing buffer solutions. Here we present an improved protocol which permits protein elution under nondenaturing conditions using chelating agents such as phosphate or compounds containing a pyrophosphate group. Furthermore, isothermal titration calorimetry experiments of the purified protein with holotransferrin have been shown to be a reliable method to assess the purity and activity of the purified material.     

Microscale purification of proteins by line immunoelectrophoresis: Application of the technique in protein biogenesis studies

A small-scale version of line immunoelectrophoresis in combination with immunoprecipitate excision is describeb as a rapid convenient technique to purify proteins on a micro scale in biogenesis studies. In the purification of pig small intestinal microvillar enzymes the method was found to be capable of a quantitative purification and to result in a higher state of purity than an isolation procedure using protein A-Sepharose. Since the method furthermore allows a simultaneous purification of several different protein antigens from the sample, it may be of interest as an alternative method to other procedures in the purification of proteins on a micro scale.     

大孔树脂纯化岩高兰多酚的工艺研究

为获得大孔树脂纯化岩高兰多酚的最佳工艺,以岩高兰的地上部分为原料,通过考察6种不同类型树脂(HPD-100、X-5、AB-8、D101、HPD-600、NKA-II)的含水率、吸附率和解吸率的大小,筛选出一种最适合纯化岩高兰多酚的树脂。在此基础上,选择对纯化工艺影响较大的4种因素(上样浓度、乙醇浓度、洗脱流速、洗脱体积),进行响应面法分析得到最佳工艺。结果表明:HPD-600型大孔树脂对岩高兰多酚的纯化效果最佳,其最优工艺参数为:上样浓度0.84 mg·mL^-1;乙醇浓度62.15%;洗脱流速0.67 mL·min^-1;洗脱体积2.71 BV。该条件下,岩高兰多酚的提取率为229.18 mg·g^-1,岩高兰多酚的纯度由8.11%提高到22.56%,回收率为67.78%。本研究为岩高兰多酚的纯化工艺提供了新的技术路线,也可为岩高兰提取物的研究和应用提供参考。     

阴离子交换晶胶层析分离质粒DNA

质粒DNA(pDNA)作为重要的基因治疗药物载体,其广泛应用受纯度和产量的限制。为了获得高纯度的pDNA,首先制备超大孔连续床晶胶基质,接枝二乙氨基乙基葡聚糖得到阴离子交换型晶胶介质;然后以pUC19质粒为例,将目标质粒转化至大肠杆菌,培养收集,碱液裂解和离心;最后用阴离子交换型晶胶介质从离心上清液中一步法层析分离pDNA。通过优化层析过程的pH值和洗脱条件,最终在pH值为6.6时,用0.5 mol/L的NaCl溶液洗脱,得到较高纯度的pDNA。整个分离过程中不使用动物源性酶,也不需常规分离中的高毒试剂,使获得pDNA的过程和产物更加安全。     

离子交换层析结合反向色谱纯化聚二氨基丙酸

ε-聚赖氨酸生产菌株Streptomyces albulus PD-1可合成一种新型非蛋白质氨基酸均聚物聚二氨基丙酸,采用离子交换层析和反向色谱,对聚二氨基丙酸的分离纯化进行研究。离子交换层析柱选用DEAE-Sepharose Fast Flow填料,50 mmol/L磷酸盐缓冲液(p H 7.5)平衡上样,含0.5 mol/L Na Cl的磷酸盐缓冲液(p H 7.5)洗脱,收集洗脱液用分子筛Sephadex G-25除去磷酸盐缓冲液。然后用C18反相色谱进一步纯化,流动相为V(甲醇)/V(0.1%磷酸)=5/95。经过离子交换层析和反向色谱,纯化得到聚二氨基丙酸纯品,回收率为39.8%,样品纯度达98.4%,为后续的聚二氨基丙酸的深入研究奠定基础。     

重组人促红细胞生成素纯化工艺的优化

采用堆积床生物反应器,用无血清培养基培养分泌重组人促红细胞生成素（rhEPO）的工程细胞株ZK9703.所收集的上清,采用阴离子交换层析－反相层析－分子筛层析三步纯化工艺路线,分别用Q－Sepharose XL-C4-S-200（方法Ⅰ）和DEAE Sepharose FF-Source-S-200(方法Ⅱ）纯化3批产品,所得EPO纯度达98％以上,体外比活性大于1.3^10^5IU/mg。方法Ⅰ、方法Ⅱ纯化过程的EPO体外活性回收率分别为23.56%和28.57%。本纯化方法Ⅱ工艺纯化日程短,分离效果好,EPO体内、体外活性回收率较高,更适合于大规模生产重组人促红细胞生成素。     

Recovery of a monoclonal antibody from hybridoma culture supernatant by affinity precipitation with Eudragit S-100

An IgG₁ monoclonal antibody (MAB) was isolated from hybridoma culture supernatant by affinity precipitation with an Eudragit S-100-based heterobifunctional ligand. Affinity binding was performed in a homogeneous aqueous phase at pH 7.5 followed by precipitation of the bound affinity complex by lowering the pH to 4.8. After two washing steps, elution of specifically bound MAB was achieved by incubating the precipitate with 0.1 M glycine.HCl pH 2.5. The influence of elution volume and time on the recovery of active MAB and the overall purification factor were studied. The best conditions enabled the recovery of 50.2% of active MAB with a purification factor of 6.2. A further dialysis against 50 mM Tris.HCl pH 8.0 increased the activity yield and the purification factor to 68.4% and 8.3, respectively. This result showed that part of the antibody activity loss during affinity precipitation was due to a reversible inactivation process, being easily recovered after a refining dialysis step.     

Statistical optimization for immobilized metal affinity purification of secreted human erythropoietin from Drosophila S2 cells

We used a novel approach to affinity purify human erythropoietin (hEPO) following its secretion from Drosophila melanogaster S2 cells. Immobilized metal affinity purification of hEPO was optimized using a two-step serial statistical optimization strategy. After determining the elution conditions (based on preliminary batch-type purification experiments), the first optimization step considered three purification factors; resin, equilibrium, and washing. The results of this analysis showed that the resin amount was the major factor influencing yield and purity in both model equations and the washing factor lowered the confidence limits of the acquired model equations. The washing conditions were then set based on the results of the first step optimization and the second step then optimized three factors; resin, equilibrium, and elution. The yield and purity of hEPO were then compared following purification using three different approaches; batch-type purification based upon the conditions determined by serial statistical optimization, batch-type purification performed in preliminary experiments, and FPLC column chromatography-type purification. We found that the serial statistical optimization approach provided the best combination of yield and purity. These findings indicate that serial statistical optimization strategies can be successfully employed for immobilized metal affinity protein purification using either batch-type or column approaches.