Scale-up of free-flow electrophoresis: II. Purification of alcohol dehydrogenase from a crude yeast extract by field step electrophoresis and combined field step-zone electrophoresis

Results of the purification of alcohol dehydrogenase (ADH) by field step electrophoresis and combined field step-zone electrophoresis are presented. In field step electrophoresis, optimization of voltage, residence time and pH of the sample solution led to a maximal purification factor of 2.8 and a yield of 89% ADH. The limit of loading capacity was reached at a protein concentration of the sample solution of approximately 4 g/L, allowing a maximal throughput of 1.14 g/h with a yield of 86% and a 2.8-fold purification in the Elphor VaP 22 apparatus. With a production scale apparatus a throughput of 2.07 g/h without any loss of separation quality could be achieved. By introducing the sample solution into the separation chamber through 3 inlets, simultaneously, the throughput was increased to 3.2 g/h with a purification factor of 2.7 and a yield of 82% ADH. For the combined field step-zone electrophoresis method a maximum purification factor of 3.6 and a yield of 80% ADH were achieved. The loading capacity was limited to a 4.13 g/L protein concentration of the sample solution, resulting in a throughput of 440 mg/h. Injecting the sample solution simultaneously into 3 inlets resulted in a maximum throughput of 1.92 g/h with 3.1-fold purification and a yield of 80% ADH. Zone electrophoresis, field step electrophoresis and a combination of both are compared with respect to resolution, throughput and the application potential in a protein purification scheme. A scale-up to 3 g/h is possible in zone electrophoresis and field step electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Scale-up of free flow electrophoresis: I. Purification of alcohol dehydrogenase from a crude yeast extract by zone electrophoresis

The potential and limitations in scaling-up free-flow electrophoresis, with emphasis on zone electrophoresis, are demonstrated. Purification of alcohol dehydrogenase (ADH) from a crude yeast extract was chosen as a model for an industrial approach to enzyme purification. In zone electrophoresis the separation quality strongly depends on the pH and conductivity of the background electrolyte, its residence time and flow rate, as well as the applied voltage. Optimization of these parameters resulted in a purification factor of 5.3 and a yield of 96% ADH, using a Tris/HCl buffer, pH 8.0, and a conductivity of 1 mS/cm, with a residence time of 10 min at 500 V. The loading capacity of the method for a laboratory-sized free-flow electrophoresis apparatus was limited to a sample throughput of about 0.4 g/h. By increasing the chamber dimensions it was possible to purify the enzyme by a purification factor of 4.7 and a yield of 93% ADH, at a throughput of about 1 g total protein/h. By simultaneously applying the sample at 3 input positions the throughput could be increased to 2.75 g/h with a purification factor of 4.7 and an overall yield of 90%.     

Separation of enzymes from Candida boidinii crude extract by continuous flow zone electrophoresis.

Separation of the enzymes formate dehydrogenase, formaldehyde dehydrogenase and methanol oxidase from Candida boidinii crude extract has been explored using continuous flow zone electrophoresis in the VaP-22 and the scaled-up VaP-220 electrophoresis apparatus. Yields up to 95% and purification factors between 3 and 7 were obtained, together with separation of cell debris from the enzymes. Multiple injections of sample were used to obtain a protein throughput of 46.2 mg/h in the VaP-22. A tenfold higher throughput was achieved using the VaP-220. Correlation of the electrophoretic mobility in continuous flow zone electrophoresis with the elution behavior in ion-exchange chromatography confirmed the primary role of net surface charge in the separation of biological molecules. Proteins and enzymes with differences greater than 0.05 M elution molarities in ion-exchange chromatography can be separated. This corresponds to a preparative scale (mg/h or g/h) separation of proteins and enzymes whose difference in apparent electrophoretic mobility is greater than 0.70 x 10(-5) cm2/(V.s).     

Purification of low-abundance lysozyme in egg white via free-flow electrophoresis with gel-filtration chromatography

As an effective separation tool, free-flow electrophoresis has not been used for purification of low-abundance protein in complex sample matrix. Herein, lysozyme in complex egg white matrix was chosen as the model protein for demonstrating the purification of low-content peptide via an FFE coupled with gel fitration chromatography (GFC). The crude lysozyme in egg while was first separated via free-flow zone electrophoresis (FFZE). After that, the fractions with lysozyme activity were condensed via lyophilization. Thereafter, the condensed fractions were further purified via a GFC of Sephadex G50. In all of the experiments, a special poly(acrylamide- co-acrylic acid) (P(AM-co-AA)) gel electrophoresis and a mass spectrometry were used for identification of lysozyme. The conditions of FFZE were optimized as follows: 130 μL/min sample flow rate, 4.9 mL/min background buffer of 20 mM pH 5.5 Tris-Acetic acid, 350 V, and 14 °C as well as 2 mg/mL protein content of crude sample. It was found that the purified lysozyme had the purity of 80% and high activity as compared with its crude sample with only 1.4% content and undetectable activity. The recoveries in the first and second separative steps were 65% and 82%, respectively, and the total recovery was about 53.3%. The reasons of low recovery might be induced by diffusion of lysozyme out off P(AM-co-AA) gel and co-removing of high-abundance egg ovalbumin. All these results indicated FFE could be used as alternative tool for purification of target solute with low abundance.     

Free-flow electrophoresis for the purification of proteins: II. Isoelectric focusing and field step electrophoresis

Two modes of continuous isoelectric focusing are described. The development of a natural pH gradient, consisting of a mixture of three buffer solutions, and the focusing behavior of human serum albumin is investigated. The advantages of isoelectric focusing in an artificial pH gradient of three buffer solutions are demonstrated on the purification of alpha-amylase from an E. coli protein extract. Furthermore the principle of field step electrophoresis is presented. The most important factors influencing the efficiency: (i) residence time, (ii) conductivity of the sample and (iii) sample zone width, are discussed. The use of a larger sized device to allow simultaneous multiple injections of the sample demonstrates the feasibility of scaling-up field step electrophoresis. This approach permits a throughput of about 20 mL sample solution per minute.     

Free flow electrophoresis for the purification of proteins: I. Zone electrophoresis and isotachophoresis

The principles and some applications of free flow zone electrophoresis and isotachophoresis are described. The influence of (i) carrier electrolyte conductivity on the migration velocity and (ii) band shape on zone electrophoresis was investigated. The technique was found convenient for studying the effect of pH on the mobility of proteins to create a mobility curve. The purification of alcohol dehydrogenase from a crude yeast extract revealed the separation power of zone electrophoresis for complex protein mixtures. Without additional steps, a purification factor of 5.4, with a recovery of 97% alcohol dehydrogenase, was achieved. Free flow isotachophoresis was applied to the purification of immunoglobulins from human serum. Disadvantages of this technique are the time-consuming development of an optimized separation system and the empirical search for suitable spacers. Also, reaching of the steady state becomes increasingly difficult as the number of sample components increases.     

高速逆流色谱在分离纯化木蝴蝶活性成分中的线性放大

利用高速逆流色谱分离纯化中草药木蝴蝶乙酸乙酯粗提物中的黄酮类活性成分,并将分离规模从分析型线性放大到制备型,以获得大量的活性成分,为进一步的药物筛选提供物质基础。实验在分析型高速逆流色谱上对分离参数进行了系统优化,并将优化条件放大到制备型高速逆流色谱上对911.6 mg木蝴蝶乙酸乙酯粗提物进行分离,得到5种化合物,经高效液相色谱、电喷雾电离质谱和核磁共振氢谱、碳谱分析鉴定,分别为白杨素(160.9 mg,纯度为97.3%)、黄芩素(130.4 mg,纯度为97.6%)、黄芩素-7-O-葡萄糖苷(314.0 mg,纯度为98.3%)、黄芩素-7-O-双葡萄糖苷(179.1 mg,纯度为99.2%)和一种新的白杨素双葡萄糖苷(21.7 mg,纯度为98.8%)。该放大过程不仅将处理量提高了53倍,还保持了在分析型设备上的分离度和分离时间。该工作为天然产物的研究提供了一个高效的分离纯化方法。     

Separation and purification of furanocoumarins from Toddalia asiatica (L.) Lam. using microwave-assisted extraction coupled with high-speed counter-current chromatography

A method of microwave-assisted extraction coupled with high-speed counter-current chromatography was established for separation and purification of isopimpinellin, pimpinellin and phellopterin from Toddalia asiatica (L.) Lam. The conditions of MAE including the extraction solvent, size of sample, solid/liquid ratio, extraction temperature and extraction time were optimized by a mono-factor test. That is, 2.0 g dried powder of T. asiatica (L.) Lam of 0.30-0.15 mm size was extracted with 20 mL (solid/liquid ratio of 1:10, g/mL) methanol under 50 °C for 1 min. The crude extract was separated and purified by high-speed counter-current chromatography with hexane-ethyl acetate-methanol-water (5:5:5.5:4.5, v/v/v/v) solvent system. 0.85 mg/g of isopimpinellin, 2.55 mg/g of pimpinellin and 0.95 mg/g of phellopterin were obtained from original sample in one-step within 240 min, the purity determined by high performance liquid chromatography was 95.0%, 99.1% and 96.4%, respectively. Their chemical structures were further identified by mass spectroscopy and nuclear magnetic resonance spectroscopy. The results demonstrated that microwave-assisted extraction coupled with high-speed counter-current chromatography was a feasible, economical and efficient technique for rapid extraction, separation and purification of effective compounds from natural products.     

Purification of low‐concentration phenazine‐1‐carboxylic acid from fermentation broth of Pseudomonas sp. M18 via free flow electrophoresis with gratis gravity

The low‐concentration phenazine‐1‐carboxylic acid (PCA) (=0.3 mM) extracted from fermentation broth of Pseudomonas sp. M18 was selected to be purified with a newly facile free flow electrophoresis (FFE) device with gratis gravity. Three factors of pH value and concentration of background buffer, and the cooling circle of FFE device were investigated for the purification of PCA in the FFE device. It was found that the pH value and concentration of background buffer had mild influences on the separation of PCA whether with cooling circle or not. However, the cooling circle had a much greater impact on the separation of PCA. The controlling of the band zone of PCA in FFE chamber would be difficult if without cooling circle, while the controlling would become easy if with cooling circle. Under the optimal conditions (10 mM pH 5.5 phosphate as background buffer, 30 mM pH 5.5 phosphate buffer as electrode solution, 5.46 mL/min background flux, 10 min residence time of injected sample, and 500 V), PCA could be continuously prepared from its impurities with relative high purity. The flux of sample injection was 115 μL/min, viz. 7 mL sample throughput per hour, and the recovery was up to 85%. All of the experiments indicated that the FFE technique was a good alternative tool for the study on natural biological control agents.     

Chiral capillary electrophoresis as predictor for separation of drug enantiomers in continuous flow zone electrophoresis

Separation of the enantiomers of chlorpheniramine and methadone in acidic buffers containing carboxymethyl-betacyclodextrin (CMCD) as chiral selector was investigated by capillary zone electrophoresis. For a range of pH and CMCD concentrations, the mobility difference and resolution of the enantiomers were determined. Then, conditions known to provide well resolved enantiomers and optimized chiral separation were applied to chiral continuous flow electrophoresis. In that approach, a thin film of fluid flowing between two parallel plates is employed as carrier for electrophoresis. The electrolytes and the sample are continuously admitted at one end of the electrophoresis chamber and are fractionated by an array of outlet tubes at the other. The number of pure enantiomeric fractions obtained by chiral continuous flow electrophoresis was found to be directly dependent on the enantiomeric mobility difference. For racemic chlorpheniramine separated in a betaine-acetic acid buffer at a total throughput of 5 mg/h, complete enantiomeric separation is shown to require a mobility difference of about 3 x 10(-9) m2/V s. Furthermore, compared to the previous investigations with hydroxypropyl-beta-cyclodextrin, CMCD was found to permit improved fractionation of methadone enantiomers. With a total racemic drug throughput of about 15 mg/h, continuous flow zone electrophoresis processing with CMCD as chiral selector is shown to have the potential of providing pure enantiomers on a mg/h scale. The results indicate that chiral capillary zone electrophoresis data can be employed as predictor for preparative scale chiral separations based upon continuous flow zone electrophoresis.     

猪肝中单胺氧化酶B的分离纯化

依据单胺氧化酶B(monoamine oxidase B, MAOB)的疏水特性,建立了一种从猪肝中分离纯化MAOB的新方法。用含有1% Triton X-100的膜蛋白裂解液制备粗酶,以饱和度为20%～50%的硫酸铵反抽提进行粗提,再利用自制的配基密度为75.7 μmol/mL的苯基疏水色谱及Sepharose Q High Performance离子交换色谱进一步分离纯化,得到纯化倍数为18.2、酶比活为135 U/mg的MAOB。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析显示为相对分子质量约60 000的单一蛋白质带。采用高效液相色谱-电喷雾串联质谱对该酶进行鉴定,证实为MAOB。本研究所用分离纯化方法可以有效纯化MAOB, 为MAOB的深入研究提供技术支撑。     

Ultra‐high‐pressure‐assisted extraction of wedelolactone and isodemethylwedelolactone from Ecliptae Herba and purification by high‐speed counter‐current chromatography

Ultra‐high‐pressure extraction combined with high‐speed counter‐current chromatography was employed to extract and purify wedelolactone and isodemethylwedelolactone from Ecliptae Herba. The operating conditions of ultra‐high‐pressure extraction were optimized using an orthogonal experimental design. The optimal conditions were 80% aqueous methanol solvent, 200 MPa pressure, 3 min extraction time and 1:20 (g/mL) solid–liquid ratio for extraction of wedelolactone and isodemethylwedelolactone. After extraction by ultra‐high pressure, the extraction solution was concentrated and subsequently extracted with ethyl acetate; a total of 2.1 g of crude sample was obtained from 100 g of Ecliptae Herba. A two‐phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (3:7:5:5, v/v) was used for high‐speed counter‐current chromatography separation, by which 23.5 mg wedelolactone, 6.8 mg isodemethylwedelolactone and 5.5 mg luteolin with purities >95% were purified from 300 mg crude sample in a one‐step separation. This research demonstrated that ultra‐high‐pressure extraction combined with high‐speed counter‐current chromatography was an efficient technique for the extraction and purification of coumestans from plant material.     

Improved separation of integral membrane proteins by continuous elution electrophoresis with simultaneous detergent exchange: application to the purification of the fusion protein of the human immunodeficiency virus type 1

We describe a protocol for preparative-scale purification of the fusion protein of the human immunodeficiency virus type 1 (HIV-1), gp41, from cells overexpressing the viral envelope proteins and from HIV-1 isolates. In the first step, the proteins were extracted from the membrane in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The extract was then subjected to separation by continuous elution electrophoresis using a nonionic or zwitterionic detergent in the mobile elution buffer, which results in the simultaneous exchange of SDS with that detergent. The separated proteins were obtained in an SDS-free buffer containing either Brij, 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or Triton X-100 and could then be subjected to subsequent purification steps like isoelectric focusing in the second dimension or immunoaffinity chromatography. The dilute protein fraction was concentrated and applied on a 10 mL immunoaffinity column packed with anti-gp41 monoclonal antibody immobilized on protein-G sepharose. The protein was eluted from the column at pH 2.7 and obtained in pure form in amounts of 30-50 micrograms that constituted a yield of 1%. The pure gp41 could not be sustained in solution in the absence of detergent and was not susceptible to proteolytic digestion by trypsin. The identification of the protein and the degree of purity was confirmed indirectly using surface enhanced laser desorption ionization-time of flight-mass spectrometry (SELDI-TOF-MS). The possible application of this approach for the isolation of integral membrane proteins with the propensity to undergo spontaneous folding and aggregation is being discussed.     

Reassemblable quasi-chip free-flow electrophoresis with simple heating dispersion for rapid micropreparation of trypsin in crude porcine pancreatin

An increasing number of small biosamples (e.g. proteins and enzymes) need micropreparation in lab. However, neither large-scale free-flow electrophoresis (LS-FFE) nor chip FFE (C-FFE) could fit the growing demands. Herein, a simple quasi-chip FFE (QC-FFE) was constructed. In contrast to C-FFE, the features of QC-FFE are as follows: (i) its separation chamber is reassemblable and rewashable avoiding discard of C-FFE due to blockage of solute precipitation in chamber; (ii) its chamber size is 45 mm × 30 mm × (80-500) μm (108-654 μL volume) having function of micropreparation; (iii) there are up to 16 outlets in QC-FFE bestowing fine fraction for micropurification. The QC-FFE was used for the micropurification of model enzyme of self-digestible trypsin in crude pancreatin. Under the given conditions, the purification factor of enzyme was 11.7, the specific activity reached 6236 U/mg, the run time for 19 μL sample purification was 45 s and the throughput of trypsin was 3.34 mg/h, and the yield of pure trypsin was 55.2%. All of the results show the feasibility of enzyme micropreparation via QC-FFE. The developed device and procedure have potential use to other micropurification of protein or peptide sample.     

三步柱色谱法从江浙蝮蛇蛇毒中分离纯化类凝血酶

建立了一种用CM Sepharose CL-6B阳离子交换、DEAE Sepharose Fast Flow阴离子交换和Sephadex G-75凝胶排阻三步柱色谱从江浙蝮蛇蛇毒中分离纯化类凝血酶的方法。在实验室小柱分离方案的基础上,对该纯化工艺进行了放大。当上样量达实验室小柱的25倍时,所得类凝血酶的质量指标与实验室小柱基本一致。采用该法所得的蝮蛇类凝血酶经Shim-pack Diol-300高效凝胶排阻柱测得其相对分子质量约为33500,用Shim-pack VP-ODS反相色谱柱检测其纯度约为96%。从江浙粗蛇毒中提取类凝血酶时,类凝血酶的总质量收率约为0.3%,总活性收率约为64%,比活可达2000 U/mg。     

Efficient Protocol for Large-Scale Purification of Naringin with High Recovery from Fructus aurantii by Macroporous Resin Column Chromatography and HSCCC

Several kinds of resins were investigated in the first step and D101 macroporous resin was selected for cleaning-up naringin (NAR), a major flavonoid glycoside from Fructus aurantii. In the subsequent column chromatography, 10% aqueous ethanol was first used to elute the column to remove the undesired constituents and 70% aqueous ethanol was used to elute the target. The content of NAR was 57.1% with 95.7% recovery in this process. In the second step, the obtained crude sample was directly isolated by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of ethyl acetate–n-butanol–water at a volume ratio of 2: 0.8: 3.2 (v/v/v), and 331 mg NAR with 98.3% purity was obtained from 600 mg crude extract in only one run. The recovery of the compound in this step was 95.0%. Thus, the total recovery of NAR was 90.9% after the two step purification. The established protocol for large-scale isolation and separation of NAR with high purity and recovery from F. aurantii was simple, efficient, and suitable for pharmace- utical and commercial use.     

利用超载洗脱技术在TSK Gel CM-5PW分析柱上进行溶菌酶百毫克级制备

本实验采用非线性色谱的展开方式之一-超载洗脱, 在普通的分析型离子交换柱TSK Gel-5PW (Φ7.5×75mm)上, 一次进样蛋白质混合样150mg, 成功地进行了溶菌酶的分离纯化, 回收率达90%。收集的馏份经透析和冰冻干燥后,通过高效毛细管电泳(HPCE)测定纯度, 得到了满意的结果, 活性回收率达88%。     

Optimization of throughput and desorbent consumption in simulated moving-bed chromatography for paclitaxel purification.

In simulated moving-bed (SMB) applications, throughput and desorbent consumption are two key factors that control process cost. For a given adsorbent volume and product purity requirements, throughput and desorbent consumption depend on desorbent composition, column configuration, column length to diameter ratio, and adsorbent particle size. In this study, these design parameters are systematically examined for paclitaxel purification. The results show that if adsorbent particle size, column dimensions and column configuration are fixed, the higher the product purity required, the lower the throughput. If product purity and yield are fixed, the larger the solute migration speed ratio, the higher the throughput, and the lower the desorbent consumption. If total bed volume and product purities are fixed, the longer the separation zones, the higher the throughput, but the higher the desorbent flow-rate. An intermediate configuration gives the minimum desorbent consumption. If there are no limits on pressure drop or zone flow-rate, the larger the column length to diameter ratio, the smaller the adsorbent particle size, the higher the throughput, and the lower the desorbent consumption. If the maximum zone flow-rate is controlled by the pressure drop limit and not by the standing waves requirement, the longer the columns, the lower the zone flow-rates and the lower the throughput. For 150 microns adsorbent particles and a maximum zone flow-rate of 300 ml/min, a design with optimal throughput and desorbent consumption is found for paclitaxel purification.     

Preparation of a monolithic column for weak cation exchange chromatography and its application in the separation of biopolymers

A procedure for the preparation of a monolithic column for weak cation exchange chromatography was presented. The structure of the monolithic column was evaluated by mercury intrusion. The hydrodynamic and chromatographic properties of the monolithic column--such as back pressures at different flow rates, effects of pH on protein retention, dynamic loading capacity, recovery, and stability--were determined under conditions typical for ion-exchange chromatography. The prepared monolithic column might be used in a relatively broad pH range from 4.0 to 12.0 and exhibited an excellent separation to five proteins at the flow rates of both 1.0 and 8.0 mL/min, respectively. In addition, the prepared column was first used in the purification and simultaneous renaturation of recombinant human interferon gamma (rhIFN-gamma) in the extract solution with 7.0 mol/L guanidine hydrochloride. The purity and specific bioactivity of the purified rhIFN-gamma in only one chromatographic step were obtained to be 93% and 7.8 x 10(7) IU/mg, respectively.     

高速逆流色谱法快速分离制备枸杞中莨菪亭

建立了用高速逆流色谱(HSCCC)从枸杞中快速分离莨菪亭的方法。将枸杞的乙醇提取物经D-101大孔树脂初步纯化后直接进行高速逆流色谱分离,用薄层色谱-荧光法考察了莨菪亭在不同溶剂体系中的分配情况。结果表明,最佳的溶剂体系为氯仿-甲醇-水(10:7:3, v/v/v),取上相为固定相,下相为流动相,在主机转速为850 r/min、流速为1.5 mL/min、检测波长为365 nm的条件下,从200 mg样品中一次性分离制备可得到10.2 mg纯度达到98.3%的莨菪亭。制备所得的莨菪亭与对照品的高效液相色谱(HPLC)保留时间一致,且经核磁共振氢谱、碳谱鉴定结构;纯度经HPLC法测定。研究发现,氯仿-甲醇-水(10:7:3, v/v/v)体系可连续二次进样而样品的峰形未受明显的影响。实验结果表明用薄层色谱-荧光法可快速选定HSCCC溶剂体系,进而可快速、简便地制备高纯度的莨菪亭。