A large-scale purification of paclitaxel from cell cultures of Taxus chinensis

A large-scale purification method was developed for producing paclitaxel, to guarantee high purity and yield from plant cell cultures. The complete method for mass production was a simple and efficient procedure, for the isolation and purification of paclitaxel from the biomass of Taxus chinensis, consisting of solvent extraction, synthetic adsorbent treatment, and two steps of precipitation, followed by two steps of high performance liquid chromatography (HPLC). The organic solvent extraction of biomass obtained crude extract containing paclitaxel. The use of synthetic adsorbent treatment and precipitation in the prepurification process allows for rapid and efficient separation of paclitaxel from interfering compounds and dramatically increases the yield and purity of crude paclitaxel for HPLC purification steps compared to alternative processes. This prepurification process serves to minimise solvent usage, size, and complexity of the HPLC operations for paclitaxel purification. The paclitaxel of over 99.5% purity can be simply obtained with high yield from crude paclitaxel by HPLC using reverse-phase separation on C18 as the first step and normal-phase separation on silica as the second step.     

Purification of NAD glycohydrolase from Agkistrodon acutus venom

NAD glycohydrolase (NADase) from Agkistrodon acutus venom was purified to electrophoretic homogeneity by a fast, reproducible 3-step procedure including Q Sepharose Fast Flow, Superdex 75, and Mono S column chromatography. This new procedure gave a 15.6-fold purification with a recovery yield of 7.9% and a specific activity of 12.8 units/mg.     

A simple purification procedure of biologically active recombinant human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture

A simple purification procedure of bioactive human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture has previously been described. In this study the protein was purified to apparent homogeneity with an overall yield of 80.1% by ammonium sulfate precipitation and a single chromatographic step involving FPLC-anion exchange chromatography. The purified hGM-CSF revealed at least five glycosylated forms ranging from 21.5∼29 kDa, and its biological activity was independent of the glycosylation pattern. This is the first purification report of recombinant hGM-CSF to apparent homogeneity from rice cell suspension cultures.     

Purification and enzyme stability of alcohol dehydrogenase from Drosophila simulans, Drosophila virilis and Drosophila melanogaster adhS.

Three alcohol dehydrogenases from Drosophila simulans, Drosophila virillis and Drosophila melanogaster adhS (which possesses an alloenzyme with slow electrophoretic mobility) were purified essentially to homogeneity. The purification procedure involves a new step of affinity chromatography, which efficiently lowers the amount of contaminants in the final preparation, producing a very stable enzyme. The purification procedure developed consists of a salmine sulphate precipitation, two CM-Sepharose CL-6B colume-chromatography steps, an affinity-chromatography step and a Sephacryl gel filtration. A minimum of 30-fold purification is obtained and the yield is not less than 34%. The isoelectric points and molar absorption coefficients were determined.     

Rapid large-scale purification of pig heart nucleoside diphosphate kinase by affinity chromatography on Cibacron Blue 3G-A Sepharose

A rapid procedure for the large-scale purification of pig heart nucleoside diphosphate kinase is described. The purification procedure involves extraction of the enzyme, absorption on cibacron Blue 3G-A Sepharose, elution with ATP, ammonium sulfate precipitation, heat treatment, and rechromatography on Cibacron Blue 3G-A Sepharose. Typically, 10–12 mg of pure nucleoside diphosphate kinase is obtained from 1 kg of heart muscle (50% yield), with a purification factor of 1200 over the extract. The specific activity is 1500 units/mg at 25°C with 8-bromoinosine 5′-diphosphate as acceptor nucleotide. This method may be easily scaled up.     

Preliminary studies on the purification of a monoclonal antibody by affinity precipitation with Eudragit S-100

A simple procedure for the purification of an IgG-type monoclonal antibody by affinity precipitation using Eudragit S-100 is presented. The ligand, a microbial lipase previously used as antigen, was coupled to the polymer at a concentration of 40 mg lipase/g Eudragit. This macroligand was reversibly precipitated by manipulating the pH at values higher and lower than 4.8. The effects of polymer concentration and dilution of hybridoma culture supernatant on the overall precipitation process were evaluated. The best purification factor was achieved with a polymer concentration of 0.1% (w/v) and a supernatant dilution of 1:3. The preliminary studies reported here enabled the purification of a monoclonal antibody in one step with an activity yield (by ELISA) of 50%-55% and a purification factor of ca 6.     

Rapid purification of fructose-1,6-bisphosphate aldolase from spinach chloroplasts

A rapid procedure for the purification of fructose-1,6-bisphosphate aldolase from spinach chloroplasts is presented which involves two steps; precipitation of bulk protein with polyethylene glycol and partitioning of remaining soluble protein in aqueous two-phase systems. A 94% pure preparation is obtained within 6h with a yield of 19%. A marked difference in the partition behaviour of the aldolase activity from whole leaf tissue suggested that the procedure is less efficient when leaf extract is used as starting material.Abbreviations EDTA Ethylenediamine Tetraacetic Acid - PEG Polyethylene Glycol - SDS Sodium Dodecyl Sulfate     

Isolation, Purification, and Crystallization of Aspartate Aminotransferase from Wheat Grain

A procedure for isolation and purification of aspartate aminotransferase from wheat grain includes chromatography on DEAE cellulose, acidification-alkalization, precipitation with protamine sulfate, fractionation with ammonium sulfate, and chromatography on hydroxyapatite. The yield of protein was 27% with 95% purity. Crystals of the enzyme (0.05 x 0.025 x 0.015 mm3) were obtained from ammonium sulfate solution.     

Two-step purification of Bacillus circulans chitinase A1 expressed in Escherichia coli periplasm

A protein purification procedure was developed to efficiently and effectively purify the target enzyme, chitinase A1 of Bacillus circulans WL-12, from Escherichia coli DH5alpha carrying the chiA gene with its natural promoter in the plasmid pNTU110. Chitinase A1 was purified to apparent homogeneity from E. coli periplasm with a final recovery of 90.6%. Two main steps were included in this protein purification procedure, ammonium sulfate precipitation (40% saturation) and anion-exchange chromatography at pH 6.0 using Q Ceramic HyperD column. The yield of chitinase A1 was estimated at 95 microg/L. A polyclonal antibody against chitinase A1 was raised by immunizing BALB/c mice with chitinase A1 purified from E. coli DH5alpha(pNTU110). As indicated by Western blot analysis, a 3000-fold diluted antibody detected purified chitinase A1 from E. coli DH5alpha(pNTU110) in an amount of at least 1 ng and specifically detected chitinase A1 produced by B. circulans WL-12.     

Purification of commercial preparations of NADP+ from AMP contamination

A method for purification of commercial preparations of NADP+ from AMP contamination is described. The purification procedure includes one-step anion-exchange chromatography on Dowex-1 (formate) and results in a highly purified salt-free coenzyme with a yield of 70-80%. The chromatography conditions have been selected allowing for complete separation of AMP from NADP+ in a HCOOH concentration gradient. This is followed by NADP+ elution with 1.5 M HCOOH containing HCOOK at a concentration at which the salt remains in solution during subsequent precipitation and washing of NADP+ with acetone. An addition of HCOOK is necessary to reduce the coenzyme elution volume that is important for further precipitation of NADP+ with acetone.     

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.     

Purification of a special estrogen-binding protein from the liver of rats by affinity chromatography on estradiol sepharose

A procedure has been developed for purification of a special rat liver estrogen-binding protein. It includes protein precipitation by ammonium sulfate, gel filtration, ion exchange chromatography, and affinity chromatography on estradiol sepharose. The protein is purified 2260-fold with a 27% yield. Upon electrophoresis in 10% PAG in the presence of sodium dodecylsulfate the protein gives one polypeptide strip with a molecular weight of 31.200 +/- 400 dalton.     

Preparation of myosin rods from myofibrils

A simple and fast procedure is described for producing myosin rods with a good yield. The method is based on the digestion of myofibrils by soluble papain. After purification by alcoholic precipitation and gel filtration, rod segments exhibited physical-chemical parameters identical to previously reported data.     

A new biochemical engineering approach to the fractional precipitation of proteins

A biochemical engineering framework for optimizing the design and operation of fractional protein precipitation has been developed. The method utilizes a fractionation diagram to represent the purification of a product protein relative to total contaminating protein. The purification factor for a single or double-cut fractional precipitation is obtained as the gradient of an appropriate operating tie-line. A computer algorithm has been devised to maximize the tie-line gradient for a given yield enabling a plot of optimum purification factor versus yield to be constructed. The recovery of the enzyme alcohol dehydrogenase from clarified bakers homogenate using saturated ammonium sulphate has been examined. Fractionation and purification versus yield diagrams were used to investigate the effects of such process parameters as pH, temperature, and initial total protein concentration on fractionation efficiency. The results are discussed in terms of the underlying solubility and mixing phenomena and the industrial application of fractional precipitation.     

A rapid method for the purification of fatty acid synthetase from the yeast Saccharomyces cerevisiae

A rapid method for the isolation and purification of small quantities of highly active fatty acid synthetase (FAS) from several strains of the yeast Saccharomyces cerevisiae, is presented. The purification procedure which is the shortest reported to this date (18 hr), involves the release of the enzyme by either cell wall digestion with Zymolyase 60000 or cell wall disruption by glass beads, followed by 35-50% ammonium sulfate fractionation, desalting by Sephadex G-25 chromatography, then calcium phosphate gel treatment, concentration by 50% ammonium sulfate precipitation, sedimentation of the enzyme in the ultracentrifuge and finally, column chromatography on DEAE Bio-Gel A. Fatty acid synthetase prepared by the cell breakage method, was found to be homogeneous according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), SDS-Tris-glycine disc gel electrophoresis and immunoelectrophoresis criteria. However, enzyme prepared from Zymolyase treated cells showed several proteolytic bands in addition to FAS bands, on SDS-PAGE. Enzyme obtained by both methods of cell breakage, showed a similar behavior throughout the purification procedure and gave a similar yield of enzyme of high specific activity (4800-7200 nmol/min/mg) that remained stable for several months at -85 degrees C.     

Purification of recombinant protein A by aqueous two-phase extraction integrated with affinity precipitation

Aqueous two-phase extraction incorporated affinity precipitation was examined as a technique for protein purification. An enteric coating polymer, Eudragit S100, was employed as a ligand carrier. Eudragit was specifically partitioned to the top phase in the aqueous two-phase systems. For application of this method to purification of recombinant protein A using human IgG coupled to Eudragit in an aqueous two-phase system, 80% of protein A added was recovered with 81% purity. The purity was enhanced 26-fold by thid method. The IgG-Eudragit could be used repeatedly for the purification process. This seperation method should be applicable to industrial-scale purification as a new purification procedure combining the advantages and compensating for the disadvantages of the aqueous two-phase method and affinity precipitation method. (c) 1992 John Wiley & Sons, Inc.     

A novel method for the purification of low soluble recombinant C-type lectin proteins

Snake venoms contain a complex mixture of many biological molecules including proteins. The purification of recombinant proteins is a key step in studying their function and structure with affinity chromatography as the common method used in their purification. In bacterial expression systems, hydrophobic recombinant proteins are usually precipitated into inclusion bodies, and contaminants are typically associated with tagged proteins after purification. The purpose of this study was to develop a procedure to purify hydrophobic recombinant proteins without an affinity tag. Snake venom mature C-type lectin-like proteins (CLPs) with a tag were cloned, expressed, and purified by repeated sonication and wash steps. The effects of the signal peptide on the expression and solubility of the recombinant protein were investigated. The CLPs in washed inclusion bodies were solubilized and refolded by dialysis. The CLPs without a tag were successfully purified with a yield 38 times higher than the traditional method, and inhibited blood platelet aggregation with an IC(50) of 100.57μM in whole blood. This novel procedure is a rapid, and inexpensive method to purify functional recombinant hydrophobic CLPs from snake venoms useful in the development of drug therapies.     

Caprylic acid‐induced impurity precipitation from protein A capture column elution pool to enable a two‐chromatography‐step process for monoclonal antibody purification

This article presents the use of caprylic acid (CA) to precipitate impurities from the protein A capture column elution pool for the purification of monoclonal antibodies (mAbs) with the objective of developing a two chromatography step antibody purification process. A CA‐induced impurity precipitation in the protein A column elution pool was evaluated as an alternative method to polishing chromatography techniques for use in the purification of mAbs. Parameters including pH, CA concentrations, mixing time, mAb concentrations, buffer systems, and incubation temperatures were evaluated on their impacts on the impurity removal, high‐molecular weight (HMW) formation and precipitation step yield. Both pH and CA concentration, but not mAb concentrations and buffer systems, are key parameters that can affect host–cell proteins (HCPs) clearance, HMW species, and yield. CA precipitation removes HCPs and some HMW species to the acceptable levels under the optimal conditions. The CA precipitation process is robust at 15–25°C. For all five mAbs tested in this study, the optimal CA concentration range is 0.5–1.0%, while the pH range is from 5.0 to 6.0. A purification process using two chromatography steps (protein A capture column and ion exchange polishing column) in combination with CA‐based impurity precipitation step can be used as a robust downstream process for mAb molecules with a broad range of isoelectric points. Residual CA can be effectively removed by the subsequent polishing cation exchange chromatography. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1515–1525, 2015     

A new method for the isolation of histatins 1, 3, and 5 from parotid secretion using zinc precipitation

Histatins, a family of small-molecular-weight, histidine-rich cationic salivary proteins, have been difficult to isolate in an efficient way by conventional procedures due to their anomalous interactions with chromatographic resins. In the present study we explored the possibility of developing a new isolation procedure based on recent observations that histatins associate with various metal ions, including zinc. Since solubility studies showed that histatin 5 forms precipitates with zinc under alkaline conditions, we investigated whether this characteristic could be exploited for the preparative isolation of histatins from salivary secretions. A fast and efficient two-step procedure was developed using zinc precipitation of histatins from human parotid secretion followed by final purification using reversed-phase high-performance liquid chromatography (HPLC). Analysis of zinc precipitates by Tricine-SDS-PAGE, cationic PAGE, HPLC, and mass spectrometry revealed the presence of the three major histatins, 1, 3, and 5, as well as statherin. The histatin yield obtained by the precipitation step was approximately 90%. Therefore, zinc precipitation of histatins from glandular salivary secretions is a novel, rapid, and effective means for the isolation of these proteins.     

Prepurification of paclitaxel by micelle and precipitation

A novel prepurification method was developed aiming at increasing yield and purity, also reducing solvent usage for purification of paclitaxel. This method was a simple and efficient procedure, for the isolation and prepurification of paclitaxel from the biomass of Taxus chinensis, consisting of micelle formation, followed by two precipitation steps. The purity and yield were 65.8 and 80% in the prepurification step, respectively. The use of a micelle and precipitation in the prepurification process allows rapid separation of paclitaxel from interfering compounds and dramatically reduces solvent usage compared to alternative methodologies. This prepurification process serves to minimize the size and complexity of the HPLC operations for paclitaxel purification. This process is readily scalable to a pilot plant and eventually to a production environment where multikilogram quantities of material are expected to be produced. As much as possible, the process has been optimized to minimize solvent usage, complexity, and operating costs.