Soybean metal-binding proteins: calmodulin purification by hydrophobic interaction with polymer 3520

Polymer 3520, a non-polar styrene divinylbenzene polymer, provides a simple way to purify calmodulin (CAM) from soybeans. This polymer, which selectively adsorbs CAM by hydrophobic interaction within the polymer matrix, contains no exchangeable groups; thus, interaction with CAM requires no Ca++ ions, and elution is achieved with 50% ethanol. Purification by this form of reversed-phase liquid chromatography is a substantial improvement over the conventional method, which requires high salt in elution buffers. CAM in soybean meal is first extracted with 80% ethanol in the presence of EGTA at room temperature and then chromatographed directly on a polymer 3520 column to yield pure CAM. Addition of non-ionic detergent (Nonidet P-40) to the ethanolic extract helps to separate extraneous proteins, lipids, sugars, and isoflavones. Such isolated CAM migrates as a single band during polyacrylamide gel electrophoresis and reversed-phase HPLC, and it retains activity stimulatory to phosphodiesterase.     

Ca2+-induced hydrophobic site on calmodulin: application for purification of calmodulin by phenyl-Sepharose affinity chromatography

Calmodulin binds quantitatively to phenyl-Sepharose and octyl-Sepharose affinity columns in the presence of micromolar concentrations of Ca²⁺. In addition to EGTA, calmodulin also can be eluted from these affinity columns with low ionic strength buffer, non-ionic detergent (i.e., 1% Triton X-100), or ethylene glycol (50%), suggesting hydrophobic interaction. Using hydrophobic interaction chromatography calmodulin can be purified to homogeneity from bovine brain homogenate in a single step. For large-scale purification the protein fraction containing calmodulin was concentrated by isoelectric precipitation prior to application to the affinity column. The yield obtained by this procedure (160–180 mg calmodulin per kg brain) is significantly greater, and the time required (～ 5 hr) is substantially less, than that of previously described procedures for calmodulin purification. It is apparent that phenyl-Sepharose offers several advantages over phenothiazine-Sepharose for affinity purification of calmodulin.     

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 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.     

Large scale purification of factor X by hydrophobic chromatography

Factor X is a critical enzyme in the blood coagulation cascade, however, in recent years the coagulation zymogen factor X has received additional interest as a selective proteinase to allow production of functional eukaryotic proteins in a prokaryotic expression system. Traditional factor X purification schemes suffer from low yields, low capacity, lengthy dialysis steps, and contamination by the autoproteolytic activated enzyme factor Xa. By incorporating a reversible inhibitor of factor X activation, we were able to recover 67% of the factor X present without any detectable activated enzyme. Six liters of plasma could be processed onto a 50 mL phenylalanine-Sepharose hydrophobic chromatography column without saturating the matrix. The final product is devoid of detectable proteolytic activity. At time of use, the zymogen is specifically activated with a Sepharose-bound activating enzyme isolated from Russell's Viper Venom, resulting in factor Xa free of other detectable proteinases.     

One-step purification of bacterial lipid macroamphiphiles by hydrophobic interaction chromatography

Bacterial lipid macroamphiphiles extracted with phenol/water can be purified in one step by hydrophobic interaction chromatography. Lipids and the major part of protein are separated from macroamphiphiles during phenol/water extraction. Coextracted nucleic acids, polysaccharides, and residual protein are effectively removed by column chromatography on octyl-Sepharose whereby macroamphiphiles are primarily adsorbed and later eluted with a buffered propanol gradient. The procedure is applicable to macroamphiphiles with various lipid structures as was demonstrated using the diacylglycerol-containing lipoglycan of Micrococcus luteus, the lipid A-containing lipopolysaccharide of Salmonella typhimurium, and the diglyceryl tetraether lipoglycans of Thermoplasma acidophilum and Thermoplasma volcanicum. On elution from octyl-Sepharose, separation into molecular species of different compositions was observed with the lipopolysaccharide of S. typhimurium and the lipoglycan of T. volcanicum. It was also shown that, after phenol/water extraction, membrane lipids are completely recoverable from the phenol layer, which makes it possible to isolate lipids along with macroamphiphiles from the same sample of bacteria.     

Native purification of biomolecules with temperature-mediated hydrophobic modulation liquid chromatography

The high-resolution purification of native enzymes is impeded by the limitations in the mobile-phase choices required for conventional hydrophobic separations such as in reverse-phase chromatography. To avoid problems associated with varying the composition of the mobile phase, we developed a stationary phase with a hydrophobicity that can be modulated by slight variations in temperature to bind and elute biomolecules. This chromatographic matrix was tested on nucleotide analogs, amino acids, and protein samples. Visualization of the temperature-dependent hydrophobic interaction with the chromatographic matrix was performed with fluorescence microscopy of CY3-ATP. Amino acids adsorbed to the column according to their known hydrophobicities, confirming the hydrophobic nature of their interaction with the matrix. Biomolecules were separated by modulating the hydrophobicity of the column matrix with slight adjustments to the running temperature between 22 and 37 degrees C without changing the mobile phase. Freedom in the choice of a mobile phase for both the loading and the elution of samples provides great practical advantages by eliminating the need for buffer-exchange steps and allowing more native conditions for purifying delicate enzymes, such as myosin.     

A simple reversed phase high-performance liquid chromatography method for polysorbate 80 quantitation in monoclonal antibody drug products

In this paper, we discuss an improved high-performance liquid chromatography (HPLC) method for the quantitation of polysorbate 80 (polyoxyethylenesorbitan monooleate), a commonly used stabilizing excipient in therapeutic drug solutions. This method is performed by quantitation of oleic acid, a hydrolysis product of polysorbate 80. Using base hydrolysis, polysorbate 80 releases the oleic acid at a 1:1 molar ratio. The oleic acid can then be separated from other polysorbate 80 hydrolysis products and matrices using reversed phase HPLC. The oleic acid is monitored without derivatization using the absorbance at 195 nm. The method was validated and also shown to be accurate for the quantitation of polysorbate 80 in a high protein concentration monoclonal antibody drug product. For the measured polysorbate 80 concentrations, the repeatability was less than 6.2% relative standard deviation of the mean (% RSD) with the day-to-day intermediate precision being less than 8.2% RSD. The accuracy of the oleic acid quantitation averaged 94–109% in different IgG₁ and IgG₄ drug solutions with variable polysorbate 80 concentrations. In this study, polyoxyethylene, a by-product of the polysorbate 80 hydrolysis was also identified. This peak was not identified by previous methods and also increased proportionally to the polysorbate 80 concentration. We have developed and qualified a method which uses common equipment found in most laboratories and is usable over a range of monoclonal antibody subclasses and protein concentrations.     

Performance of hydrophobic chromatography in purification of alpha-amylase

Hydrophobic ligands were introduced onto agarose beads, and the adsorption capacity of the beads was measured. The adsorption capacity increased with increase in the carbon number of the ligand, ionic strength of the buffer solution, and temperature. Crude alpha-amylase was purified with these hydrophobic adsorbents and the breakthrough and elution curves were estimated based on the mass transfer theory. Under strongly hydrophobic conditions, impurities contained in crude feeds and the lack of uniformity of packing caused by aggregation of beads affected adsorption and elution behaviors.     

Refolding and purification of interferon-gamma in industry by hydrophobic interaction chromatography

A new technology for renaturation with simultaneous purification of the recombinant human interferon-gamma (rhIFN-gamma) in downstream of biotechnology is presented. The strategies to develop the new technology in industry scale were suggested. Based on chemical equilibrium and molecular interactions, the principle of rhIFN-gamma refolding by HPHIC was described. The kind of stationary and mobile phases were evaluated and found the former to contribute to the rhIFN-gamma refolding more than the latter. The extract containing the rhIFN-gamma in gram scale in 7.0 mol L(-1) guanidine hydrochloride solution of 700 mL was directly pumped into a unit of simultaneous renaturation and purification of proteins (USRPP, 10 x 300 mm i.d.) packed by small particle packings of hydrophobic interaction chromatography and a satisfactory recovery of bioactivity and mass of the rhIFN-gamma was obtained. With flow rate 100 mL min(-1) and a gradient elution by only one step in 4h, the purity and specific bioactivity approach to 95% and 8.7 x 10(7) IU(-1) mg, respectively. To evaluate the goodness of the presented new technology in this study, a usual method with the renaturation by dilution method firstly and then purification with a series of LC in literature was employed to compare with each other. The obtained result in terms of purity, recoveries of mass and bioactivity, cost time as well as expenses, the former is much better than the latter. Comparing the total bioactivity of rhIFN-gamma in the extract before to that after the renaturation by the USRPP, the total bioactivity of rhIFN-gamma increased 62-fold.     

Penicillin acylase purification with the aid of hydrophobic charge induction chromatography

The aim of this work was to test a chromatographic support, 4-mercaptoethyl pyridine (4-MEP) Hypercel, for penicillin acylase purification by using pure penicillin acylase and crude extract. Two equilibration buffers with various salt concentrations and different flow rates were tested. The relationships between electrostatic and hydrophobic interactions and proteins are demonstrated. (NH4)2SO4 proved preferable because no salting-in occurred, contrary to NaCl. The recovery and purification fold were similar to those obtained in pseudo-affinity chromatography with a three-fold reduction of the (NH4)2SO4 concentration.     

Evaluation of exosome loading characteristics in their purification via a glycerol-assisted hydrophobic interaction chromatography method on a polyester,capillary-channeled polymer fiber phase

Exosomes are membrane-secreted vesicles, with sizes ranging from 30 to 150 nm, which play key roles in intercellular communication. There is intense interest in developing methods to isolate and quantify exosomes toward clinical diagnostics, fundamental studies of intercellular processes, and use of exosomes as delivery vehicles for therapeutic agents. Current methods for exosomes isolation and quantification are time consuming and have operational high costs; few combine isolation and quantification into a singular operation unit. This report describes the use of hydrophobic interaction chromatography on a polyester capillary-channeled polymer fiber column, employing a step gradient for exosome elution, including use of glycerol as a solvent modifier. The entire procedure is completed in 8 min, while maintaining the structural integrity and biological activity of the isolated exosomes. Electron microscopy was used to verify the size and structural fidelity of single exosomes. Absorbance response curves for a commercial exosome sample were used for exosome quantification in the chromatographic separations. In order to determine the dynamic loading capacity for exosomes, different volumes of Dictyostelium discoideum cell culture milieu supernatant were loaded at different column lengths (5–30 cm) and loading flow rates (0.2–0.5 ml/min). A loading capacity of 5.4 × 10¹² exosomes derived from D. discoideum milieu was obtained on a 0.8 × 300 mm column; yielding recoveries of over 80%. It is believed that this isolation and purification strategy holds many advantages toward the use of exosomes across a wide breadth of medical and biotechnology applications.     

Calcium-dependent hydrophobic chromatography of calmodulin, S-100 protein and troponin-C

We have demonstrated calcium-dependent hydrophobic interactions among calmodulin, S-100 protein and troponin-C and a homologous series of omega-aminoalkyl-agaroses. The three Ca2+-binding proteins were retained on the column of agarose substituted with omega- aminooctyl or even longer with alkylamine, in the presence of Ca2+ and 0.15 M NaCl. As these proteins were not retained on the column with shorter alkylamine 'arms' (N = 2, 4), they are probably successively absorbed with a higher affinity to the hydrophobic agarose column. Calmodulin and S-100 protein were eluted from the aminoocytl -agarose column with 1 mM EGTA in the presence of 0.15 M NaCl and the elution of troponin-C was Ca2+-independently carried out with 0.3 M NaCl. On the other hand, S-100 and troponin-C were eluted Ca2+-dependently from aminodecyl -agarose in the presence of 1 M NaCl and half the amount of the calmodulin applied was eluted with 1 M NaCl. As there are obvious differences among the three Ca2+-binding proteins with regard to chromatographic behavior on omega-aminoalkyl-agarose columns, our results suggest that these three proteins expose different hydrophobic regions following Ca2+-induced conformational changes and, if so, such would explain the interaction with aminoalkyl-agaroses.     

Rapid purification of calmodulin and S-100 protein by affinity chromatography with melittin immobilized to sepharose

Melittin-Sepharose was prepared for Ca2+-dependent affinity chromatography of calmodulin and S-100 protein. This matrix exhibits extremely high capacity (approximately 10 mg calmodulin/ml gel), low nonspecific binding, and excellent recovery (greater than 90%) under optimal conditions. Recovery of calmodulin from melittin-Sepharose was related to the degree of saturation of column capacity with lower yields when only partial saturation was achieved. Large-scale, simultaneous purification of calmodulin and S-100 protein from brain was carried out using selective adsorption to organomercurial agarose followed by melittin-Sepharose chromatography; yields were 250-300 mg of calmodulin and 200-300 mg of S-100 per kg tissue. Calmodulin also was purified in a single step from bovine testis supernatant using melittin-Sepharose in yields comparable to those from brain.     

A rapid method for the purification of D-amino acid oxidase of Trigonopsis variabilis by hydrophobic chromatography

Summary A relatively simple method has been described for the rapid purification of D-amino acid oxidase from Trigonopsis variabilis by hydrophobic chromatography on Phenyl-Sepharose CL-4B and negative adsorption on DEAE-cellulose. The purified enzyme had a specific activity of 22–24 units at 25°C and exhibited three bands on enzymatic staining.     

Separation and quantification of alkylphosphocholines by reversed phase high performance liquid chromatography

Alkylphosphocholines represent a new class of drugs with remarkable antineoplastic and antiprotozoal activity. For instance, hexadecylphosphocholine has been approved for the topical treatment of skin metastasis. In addition, it was successfully studied in India for the treatment of leishmaniasis. Different phase-I and phase-II-trials resulted in cure rates of more than 97%. To optimize antitumor or antiprotozoal activity, we have prepared alkylphosphocholines differing in chain length and unsaturation. For the qualitative and quantitative analysis of these longer chain analogues, we have used isocratic high performance liquid chromatography. The separation of the alkylphosphocholines with different chain lengths in this reversed phase HPLC system was achieved on a YMC-TMS column with a mobile phase consisting of methanol-water (85:15; v/v) at a flow rate of 1.0 ml/min. Furthermore the cis-/trans-isomers such as oleylphosphocholine and elaidylphosphocholine were clearly separated on a YMC-C8 column with a methanol-water mixture (80:20; v/v) as mobile phase. In the described reversed phase HPLC systems simple refractive index detection and UV detection allow the sensitive and quantitative determination of alkylphosphocholines. These methods are very important for reproducible identification and quantitative determination of saturated and mono-unsaturated alkylphosphocholines with alkyl residues containing up to 25 carbon atoms.