Purification of carbonic anhydrase isoenzymes by high-performance affinity chromatography and hydrophobic interaction chromatography

Isoenzymes of carbonic anhydrase were purified by a combination of affinity chromatography and hydrophobic interaction chromatography. Immobilization of sulfonamides on an epoxy-activated support provided a stationary phase for affinity chromatography which was stable to hydrolysis by carbonic anhydrase. A first purification step allowed the isolation of enzymes directly from homogenates of human erythrocytes and rat stomach. Without any further preparation, except the addition of ammonium sulfate to the eluate from affinity chromatography, the isoenzymes could be separated by hydrophobic interaction chromatography with very high recovery of protein and retention of enzymatic activity.     

Hydrophobic displacement chromatography of proteins

Displacement chromatography of proteins was successfully carried out in both hydrophobic interaction and reversed-phase chromatographic systems using low-molecular weight displacers. The displacers employed for hydrophobic displacement chromatography were water soluble, charged molecules containing several short alkyl and/or aryl groups. Spectroscopy was employed to verify the absence of structural changes to the proteins displaced on these hydrophobic supports. Displacement chromatography on a reversed-phase material was employed to purify a growth factor protein from its closely related variants, demonstrating the high resolutions that can be achieved by hydrophobic displacement chromatography. This process combines the high-resolution/high-throughput characteristics of displacement chromatography with the unique selectivity of these hydrophobic supports and offers the chromatographic engineer a powerful tool for the preparative purification of proteins.     

Evaluation of radial chromatography versus axial chromatography, practical approach

Developments in packing and packing port design of radial columns in recent years have resulted in a claimed significant increase in performance of this process chromatography technology. In this first study, the main chromatographic parameters as efficiency, capacity factor, asymmetry and resolution were evaluated in a unique one-to-one comparison between a 120 ml bed-volume and 6 cm bed length radial chromatography mini-process column against a 50 mm diameter, 6 cm bed height and 120 ml bed-volume axial chromatography column. Radial chromatography showed an increase in efficiency by 31% in the number of plates per meter while the equilibration could be reduced by 0.4-0.5 column volumes. The asymmetry factor for bovine serum albumin in radial chromatography showed a reduction of 20% while the reduction of the asymmetry factor of the smaller protein ovotransferrin decreased even by 46% in comparison to the performance of the comparative axial chromatography column. Therefore in radial chromatography resolution improved up to 20%. The retention volume was similar in both cases. For radial chromatography, the decrease in "width at half height" at Height Equivalent of Theoretical Plates (HETP) measurements was 40% while the decrease of the over-all width of the peak was 27%. For adsorbed/desorbed proteins, the elution peak showed similar results: "width at half height" decreased to 45% while the over-all width of the peak decreased by 28%. The concentration of the non-retained protein in the flow-through (lysozyme), increased by 35% while the concentration of the eluted fraction (serum albumin bovine), increased with 40% in the radial chromatography columns. The better results obtained with the radial column were probably the consequence of the geometrical design of this device (larger inlet surface area and small outlet surface area which concentrate the eluted fraction).     

Applications of affinity chromatography in proteomics

Affinity chromatography is a powerful protein separation method that is based on the specific interaction between immobilized ligands and target proteins. Peptides can also be separated effectively by affinity chromatography through the use of peptide-specific ligands. Both two-dimensional electrophoresis (2-DE)- and non-2-DE-based proteomic approaches benefit from the application of affinity chromatography. Before protein separation by 2-DE, affinity separation is used primarily for preconcentration and pretreatment of samples. Those applications entail the removal of one protein or a class of proteins that might interfere with 2-DE resolution, the concentration of low-abundance proteins to enable them to be visualized in the gel, and the classification of total protein into two or more groups for further separation by gel electrophoresis. Non-2-DE-based approaches have extensively employed affinity chromatography to reduce the complexity of protein and peptide mixtures. Prior to mass spectrometry (MS), preconcentration and capture of specific proteins or peptides to enhance sensitivity can be accomplished by using affinity adsorption. Affinity purification of protein complexes followed by identification of proteins by MS serves as a powerful tool for generating a map of protein-protein interactions and cellular locations of complexes. Affinity chromatography of peptide mixtures, coupled with mass spectrometry, provides a tool for the study of protein posttranslational modification (PTM) sites and quantitative proteomics. Quantitation of proteomes is possible via the use of isotope-coded affinity tags and isolation of proteolytic peptides by affinity chromatography. An emerging area of proteomics technology development is miniaturization. Affinity chromatography is becoming more widely used for exploring PTM and protein-protein interactions, especially with a view toward developing new general tag systems and strategies of chemical derivatization on peptides for affinity selection. More applications of affinity-based purification can be expected, including increasing the resolution in 2-DE, improving the sensitivity of MS quantification, and incorporating purification as part of multidimensional liquid chromatography experiments.     

Continuous removal of protein aggregates by annular chromatography

The removal of polymeric proteins from their monomers is a frequently encountered separation task, especially in the polishing step of therapeutic proteins. Continuous separation of protein polymers from monomers by annular chromatography using size exclusion chromatography has been studied regarding the resolution, recovery, fouling, and productivity and has been compared to conventional chromatography. An IgG preparation rich in aggregates was used as a model protein mixture. Under conditions that maximized the throughput, the polymers could be separated from the monomers, but baseline separation could not be achieved. Baseline separation was also not possible in batch mode using equivalent conditions, which was also confirmed by computer simulation. For separation of the aggregates from the product the entire available separation space (360 degrees ) was indispensable. Therefore only cyclic, discontinuous regeneration could be carried out. Loading was identified as a critical step, since the concentrated protein solution evaded into the headspace instead of migrating into the gel where viscous fingering often occurs in conventional chromatography. The productivity of annular chromatography was two times higher than that of the conventional batch chromatography, and the buffer consumption was reduced to half the conventional value. These two benefits are especially important for protein separation processes that suffer from low loadability, such as size exclusion chromatography. We have demonstrated that size exclusion can be performed on an industrial scale when it is run continuously with the aid of a pressurized annular chromatograph.     

Urinary protein profiling by high-performance gel permeation chromatography

We describe a new application of high-performance aqueous gel permeation chromatography for the analysis of human proteinuria. Separations of urinary proteins from normal subjects and patients with renal impairment were performed with TSK G 3000 SW columns. The effects of pH and icnic strength of the eluent on the separation of urinary proteins were investigated. Albumins were selectively separated from urine by affinity chromatography on Blue Sepharose CL-6B. According to the results of clinical investigations, urinary protein pattern derived from gel permeation chromatography revealed a good prediction of the site of renal involvement. Predominant excretion of proteins with lower molecular weight than albumin correlated with tubular damage. Albumin and higher molecular weight protein patterns were associated with glomerular disease. Absorbance measurements of the eluent at 280 nm were used for quantitative determination of total urinary protein. Gel permeation chromatography was compared to sodium dodecyl sulfate—polyacrylamide gel electrophoresis and the resulting protein patterns are in good agreement.     

Purification of prenylated proteins by affinity chromatography on cyclodextrin-modified agarose

Although protein prenylation is widely studied, there are few good methods for isolating prenylated proteins from their nonprenylated relatives. We report that crosslinked agarose (e.g., Sepharose) chromatography medium that has been chemically functionalized with β-cyclodextrin (β-CD) is extremely effective in affinity chromatography of prenylated proteins. In this study, a variety of proteins with C-terminal prenylation target (“CAAX box”) sequences were enzymatically prenylated in vitro with natural and nonnatural prenyl diphosphate substrates. The prenylated protein products could then be isolated from starting materials by gravity chromatography or fast protein liquid chromatography (FPLC) on a β-CD-Sepharose column. One particular prenylation reaction, farnesylation of an mCherry-CAAX fusion construct, was studied in detail. In this case, purified farnesylated product was unambiguously identified by electrospray mass spectrometry. In addition, when mCherry-CAAX was prenylated with a nonnatural, functional isoprenoid substrate, the functional group was maintained by chromatography on β-CD-Sepharose, such that the resulting protein could be selectively bound at its C terminus to complementary functionality on a solid substrate. Finally, β-CD-Sepharose FPLC was used to isolate prenylated mCherry-CAAX from crude HeLa cell lysate as a model for purifying prenylated proteins from cell extracts. We propose that this method could be generally useful to the community of researchers studying protein prenylation.     

Separation of proteins by high-speed pressure liquid chromatography

Two chromatographic systems for separation of proteins by high-speed pressure liquid chromatography are described. Molecular size exclusion chromatography was achieved by the use of porous silica deactivated by Carbowax-20M to prevent protein adsorption. Protein separations were successful provided the salt concentration in the eluting buffer was relatively high.The second system described is adsorption chromatography of proteins on deactivated Porasil. This technique involves elution of the proteins from the gel by means of a salt and pH gradient. In both systems the total time required for the chromatography is less than 1 hr.     

Adsorption chromatography of proteins

A method for adsorption chromatography of proteins is proposed. A protein solution is passed through a cellulose column at a pH value corresponding to an isoelectric point of the protein. Depending on the charge of unwanted proteins, they either remain at the origin (if charges of protein and ion-exchanger are opposite) or are released from the column (if charges of protein and ion-exchanger coincide). Elution volume of the purified protein is higher than for the second group of unwanted proteins because movement of the uncharged protein of interest includes its adsorption on cellulose followed by subsequent desorption caused by the elution buffer. Problems of optimization of buffers and adsorbents are discussed. Applicability of the method of adsorption chromatography is illustrated using purification of horseradish peroxidase as an example.     

Extension of the selection of protein chromatography and the rate model to affinity chromatography

The rational selection of optimal protein purification sequences, as well as mathematical models that simulate and allow optimization of chromatographic protein purification processes have been developed for purification procedures such as ion-exchange, hydrophobic interaction and gel filtration chromatography. This paper investigates the extension of such analysis to affinity chromatography both in the selection of chromatographic processes and in the use of the rate model for mathematical modelling and simulation. Two affinity systems were used: Blue Sepharose and Protein A. The extension of the theory developed previously for ion-exchange and HIC chromatography to affinity separations is analyzed in this paper. For the selection of operations two algorithms are used. In the first, the value of η, which corresponds to the efficiency (resolution) of the actual chromatography and, Σ, which determines the amount of a particular contaminant eliminated after each separation step, which determines the purity, have to be determined. It was found that the value of both these parameters is not generic for affinity separations but will depend on the type of affinity system used and will have to be determined on a case by case basis. With Blue Sepharose a salt gradient was used and with Protein A, a pH gradient. Parameters were determined with individual proteins and simulations of the protein mixtures were done. This approach allows investigation of chromatographic protein purification in a holistic manner that includes ion-exchange, HIC, gel filtration and affinity separations for the first time.     

Purification of ficin by affinity chromatography

The sulfhydryl proteinase ficin (EC 3.4.4.12) was purified by chromatography on an agarose-mercurial column. Two separate protein fractions were eluted, ficin and mercurificin, both exhibiting enzymatic activity upon activation by excess thiol.     

应用柱层析法纯制Vero细胞肾综合征出血热疫苗

从Vero细胞培养液中提纯培养的汉滩病毒,将细胞冻融后上清液过Sepharose4FF凝胶层析柱,经紫外线280nm波长检测到三个吸收峰,RPHA证实仅第一峰为病毒抗原峰,另二个峰为杂蛋白,实验说明Sepharose4FF凝胶过滤对于提纯HFRS汉滩病毒是非常有效的,能去除98%的杂蛋白。     

Recent development of multi-dimensional chromatography strategies in proteome research

As a complementary approach to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), multi-dimensional chromatography separation methods have been widely applied in all kinds of biological sample investigations. Multi-dimensional liquid chromatography (MDLC) coupled with bio-mass spectrometry (MS) is playing important roles in proteome research due to its high speed, high resolution and high sensitivity. Proteome analysis strategies mainly include bottom-up and top-down approaches which carry out biological sample separation based on peptide and protein levels, respectively. Electrophoretic methods combined with liquid chromatography like IEF-HPLC and HPLC-SDS-PAGE have been successful applied for protein separations. As for MDLC strategy, ion-exchange chromatography (IEX) together with reversed phase liquid chromatography (RPLC) is still a most widely used chromatography in proteome analysis, other chromatographic methods are also frequently used in protein pre-fractionations, while affinity chromatography is usually adopted for specific functional protein analysis. Recent MDLC technologies and applications to variety of proteome analysis have been achieved great development. A digest peptide-based approach as so-called "bottom-up" and intact protein-based approach "top-down" analysis of proteome samples were briefly reviewed in this paper. The diversity of combinations of different chromatography modes to set up MDLC systems was demonstrated and discussed. Novel developments of MDLC techniques such as high-abundance protein depletion and chromatography array were also included in this review.     

Virus clearance validation across continuous capture chromatography

Multicolumn capture chromatography is gaining increased attention lately due to the significant economic and process advantages it offers compared with traditional batch mode chromatography. However, for wide adoption of this technology in clinical and commercial space, it requires scalable models for executing viral validation studies. In this study, viral validation studies were conducted under cGLP guidelines to assess retro- (X-MuLV) and parvo-virus (MVM) clearance across twin-column continuous capture chromatography (CaptureSMB). A surrogate model was also developed using standard batch mode chromatography based on flow path modifications to mimic the loading strategy used in CaptureSMB. The results show that a steady state was achieved by the second cycle for both antibody binding and virus clearance and that the surrogate model using batch mode chromatography equipment provided impurity clearance that was comparable to that obtained during cyclical operation of CaptureSMB. Further, the log reduction values (LRVs) achieved during CaptureSMB were also comparable to the LRVs obtained using standard batch capture chromatography. This was expected since the mode of virus separation during protein A chromatography is primarily based on removal during the flow through and wash steps. Finally, this study also presents assessments on the resin cleaning strategy during continuous chromatography and how the duration of clean-in-place solution exposure impacts virus carryover.     

Purification of a brain-specific astroglial protein by immunoaffinity chromatography

An immunoaffinity chromatography procedure for the isolation of bovine glial fibrillary acidic (GFA) protein is described. Degraded GFA protein isolated by hydroxyapatite chromatography from human spinal cord was used to prepare the antiserum. The immunoglogulin G fraction of the antiserum was covalently linked to CNBr-activated Sepharose, and columns of the immuno-affinity gel were used to adsorb bovine GFA protein from brain extracts. Elution was accomplished with a solution of 1 m acetic acid, 5 m urea, 0.8 m sodium chloride, pH 2.5. The yield of about 0.5 mg of highly purified protein/g of cerebral white matter could be increased to 1.5 mg/g of tissue by lowering the ionic strength of the extracting buffer from 50 mm to 1 mm sodium phosphate. Isolation in the presence of EDTA prevented the formation of an oxidation product migrating as a dimer of the monomeric species on SDS-polyacrylamide gel electrophoresis.     

Hydrophobic interaction chromatography in alkaline pH

Hydrophobic interaction chromatography is a very powerful protein purification technique which is dependent on strong salting-out salts to increase the hydrophobic interactions between the protein and the ligand. Ammonium sulfate is the salt most commonly used for this purpose, but it cannot be used at very alkaline pH. Monosodium glutamate was therefore tested as a salt for hydrophobic interaction chromatography at pH 9.5. When ribonuclease A, ovalbumin, and beta-lactoglobulin were individually applied to a phenyl superose column in 2 M monosodium glutamate, all three proteins bound to the column and could be subsequently eluted by decreasing the salt concentration. Using this salt, it was possible to separate commercially obtained beta-lactoglobulin into authentic protein and contaminants and to purify the individual proteins from a mixture of ovalbumin and beta-lactoglobulin. These results demonstrate that monosodium glutamate is a useful salt for hydrophobic interaction chromatography. Guanidine and sodium sulfate and sodium aspartate were also examined at the same pH, demonstrating that they also resulted in the binding and elution of the proteins examined.     

Fractionation and recovery of whey proteins by hydrophobic interaction chromatography

A method for the recovery and fractionation of whey proteins from a whey protein concentrate (80%, w/w) by hydrophobic interaction chromatography is proposed. Standard proteins and WPC 80 dissolved in phosphate buffer with ammonium sulfate 1 M were loaded in a HiPrep Octyl Sepharose FF column coupled to a fast protein liquid chromatography (FPLC) system and eluted by decreasing the ionic strength of the buffer using a salt gradient. The results showed that the most hydrophobic protein from whey is α-lactalbumin and the less hydrophobic is lactoferrin. It was possible to recover 45.2% of β-lactoglobulin using the HiPrep Octyl Sepharose FF column from the whey protein concentrate mixture with 99.6% purity on total protein basis.     

Affinity chromatography of neoglycoproteins

Glycoproteins, as a class of biomolecules, exhibit much more heterogeneous structures than non-glycosylated proteins. They present a challenging area of research. Model glycoproteins with well-defined protein and carbohydrate structures are helpful in the search for high-resolution methods for the separation of glycoproteins. Neoglycoproteins, maltose-modified chymotrypsin and lactose-modified chymotrypsin, were synthesised by modifying chymotrypsin with maltose and lactose, respectively, using the reductive amination method. Boronate chromatography was applied to isolate the neoglycoproteins from non-glycosylated substances. The use of Tris–HCl as a shielding reagent during the boronate chromatography proved to be efficient in eliminating unwanted interactions between the boronate ligand and the peptide backbone of chymotrypsin. The retention time of neoglycoproteins on the boronate column was increased with increasing the degree of modification.     

Surfactant-aided size exclusion chromatography

The flexibility and selectivity of size exclusion chromatography (SEC) for protein purification can be modified by adding non-ionic micelle-forming surfactants to the mobile phase. The micelles exclude proteins from a liquid phase similar to the exclusion effect of the polymer fibers of the size exclusion resin. This surfactant-aided size exclusion chromatography technology (SASEC) is demonstrated on the separation of two model proteins; bovine serum albumin (BSA) and myoglobin (Myo). The effect of the added surfactants on the distribution behavior of the proteins is predicted adequately by a size exclusion model presented in this paper.     

Purification of L-glutamate decarboxylase by affinity chromatography

L-Glutamate decarboxylase (L-glutamate 1-carboxy-lyase, EC 4.1.1.15) from rat brain synaptosomal extract was partially purified by affinity chromatography. On further purification by DEAE-Sephadex A 50 and Sephadex G-200, L-glutamate decarboxylase was purified to greater extent. It was found that a single affinity chromatography by appropriate elution gave a highly purified protein giving a single band of high specific activity on polyacrylamide gradient gel slab electrophoresis with minimal contamination. Substrate specificity of the purified enzyme was modified in the presence of 6-azauracil or phenylalanine resulting in decreased specificity to L-glutamate and increased specificity to L-aspartate.