Isoelectric protein purification by orthogonally coupled hydraulic and electric transports in a segmented immobilized pH gradient

A new method is described for preparative protein purification, based on isoelectric focusing on immobilized pH gradients. The principle is entirely new, as it is based on keeping the protein of interest isoelectric, in a flow-chamber, and focusing the impurities in the Immobiline gel. For this, a hydraulic flow is coupled orthogonally to an electric flow, sweeping away the non-isoelectric impurities from the recycling chamber. The sample flow-chamber is built in the centre of the apparatus, and is coupled to an upper and lower segment of an immobilized pH gradient. The protein to be purified is kept isoelectric in the flow-chamber and prevented from leaving it by arranging for the extremities of the immobilized pH gradient, forming the ceiling and the floor of this chamber, to have isoelectric points just higher (e.g. +0.05 pH units, on the cathodic side) and just lower (e.g. -0.05 pH units, on the anodic side) than the known pI of the species of interest. Macromolecules and small ions leave the flow chamber at a rate corresponding to a first order reaction kinetics (the plot of log C vs. time being linear). In general, for macromolecules, 12 h of recycling under current allow removal of 95% impurities. After 24 h of recycling, the protein of interest is more than 99.5% pure. The recoveries are very high (approaching 100%) as the sample under purification never enters the Immobiline gel and thus does not have to be extracted from a hydrophilic matrix, as typical of preparative gel electrophoresis.     

A horizontal apparatus for isoelectric protein purification in a segmented immobilized pH gradient

A modification of the previously described apparatus (Faupel et al. (1987) J. Biochem. Biophys. Methods 15, 147-162), for recycling isoelectric focusing in a segmented immobilized pH gradient, is here reported. The most important improvements are: (1) a horizontal, vs. the previously vertical assembly; (2) a reduction of the thickness of the central flow chamber to 6 mm, vs. the previous 3 cm length and (3) the introduction, at both gel extremities of each Immobiline segment, of polypropylene filters, thus efficiently blocking the gel in situ. The advantages are: (i) the spontaneous removal of air bubbles, which in the vertical apparatus tend to accumulate in the ceiling of the flow chamber and to obstruct the flow of electric current; (ii) a more efficient hydraulic flow with a reduced chance of heating the liquid stream in the flow chamber, due to its reduced length along the separation path and (iii) a reduced risk of gel detachment from the tube walls, due to osmotic swelling caused by focused protein zones in the gel phase and by the fixed Immobiline charges in the polyacrylamide matrix.     

Isoelectric protein purification in segmented immobilized pH gradients. Effect of salts on rate of contaminants' removal

A method is described for keeping a constant salt background during protein purification in a segmented immobilized pH gradient. It is based on an external hydraulic flow replenishing the salt loss due to combined electric and diffusional mass transport (similar to the concept of Ribes' steady-state rheoelectrolysis). Such a minimum of ionic strength might be needed for proteins which tend to precipitate and aggregate at or in vicinity of the isoelectric point. However, it is found that any salt level in the sample feed (already at 1 mM concentration) deteriorates transport of non-isoelectric proteins, because of the much larger current fraction carried by the ions themselves as opposed to proteins. In addition, high salt levels in the sample reservoir might form cathodic and anodic ion boundaries, alkaline and acidic, respectively, which might hamper protein migration and even induce denaturation. Thus, when high salt backgrounds are needed in the sample feed, external pH control should be exerted, e.g. with a pH-stat. Three parameters influence protein transport in the segmented IPG chamber: (a) cross-sectional area of the Immobiline membranes; (b) delta pI between the isoelectric protein and the contaminants and (c) salt molarity in the sample reservoir. The first 2 show a positive, the last a negative correlation.     

Preparative immobiline isoelectric focusing of plasma apolipoproteins on vertical slab gels

An effective preparative isoelectric focusing method has been developed using the LKB Immobiline system in a vertical slab gel apparatus. Advantages of this procedure are ease of sample application, excellent resolution, and the direct visualization of focused bands. Narrow pH gradients have been used to separate apolipoprotein E3 isoforms (pH gradient 4.9-5.9) and to resolve the apolipoprotein C mixture (pH gradient 4.0-5.0). Recoveries ranged from 40 to 70%. The method should be valuable for protein and isoform purification.     

Isoelectric focusing in immobilized pH gradients in the pH 10-11 range

With the synthesis of a new, strongly basic Immobiline (pK 10.3 at 10 degrees C) it has been possible to formulate a new pH 10-11 recipe for focusing very alkaline proteins, not amenable to fractionation with conventional isoelectric focusing in carrier ampholyte buffers. In this formulation, water is added as an acidic Immobiline having pK = 14 and a unit molar concentration (or with a pK = 15.74 and standard 55.56 molarity) since around pH 11 its buffering power becomes significant. The gel contains a 'conductivity quencher', i.e. a density gradient incorporated in the matrix, with the dense region located on the cathodic side (pH 11) for (a) smoothing the voltage gradient on the separation cell and (b) reducing the anodic electrosmotic flow due to the net positive charge acquired by the matrix at pH 11 (1 mM excess protonated amino groups to act as counterions to the 1 mm OH- groups in the bulk water solution generated by the local value of pH 11). Excellent focusing is obtained for such alkaline proteins as lysozyme (pI 10.55), So-6 (a leaf protein, pI 10.49), cytochrome c (pI 10.45) and ribonuclease (pI 10.12).     

Direct recovery of proteins into a free-liquid phase after preparative isoelectric focusing in immobilized pH gradients

A new method for electrophoretic retrieval of protein zones from Immobiline matrices is described, based on elution directly in a free liquid phase, rather than in ion-exchange beads or molecular sieves, as previously described. The chopped Immobiline gel is loaded on top of a 5% T stacking gel, 6-10 mm in height, and forced to transverse it and collect into a chamber, filled with 20% sucrose solution, closed on its anodic side by a dialysis sac. The transfer is practically quantitative, for most proteins, after 30-60 min of zone electrophoresis at 10 W (300 V potential differential). Recovery of protein mass is in general better than 90%, while for enzyme activity is in the range of 60-80%. For preserving enzyme integrity, the following precautions are recommended: short electrophoretic times; avoidance of anodic oxidation; chilling of the buffer in the anodic chamber; and use of low levels (2-5 mM) of the specific enzyme substrate throughout the entire electrophoretic system (cathode, anode and gel plug).     

Covalent immobilization of proteins to N-hydroxysuccinimide ester derivatives of agarose. Effect of protein charge on immobilization

An uncharged N-hydroxysuccinimide ester derivative of agarose, Affi-Gel 10, exhibited excellent capacity for immobilization, at pH 7.5, of proteins having isoelectric points of 6.5--11.0. Under identical conditions, acidic proteins with isoelectric points of 3.3--5.9 did not couple well to this activated gel. Immobilization of acidic proteins increased in the presence of 80 mM CaCl2, or at a pH equal to or less than the isoelectric point. Affi-Gel 15, a new N-hydroxysuccinimide ester derivative of agarose containing a tertiary amine in the spacer arm, coupled acidic proteins efficiently at pH 7.5 but basic proteins coupled poorly. The immobilization of basic proteins to Affi-Gel 15 was increased to useful levels by increasing the ionic strength, or the pH, of the reaction medium. The lectin concanavalin A was efficiently immobilized using either activated gel, and the concanavalin A-agarose derivatives bound 3.9--4.1 mg ovalbumin/ml gel. These studies demonstrate that the charge of the protein relative to the charge of the gel is an important factor affecting the level of protein immobilization to active ester gels.     

Protein purification by Off-Gel electrophoresis

A novel free-flow protein purification technique based on isoelectric electrophoresis is presented, where the proteins are purified in solution without the need of carrier ampholytes. The gist of the method is to flow protein solutions under an immobilised pH gradient gel (IPG) through which an electric field is applied perpendicular to the direction of the flow. Due to the buffering capacity of the IPG gel, proteins with an isoelectric point (pI) close to pH of the gel in contact with the flow chamber stay in solution because they are neutral and therefore not extracted by the electric field. Other proteins will be charged when approaching the IPG gel and are extracted into the gel by the electric field. Both a demonstration experiment with pI markers and a simulation of the electric field distribution are presented to highlight the principle of the system. In addition, an isoelectric fractionation of an Escherichia coli extract is shown to illustrate the possible applications.     

Purification of glucose oxidase from complex fermentation medium using tandem chromatography

A fast and efficient purification method for recombinant glucose oxidase (rGOx) for flask fermentation scale (up to 2L) was designed for the purposes of characterization of rGOx mutants during directed protein evolution. The Aspergillus niger GOx was cloned into a pYES2-alphaMF-GOx construct and expressed extracellularly in yeast Saccharomyces cerevisiae. Hydrophobic interaction (HIC)/size exclusion (SEC)-tandem chromatographic system was designed for direct purification of rGOx from a conditioned complex expression medium with minimum preceding sample preparation (only adjustments to conductivity, pH and coarse filtering). HIC on Butyl 650s (50 mM ammonium acetate pH 5.5 and 1.5 M ammonium sulphate) absorbs GOx from the medium and later it is eluted by 100% stepwise gradient with salt free buffer directly into SEC column (Sephadex 200) for desalting and final polishing separation. The electrophoretic and UV-vis spectrophotometric analyses have proven enzyme purity after purification.     

Isoelectric focusing with reduced cathodic drift and migration into the anode chamber

An apparatus has been developed to reduce cathodic drift and migration into the anode chamber in vertical gel rod isoelectric focusing (IEF). In contrast to commercially available apparatuses, this apparatus can easily handle many more gels at one time, and the length, diameter and shape of its gel can be arbitrarily changed. In addition, high concentrations of detergent can be used to dissolve the protein samples, and removal of the gel cylinders from the glass tubes is easy.     

Characterization of immobiline membranes for application in a multicompartment electrolyzer for protein purification.

The efficient use of preparative protein purification in a multicompartment electrolyzer with Immobiline membranes depends on the knowledge of membrane characteristics. For that purpose, an experimental investigation of the effects of ionic charges on the membrane characteristics has been carried out through the measurements of membrane swelling and conductance. We also investigated the effects on the electrolyzer behaviour of operating parameters such as the Immobiline concentration and the presence of ion-exchange membranes. Data show that polyacrylamide gel degree of swelling is strongly dependent upon the pH and the ionic strength of the bathing solution as well as on the type and molarity of charges incorporated in the gel. The conductance of supported Immobiline gels in contact with uni-univalent chloride solutions has been measured by means of a mercury cell. The membrane conductance is also influenced by the ionic strength of the equilibrium solution and the presence of weak ionizable groups in the gel matrix. This study has demonstrated the close link between electrochemical and electromechanical properties of Immobiline membranes.     

The use of micropreparative electrophoresis of protein/peptide isolations for primary structure determinations

High-performance electrophoresis chromatography (HPEC) is a recent development that features continuous-elution gel electrophoresis for isolating proteins or peptides in range of 1 to 300 microgram quantities. Column gel electrophoresis is conducted under thermostated conditions, and the field voltage can be varied within a run with a programmable power supply. Applications of this apparatus in protein purification are presented to demonstrate the utility of the (Model 230A) HPEC. These examples include on-line detection with direct analyte recovery of highly purified sample, which mimics high-performance liquid chromatography, for subsequent structure-function characterization. A method to remove salts from sodium dodecyl sulfate electrophoresed samples for subsequent sequencing or amino acid analysis is described. This desalting procedure recovers from 90%-95% of the sample and employs a low molecular weight cut-off membrane during sample centrifugation onto a polyvinylidene difluoride membrane. Subsequent washings are performed to efficiently remove salts, free amino acids and detergents that are known to interfere with sequence analysis. Sequence information such as initial recovery, repetitive yields and chromatogram comparisons are presented to demonstrate the utility of this procedure when used following isolation of sample with HPEC.     

Salt tolerant membrane adsorbers for robust impurity clearance

Clearance of impurities such as viruses, host cell protein (HCP), and DNA is a critical purification design consideration for manufacture of monoclonal antibody therapeutics. Anion exchange chromatography has frequently been utilized to accomplish this goal; however, anion exchange adsorbents based on the traditional quaternary amine (Q) ligand are sensitive to salt concentration, leading to reduced clearance levels of impurities at moderate salt concentrations (50–150 mM). In this report, membrane adsorbers incorporating four alternative salt tolerant anion exchange ligands were examined for impurity clearance: agmatine, tris‐2‐aminoethyl amine, polyhexamethylene biguanide (PHMB), and polyethyleneimine. Each of these ligands provided greater than 5 log reduction value (LRV) for viral clearance of phage ?X174 (pI ～ 6.7) at pH 7.5 and phage PR772 (pI ～ 4) at pH 4.2 in the presence of salt. Under these same conditions, the commercial Q membrane adsorber provided no clearance (zero LRV). Clearance of host‐cell protein at pH 7.5 was the most challenging test case; only PHMB maintained 1.5 LRV in 150 mM salt. The salt tolerance of PHMB was attributed to its large positive net charge through the presence of multiple biguanide groups that participated in electrostatic and hydrogen bonding interactions with the impurity molecules. On the basis of the results of this study, membrane adsorbers that incorporate salt tolerant anion exchange ligands provide a robust approach to impurity clearance during the purification of monoclonal antibodies. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A Novel Method for Separation of Caseins from Milk by Phosphates Precipitation

Milk protein of farm animals is difficult to isolate because of the presence of casein micelles, which are hard to separate from whey by using centrifugation or filtration. Insoluble casein micelles also create an obstacle for purification instruments to operate efficiently. The conventional method, to precipitate caseins by lowering pH to 4.6 and then recover the whey fraction for further purification using chromatography techniques, is not applicable to proteins having an isoelectric point similar to caseins. In addition, the acid condition used for casein removal usually leads to significantly poor yields and reduced biological activities. In this study, a novel method of precipitating caseins under neutral or weak acidic conditions is presented. The method employs a phosphate salt and a freeze–thaw procedure to obtain a casein-free whey protein fraction. This fraction contains more than 90% yield with little loss of bioactivity of the target protein, and is readily available for further chromatographic purification. This method was successfully applied to purify recombinant human factor IX and recombinant hirudin from the milk of transgenic pigs in the presented study. It is an efficient pretreatment approach prior to chromatographic purification of milk protein from farm animals and particularly of great value to collect those recombinants secreted from transgenic livestock.     

New polyacrylamide matrices for drift-free isoelectric focusing

The major cause for pH gradient decay and cathodic drift during isoelectric focusing in polyacrylamide gels has been found to be electroendo-osmotic flow generated by fixed charges in the gel matrix. These charges have the following causes: (a) trace impurities of acrylic acid in the co-monomers; (b) covalent incorporation of catalysts (persulphate and riboflavin 5'-phosphate) as terminal groups in polyacrylamide chains; (c) hydrolysis of amide groups to acrylic acid in the gel layer underneath the cathodic filter paper strip. The result of these fixed negative charges in the matrix is a movement of counter-ions with hydration water towards the cathode (i.e. electroendo-osmosis) with concomitant drift of pH gradient and focused protein zones in the same direction. It is impossible to cure the cathodic drift by increasing the pH of the anolyte, or decreasing the pH of the catholyte, or both, as previously suggested. One way to reduce the cathodic drift efficiently is to incorporate covalently in the matrix tertiary or quaternary groups (3-dimethylaminopropylmethacrylamide) in stoichiometric amounts as compared with the negative charges.This ‘balanced’ polyacrylamide displays zero drift for at least 5000V·h, which is considered to be an ample time for equilibrium separation of protein species in isoelectric focusing.     

Focusing of pepsin in strongly acidic immobilized pH gradients

A new acrylamido buffer has been synthesized, for use in isoelectric focusing in immobilized pH gradients. This compound (2-acrylamido glycolic acid) has a pK = 3.1 (at 25 degrees C, 20 mM concentration during titration) and is used, by titration with the pK 9.3 Immobiline, to produce a linear pH gradient in the pH 2.5-3.5 interval. Pepsin (from pig stomach) focused in this acidic pH gradient is resolved into four components, two major (with pI values 2.76 and 2.78) and two minor (having pI values 2.89 and 2.90). This is the first time that such strongly acidic proteins could be focused in an immobilized pH gradient. Even in conventional isoelectric focusing in amphoteric buffers it has been impossible to focus reproducibly very-low-pI macromolecules.     

Large-scale electrophoresis for protein purification: exploiting isoelectricity

Preparative electrophoresis methods (including isoelectric focusing in immobilized pH gradients) in gel phases are characterized by low loadings barely a few mg protein per ml matrix), low recoveries (rarely exceeding 70%), and heavy contamination from neurotoxic gel materials (the unreacted gel monomers and ungrafted oligomers). These drawbacks can be minimized by a version of isoelectric focusing in which the need for protein of interest to pass the gel is eliminated: only the contaminants traverse the gel. This is achieved by circulating a liquid sample between two gels held at controlled pHs. The method can provide: (1) high rate of sample processing (up to 1 g h⁻¹); (2) high purification (in general to charge homogeneity); and (3) high recoveries (>95%). A large-scale membrane apparatus has been built, with a cross- sectional diameter of 9 cm. Large Pt electrode disks provide even current flow. In this electrolyser, 10 g of Eglin C (produced by recombinant DNA technology) have been purified to homogeneity in around 10 h from 1 l of a partially enriched preparation.     

Capture of human monoclonal antibodies from cell culture supernatant by ion exchange media exhibiting high charge density

A shortcut purification sequence for therapeutic proteins should consist of three steps: capture, purification, and polishing. Special emphasis has been put on direct capture of human monoclonal antibodies from culture supernatants with ion-exchangers avoiding pretreatment steps such as desalting, dilution, and other means to reduce the ionic strength. CM-HyperD, a cation-exchanger composed of an inorganic macroporous support filled with a viscoelastic gel with a high charge density was used. Capture of monoclonal antibodies from clarified hybridoma cell culture grown in media supplemented with fetal calf serum was investigated. Screening of different pH conditions and buffers for the load step showed that monoclonal antibodies were efficiently bound by CM-HyperD at pH 4.0 and 5.0 at an ionic strength equivalent to culture supernatant. Combination of negative purification with Q-Sepharose FF and capturing with CM-HyperD gave sufficient yield and resolution. Implementation of wash steps with higher conductivity did not improve the purity, but decreased the yield. Interestingly, high flow rates improved the purity. When antibodies were captured from serumfree culture supernatant the antibody could be eluted in a single peak with substantial reduction of contaminants. Capturing of antibodies by ion-exchange sorbents from culture supernatant is possible despite the high salt content.     

Two-dimensional gel electrophoretic resolution of the polypeptides of rat liver mitochondria and the outer membrane

The proteins of highly purified rat liver mitochondria were resolved by two-dimensional polyacrylamide gel electrophoresis, and detected by staining with either Coomassie blue or silver. Approximately 250 polypeptides were detected with silver staining which is 2- to 3-times that observed with Coomassie blue. Silver staining was especially more effective than Coomassie blue for detecting polypeptides of less than 50 000 daltons. A two-dimensional gel pattern of rat liver microsomes was distinct from that of the mitochondria. The mitochondrial outer membrane was prepared from purified mitochondria either with digitonin or by swelling in a hypotonic medium. As assessed by marker enzymes, the latter method yielded a considerably purer outer membrane preparation (20-fold purification) than the former (2.6-fold purification). Approximately 50 polypeptides were observed in a two-dimensional gel (pH 3-10) of the highly purified outer membrane fraction. Three isoelectric forms of the pore (VDAC) protein were observed with pI values of 8.2, 7.8 and 7.1. Monoamine oxidase was identified as a polypeptide of Mr 60 000. About 50 polypeptides were also resolved in a reverse polarity non-equilibrium pH gradient electrophoresis gel of the outer membrane, pH 3-10, with at least six isoelectric forms of the VDAC protein observed under these conditions. The six isoforms of the VDAC protein were also observed in a non-equilibrium gel with 2 micrograms of the purified protein.