Production of milligram quantities of affinity tagged-proteins using automated multistep chromatographic purification

A new chromatography system, AKTAxpress (GE Healthcare, Amersham Biosciences, Uppsala, Sweden) has been designed to meet the demand for high-throughput purification of proteins in structural genomics and drug discovery. The system offers a number of automated multistep purification protocols for affinity-tagged proteins. All protocols start with affinity chromatography followed by combinations of desalting, ion exchange chromatography and gel filtration. As an option, tag removal can be included in the purification protocols. Up to 16 proteins can be purified per day and the yield can be as high as 50 mg of each protein at > 90% purity. To highlight the versatility of the system, this paper presents several case studies; purifications of hexahistidine- and glutathione S-transferase-tagged proteins using different protocols, automated on-column tag cleavage and optimization studies for a hexahistidine-tagged kinase.     

Bovine lactoferrin purification from whey using Yellow HE‐4R as the chromatographic affinity ligand

The worldwide production of whey increases by around 186 million tons each year and it is generally considered as a waste, even when several whey proteins have important economic relevance. For its valorization, inexpensive ligands and integrated chromatography methods need to be developed for specific and low‐cost protein purification. Here, we describe a novel affinity process with the dye Yellow HE‐4R immobilized on Sepharose for bovine lactoferrin purification. This approach based on a low‐cost ligand showed an efficient performance for the recovery and purification of bovine lactoferrin directly from whey, with a yield of 71% and a purification factor of 61.     

Efficient high-performance liquid chromatographic system for protein purification

An efficient high-performance liquid chromatographic system, consisting of an affinity column and a high-performance size-exclusion column, was developed and applied to the purification of growth hormone receptors from rabbit livers. When a 6-ml sample of Triton X-100 extracts containing 16 mg of protein was applied to the system, 1200-fold purified receptor with a 10% recovery of binding activity from homogenates was obtained within 3-4 h. The purified receptor exhibited one main band on sodium dodecyl sulphate polyacrylamide gel electrophoresis, and the affinity constant (Ka = 6.0.10(9) M-1) was found to be comparable with that of 1% Triton X-100 extract (4.4.10(9) M-1). The injection of 1 ml of 3 M urea solution prior to receptor elution with 10 ml of 6 M urea solution was effective in removing non-specific binding proteins.     

Investigation of protein binding affinity in multimodal chromatographic systems using a homologous protein library

A library of cold shock protein B mutant variants was employed to examine differences in protein binding behavior in ion exchange and multimodal chromatography. Single site mutations introduced at charged amino acids on the protein surface resulted in a homologous protein set with varying charge density and distribution. The retention times of the mutants varied significantly during linear gradient chromatography in both systems. The majority of the proteins were more strongly retained on the multimodal cation exchange resin as compared to the traditional cation exchanger. Further, the elution order of the mutants on the multimodal resin was different from that obtained with the ion exchanger. Quantitative structure–property relationship models generated using a support vector regression technique were shown to provide good predictions for the retention times of protein mutants on the multimodal resin. A coarse-grained ligand docking package was employed to examine the various interactions between the proteins and ligands in free solution. The multimodal ligand was shown to utilize multiple interaction types to achieve stronger retention on the protein surface. The use of this protein library in concert with the qualitative and quantitative analyses presented in this paper provides an improved understanding of protein behavior in multimodal chromatographic systems.     

Novel ion chromatographic stationary phases for the analysis of complex matrices

Ion chromatography (IC) has a proven track record in the determination of inorganic and organic anions and cations in complex matrices. Recently, application of IC to the separation and determination of bio-molecules such as amino acids, carbohydrates, nucleotides, proteins and peptides has also received much attention. The key to the determination of all of the above species in the most analytically challenging complex matrices is the ability to manipulate selectivity through control of stationary phase chemistry, mobile phase chemistry and the choice of detection method. This Tutorial Review summarises some of the most significant recent advances made in IC stationary phase technology. In particular, the review details stationary phases specifically designed for ion analysis in complex sample matrices, and considers in which direction future stationary phase development might proceed.     

High-performance liquid affinity chromatography for the purification of immunoglobulin A from human serum using jacalin

A high-performance liquid affinity chromatographic method for the purification of serum immunoglobulin A (IgA) using a jacalin column is described. The automated procedure takes about 2 with minimal manipulation. The yields of the isolated IgA and of its IgG and IgM contamination were studied by enzyme-linked immunosorbent assay (ELISA) of 30 sera. Purity was assured by immunoelectrophoresis. The ratio of IgA1 to total IgA was unchanged after purification, as verified by ELISA. The results showed that greater than 90% IgA could be recovered with less than 0.5% total IgG and greater than 2.0% total IgM remaining in the fractions containing purified IgA.     

Rapid affinity purification of erythropoietin from biological samples using disposable monoliths

Identification of post-translational modifications of proteins in biological samples often requires access to preanalytical purification and concentration methods. In the purification step high or low molecular weight substances can be removed by size exclusion filters, and high abundant proteins can be removed, or low abundant proteins can be enriched, by specific capturing tools. In this paper is described the experience and results obtained with a recently emerged and easy-to-use affinity purification kit for enrichment of the low amounts of EPO found in urine and plasma specimens. The kit can be used as a pre-step in the EPO doping control procedure, as an alternative to the commonly used ultrafiltration, for detecting aberrantly glycosylated isoforms. The commercially available affinity purification kit contains small disposable anti-EPO monolith columns (6 μL volume, Ø7 mm, length 0.15 mm) together with all required buffers. A 24-channel vacuum manifold was used for simultaneous processing of samples. The column concentrated EPO from 20 mL urine down to 55 μL eluate with a concentration factor of 240 times, while roughly 99.7% of non-relevant urine proteins were removed. The recoveries of Neorecormon (epoetin beta), and the EPO analogues Aranesp and Mircera applied to buffer were high, 76%, 67% and 57%, respectively. The recovery of endogenous EPO from human urine was 65%. High recoveries were also obtained when purifying human, mouse and equine EPO from serum, and human EPO from cerebrospinal fluid. Evaluation with the accredited EPO doping control method based on isoelectric focusing (IEF) showed that the affinity purification procedure did not change the isoform distribution for rhEPO, Aranesp, Mircera or endogenous EPO. The kit should be particularly useful for applications in which it is essential to avoid carry-over effects, a problem commonly encountered with conventional particle-based affinity columns. The encouraging results with EPO propose that similar affinity monoliths, with the appropriate antibodies, should constitute useful tools for general applications in sample preparation, not only for doping control of EPO and other hormones such as growth hormone and insulin but also for the study of post-translational modifications of other low abundance proteins in biological and clinical research, and for sample preparation prior to in vitro diagnostics.     

Novel affinity ligands for chromatography using combinatorial chemistry

Spatially addressable combinatorial libraries were synthesized by solution phase chemistry and screened for binding to human serum albumin. Members of arylidene diamide libraries were among the best hits found, having submicromolar binding affinities. The results were analyzed by the frequency with which particular substituents appeared among the most potent compounds. After immobilization of the ligands either through the oxazolone or the amine substituent, characterization by surface plasmon resonance showed that ibuprofen affected the binding kinetics, but phenylbutazone did not. It is therefore likely that these compounds bind to Site 2 in sub domain IIIA of human serum albumin (HSA).     

Dual affinity method for plasmid DNA purification in aqueous two-phase systems

The DNA binding fusion protein, LacI–His₆–GFP, together with the conjugate PEG–IDA–Cu(II) (10 kDa) was evaluated as a dual affinity system for the pUC19 plasmid extraction from an alkaline bacterial cell lysate in poly(ethylene glycol) (PEG)/dextran (DEX) aqueous two-phase systems (ATPS). In a PEG 600–DEX 40 ATPS containing 0.273 nmol of LacI fusion protein and 0.14% (w/w) of the functionalised PEG–IDA–Cu(II), more than 72% of the plasmid DNA partitioned to the PEG phase, without RNA or genomic DNA contamination as evaluated by agarose gel electrophoresis. In a second extraction stage, the elution of pDNA from the LacI binding complex proved difficult using either dextran or phosphate buffer as second phase, though more than 75% of the overall protein was removed in both systems. A maximum recovery of approximately 27% of the pCU19 plasmid was achieved using the PEG–dextran system as a second extraction system, with 80–90% of pDNA partitioning to the bottom phase. This represents about 7.4 μg of pDNA extracted per 1 mL of pUC19 desalted lysate.     

Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography

In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu‐iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14‐atom spacer arm. The absorption analysis of this medium revealed a desorption constant K_d of 21.5 μg/mL and a theoretical maximum absorption Q_max of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high‐performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases.     

Novel affinity chromatography method for the efficient purification of recombinant Binder of SPerm homolog proteins

In mammalian species, a family of proteins named the Binder of SPerm proteins, which are expressed in the male reproductive tract, have been shown to play a role in epididymal sperm maturation and sperm capacitation. Recently, one homolog from human and two homologs from mouse were characterized. In order to further investigate the biochemical activity of these proteins, efficient purification procedures are required to isolate the proteins. Since these proteins are produced in very minute quantities, we exploited the high capacity of Escherichia coli to produce larger quantities of recombinant proteins that were subsequently purified using affinity chromatography on a diethylaminoethyl‐Sephadex A‐25 column. Binder of SPerm proteins have been shown to interact with pseudo‐choline groups such as diethylaminoethyl through affinity rather than ionic interactions. The aim of the current study was to develop a novel method for purifying these recombinant proteins, produced in Escherichia coli cells. Diethylaminoethyl is positively charged and is a weak anion exchanger, but binder of sperm proteins interacts with affinity to this resin. This study presents a new, rapid, and cost‐effective purification method that provides with an exceptional purity level, which can be used to study their roles in mammalian fertilization.     

Novel affinity purification of monomeric sarcosine oxidase expressed in Escherichia coli

An efficient affinity‐purification protocol for Bacillus monomeric sarcosine oxidase (SOX) expressed in Escherichia coli BL21 (DE3) was developed. 4‐Aminopyrrole‐2‐carboxylic acid was chosen as the affinity ligand, which was coupled with Sepharose CL 4B via spacers composed of epichlorohydrin and ethylenediamine. With the affinity medium, the purification process consisted of only one affinity chromatography step to capture monomeric SOX. The purified SOX was 94 and 96% pure when analyzed on an HPLC Vydac C₈ column and reducing SDS‐PAGE. Meanwhile, the recoveries of typical SOX activity and protein were 90.8 and 37.5%, respectively, which were higher than other reported traditional protocols. Reducing SDS‐PAGE analysis revealed that the enzyme was a single polypeptide with the mass of ～46 kDa. The desorption constant K_d and theoretical maximum absorption Q_max were 35 μg/mL and 52.7 mg/g, respectively, in absorption analysis. All results indicated that the method would be of great potential for purifying monomeric SOX on an industrial scale.     

Improved affinity chromatographic purification of D-mannose-N-acetyl-D-glucosamine-specific lectin from the bark of Sophora japonica eliminating the loss by sugar specific self-aggregation.

A novel D-mannose-N-acetyl-D-glucosamine-specific lectin of Sophora japonica bark, B-SJA-II, which showed self-aggregation based on sugar specificity, was purified by affinity chromatography on maltamyl-Sepharose subsequent to chromatographic separation on lactamyl-Sepharose to remove a major D-galactose-N-acetyl-D-galactosamine specific lectin, B-SJA-I. However, the yield of this method was low as a result of the sugar-specific precipitation and binding to other glycoproteins. A modified method was developed to circumvent this problem. All the purification procedures, except for the final chromatographic separation, were carried out in the presence of the haptenic sugar and the sugar-specific adsorption of B-SJA-II onto the adsorbent was carried out in a dialysis bag by gradually removing the sugar. This method gave a yield eight times higher than the original method.     

Potentiometric investigation of sparingly soluble metal-ligand systems using metal-ion buffers

A novel approach based on metal-ion buffers has been used to determine metal-ligand formation constants for a variety of sparingly soluble systems. It is suggested that the approach will be particularly useful in the study of metal binding by pharmaceuticals.     

Affinity chromatographic purification of lentil lectin using immobilized yeast cells

A model system for evaluating macroporous supports containing immobilized whole cells in affinity Chromatographic applications is described. Whole cells were immobilized in a polyacrylamide network in a two-step polymerization process. The affinity system discussed consists of immobilized cells ofSaccharomyces cervisiae in the purification of lentil lectin fromLens culinaris.     

Comparison of affinity membranes for the purification of immunoglobulins

The purification of immunoglobulins was studied by comparing 10 different affinity membranes, prepared by coupling various affinity ligands to different microfiltration membranes. Membranes carrying the synthetic peptide TG19318, histidine, the thiophilic ligand and iminodiacetic acid complexed with Zn(II) showed a weak affinity for human IgG, as expressed by apparent association constants (K_A) in the order of 10⁵ M⁻¹. Human IgM and rat IgG bound with high affinity to TG19318 membranes, thus, demonstrating the potential of this sorbent for the purification of immunoglobulins other than human IgG. When carrying Protein-A ligands, membranes based on Nylon 66 coated with low-molar-mass dextran or poly(vinylalcohol), as well as commercial pre-activated polysulfone (Ultrabind^®) and regenerated cellulose (Sartobind^®) membranes, showed high affinity for human IgG (K_A≈10⁶ M⁻¹). In contrast, a nylon membrane coated with high-molar-mass dextran yielded only K_A≈10⁵ M⁻¹, which was attributed to a low accessibility of the immobilized ligand. Besides the high association constants, Protein-A adsorbers based on polysulfone and regenerated cellulose membranes showed several other advantages, such as enhanced charge-to-charge consistency, simpler preparation procedure, membrane sterilisability, good selectivity for IgG purification from cell culture supernatant and good stability throughout repeated adsorption–elution cycles.     

Immobilised metal ion affinity chromatography purification of alcohol dehydrogenase from baker’s yeast using an expanded bed adsorption system

Alcohol dehydrogenase (ADH) from solutions of homogenised packed bakers’ yeast has been successfully purified using immobilised metal-ion affinity chromatography in an expanded bed. Method scouting carried out using pure ADH solutions loaded onto 5-ml HiTrap columns charged with Zn²⁺, Ni²⁺ and Cu²⁺ and eluted using 0–50 mM EDTA gradient found that charging with Zn²⁺ gave the highest recovery and the lowest EDTA concentration required for elution. These results were used to develop a protocol for the expanded bed system and further tested using clarified yeast homogenate loaded onto XK16/20 packed beds (approximately 30 ml) packed with Chelating Sepharose FastFlow matrix in order to determine the optimum elution conditions using EDTA. The ADH was found to elute at 5 mM EDTA and the dynamic and total binding capacities of Streamline chelating for ADH were found to be 235 U/ml and 1075 U/ml matrix, respectively. Expanded bed work based on a step EDTA elution protocol demonstrated that ADH could be successfully eluted from unclarified homogenised bakers’ yeast diluted to 10 mg/ml total protein content with a recovery of 80–100% that was maintained over five consecutive runs with a vigorous clean-in-place procedure between each run.     

Phosphorylcholine (PC)-bonded protein A affinity chromatographic medium for high-throughput purification with reduced non-specific protein adsorption

A protein A affinity chromatographic medium based on porous silica modified with phosphorylcholine (PC) groups and amino groups (PNSP) was synthesized. The PC groups functioned as suppressors of non-specific protein adsorption. Recombinant protein A was bound to the amino groups on PNSP with a glutaraldehyde used as a spacer (PNSP-PA). The PC groups and amino groups were immobilized on porous-silica particles using two silane coupling reagents, PC-bound silane, and 3-aminopropyltrimethoxysilane. After optimizing various factors in the synthetic process, the resultant protein A medium showed improvements in non-specific protein adsorption, dynamic binding capacity, and chemical stability under basic conditions compared with conventional protein A affinity media.     

Novel affinity separations based on perfluorocarbon emulsions. Use of a perfluorocarbon affinity emulsion for the purification of human serum albumin from blood plasma in a fluidised bed.

A perfluorocarbon affinity emulsion has been generated by homogenisation of a saturated perfluorocarbon oil with a polymeric fluorosurfactant based on poly(vinyl alcohol) (relative molecular mass 9000-10,000) previously derivatised with the triazine dye CI Reactive Blue 4. This affinity emulsion has subsequently been cross-linked in situ and used in a fluidised bed for the purification of human serum albumin (HSA) from blood plasma. HSA was quantitatively recovered in a semi-continuous fashion from plasma at an average purity of 90 +/- 3.3%. The albumin binding capacity of the emulsion has been shown to be 0.59 mg/ml by frontal analysis corresponding to a mol/mol ligand usage of 13.5%. In all regards, when used in a fluidised bed, the emulsions have been shown to behave as a normal chromatographic material. They are stable under operational conditions with no coalescence being observed for periods greater than 1 year. These novel liquid affinity supports present an exciting opportunity to develop a range of unit operations for the continuous purification of proteins.