High-Performance Liquid Chromatography of Proteins on Deformed Nonporous Agarose Beads. Affinity Chromatography of Dehydrogenases Based on Cibacron Blue-Derivatized Agarose

Nonporous agarose beads, prepared by shrinkage and cross-linking in organic solvents, were derivatized with Cibacron Blue F3G-A. A compressed bed of these beads was used for purification of dehydrogenases (glucose-6-phosphate dehydrogenase, lactate dehydrogenase and alcohol dehydrogenase). The chromatographic conditions for the purification of glucose-6-phosphate dehydrogenase were optimized by varying the pH of the buffer; the concentrations of eluting agents, i.e. NADP (specific elution) and sodium chloride (nonspecific elution); flow rate; residence time of the protein on the column bed; and protein load. Specific elution with NADP (2 mM in 0.025 M Tris-HCl, pH 8.0) gave the highest recovery (140%) and highest purification factor (200-fold) of the enzyme. The ability of the compressed bed of nonporous agarose beads to tolerate high flow rates was essential, since the recovery of the enzyme activity increased with an increase in flow rate.     

Expanded bed affinity chromatography of dehydrogenases from bakers' yeast using dye-ligand perfluoropolymer supports

Malate dehydrogenase (MDH) and glucose 6-phosphate dehydrogenase (G6PDH) have been partially purified from preparations of homogenized yeast cells using Procion Yellow H-E3G and Procion Red H-E7B, respectively, immobilized on solid perfluoropolymer supports in an expanded bed. A series of pilot experiments were carried out in small packed beds using clarified homogenate to determine the optimal elution conditions for both MDH and G6PDH. Selective elution of MDH using NADH was effective but the yields obtained were dependent on the concentration of NADH used. Selective elution was found to be most effective when a low concentration of NaCl (0.1 M) was present. MDH could be recovered in 84% yield with a purification factor of 94 when this strategy was adopted. In the case of G6PDH, specific elution using NADP(+) was successful in purifying G6PDH 178-fold in 96% yield. The dynamic capacity of both affinity supports was estimated by frontal analysis, in an expanded bed with unclarified homogenate, and corresponded to 17 U MDH/mL of settled Procion Yellow H-E3G perfluoropolymer support and 7.7 U H6PDH/mL of settled Procion Red H-E7B perfluoropolymer support. Expanded bed affinity chromatography of MDH resulted in an eluted fraction containing 89% of the applied activity with a purification factor of 113. Expanded bed affinity chromatography of G6PDH resulted in an eluted fraction containing 84% of the applied activity with a purification factor of 172. With both enzymes, the overall recovery of enzyme activity was greater than 94%, showing that the expanded bed approach to purification was nondenaturing. (c) 1995 John Wiley & Sons, Inc.     

Purification of equine chorionic gonadotropin (eCG) using magnetic ion exchange adsorbents in combination with high‐gradient magnetic separation

Current purification of the glycoprotein equine chorionic gonadotropin (eCG) from horse serum includes consecutive precipitation steps beginning with metaphosphoric acid pH fractionation, two ethanol precipitation steps, and dialysis followed by a numerous of fixed‐bed chromatography steps up to the specific activity required. A promising procedure for a more economic purification procedure represents a simplified precipitation process requiring only onethird of the solvent, followed by the usage of magnetic ion exchange adsorbents employed together with a newly designed ‘rotor‐stator’ type High Gradient Magnetic Fishing (HGMF) system for large‐scale application, currently up to 100 g of magnetic adsorbents. Initially, the separation process design was optimized for binding and elution conditions for the target protein in mL scale. Subsequently, the magnetic filter for particle separation was characterized. Based on these results, a purification process for eCG was designed consisting of (i) pretreatment of the horse serum; (ii) binding of the target protein to magnetic ion exchange adsorbents in a batch reactor; (iii) recovery of loaded functionalized adsorbents from the pretreated solution using HGMF; (iv) washing of loaded adsorbents to remove unbound proteins; (v) elution of the target protein. Finally, the complete HGMF process was automated and conducted with either multiple single‐cycles or multicycle operation of four sequential cycles, using batches of pretreated serum of up to 20 L. eCG purification with yields of approximately 53% from single HGMF cycles and up to 80% from multicycle experiments were reached, with purification and concentration factors of around 2,500 and 6.7, respectively. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:78–89, 2015     

Vector engineering anomalies: impact on fusion protein purification performance

Recombinant protein purification using IMAC is often carried out by protein fusion to affinity tags. We have identified several tags useful for protein purification on Zn(II)-IDA columns. These tags were fused to the green fluorescent protein (rGFPuv) using the vector pGFPuv distributed by Clontech Lab (Palo Alto, CA) and analyzed for purification on Zn(II)-IDA. Each fusion protein exhibited elution heterogeneity (elution in two distinct pHs) from Zn(II)-IDA columns This led us to believe that two populations of fluorescent proteins were being expressed: one without the tag coeluting with Escherichia coli proteins at pH 7.5 and one bearing the tag eluting at a pH lower than pH 7.5. Assessment of the constructs revealed the possibility of a ribosomal binding site and start codon between the fusion tag and the rGFPuv sequence which might be used as a secondary translation start site. This hypothesis was confirmed by changing the second ATG (methionine) codon to an ACG (threonine) codon. The protein produced from this new construct eluted in a single fraction from a Zn(II)-IDA column. Thus, vector irregularities (along with other possibilities) should be examined when searching for the cause of elution heterogeneity of a target protein.     

High-throughput protein purification under denaturating conditions by the use of cation exchange chromatography

A high-throughput protein purification strategy using the polycationic Z(basic) tag has been developed. In order for the strategy to be useful both for soluble and less soluble proteins, a denaturating agent, urea, was used in all purification steps. First, four target proteins were genetically fused to the purification tag, Z(basic). These protein constructs were purified by cation exchange chromatography and eluted using a salt gradient. From the data achieved, a purification strategy was planned including stepwise elution to enable parallel protein purification using a laboratory robot. A protocol that includes all steps, equilibration of the chromatography resin, load of sample, wash, and elution, all without any manual handling steps, was handled by the laboratory robot. The program allows automated purification giving milligram amounts of pure recombinant protein of up to 60 cell lysates. In this study 22 different protein constructs, with different characteristics regarding pI and solubility, were successfully purified by the laboratory robot. The data show that Z(basic) can be used as a general purification tag also under denaturating conditions. Moreover, the strategy enables purification of proteins with different pI and solubility using ion exchange chromatography (IEXC). The procedure is highly reproducible and allows for high protein yield and purity and is therefore a good complement to the commonly used His(6)-tag.     

Purification from goat antiserum of immunoglobulins against G6PD from rabbits]

DEAE Affi-Gel Blue (Bio-Rad) provides an efficient and rapid fractionation of human serum proteins by a single chromatographic step. When goat serum is applied to the matrix and chromatography is performed following the procedure utilized for the human serum proteins, the elution pattern changes and the Ig purification is not satisfactory. We achieved a better Ig purification from goat serum by the following improved procedure. We performed first an AS-40 fractionation followed by extensive dialysis in 50 mM Na-citrate pH 5.7. The sample was then loaded onto a P11 column equilibrated in the same buffer. The fraction eluted at Vo contained total IgG and the other serum proteins, except beta-globulins which were eluted with 0.24 M phosphate. Peak 1 concentrated and dialyzed in 20 mM phosphate buffer pH 8 was then applied to a DEAE Affi-Gel Blue column, equilibrated in the same buffer. Two protein peaks were eluted from this column and electrophoretically characterized as: peak 1, containing a pure Ig fraction (70% yield), peak 2 with albumin and other contaminating serum proteins. When goat antiserum is obtained against a specific protein, our technique may be suitably employed to purify polyclonal antibodies for immunoprecipitation studies.     

Development of an expanded bed technique for an affinity purification of G6PDH from unclarified yeast cell homogenates

The use of an expanded bed of STREAMLINE Red H-7B for the purification of the intracellular glycolytic enzyme glucose 6-phosphate dehydrogenase (G6PDH) directly from untreated preparations of disrupted yeast cells has been investigated. Small-scale experiments, carried out in packed beds, have shown that the optimal pH for adsorption is 6.0 and have enabled optimization of elution conditions using a series of eluents. The dynamic capacity of the adsorbent for G6PDH was determined in a small expanded bed to be 28 units/mL. These results were used to develop a preparative scale separation of G6PDH in a STREAMLINE 50 expanded bed column. G6PDH was purified directly from an unclarified yeast homogenate in 99% yield with an average purification factor in the eluted fraction of 103. Cleaning-in-place (CIP) procedures using 0.5 M NaOH and 4M urea in 60% (v/v) ethanol have demonstrated that the adsorbent can be regenerated with no loss of adsorption capacity of alteration of bed expansion characteristics after many cycles of operation. (c) 1995 John Wiley & Sons, Inc.     

Raw starch adsorption-desorption purification of a thermostable beta-amylase from Clostridium thermosulfurogenes

The beta-amylase from Clostridium thermosulfurogenes was readily adsorbed onto raw starch. The adsorbed beta-amylase was eluted from raw starch by using boiled soluble starch solution as an elutant. The soluble starch treated beta-amylase could not adsorb onto raw starch which indicates that the soluble and insoluble substrate binding sites of the beta-amylase may be the same. The beta-amylase was purified to homogeneity by raw starch adsorption-desorption techniques and octyl-Sepharose chromatography. It had a specific activity of 4188 units/mg protein. The insoluble substrate adsorption-desorption technique may be used for the purification of other enzymes.     

Partial purification of a specific inhibitor of the insulin-like growth factors by reversed-phase high-performance liquid chromatography

We report here preliminary data using reversed-phase high-performance liquid chromatography for the purification of a specific inhibitor (a molecular weight 16,000–18,000 protein) of the insulin-like growth factor (IGF) or somatomedin family. Crude inhibitor prepared from Cohn fraction IV-1 of human serum was first partially purified using an IGF/CH-Sepharose 4B affinity column. Following elution of the bound inhibitor and resuspension in 0.1% aqueous trifluroacetic acid (mobile phase A), it was injected (100 μl; 2.0 mg protein) onto a Brownlee Aquapore RP-300 column. Application of a linear gradient from 0% to 100% mobile phase B (45% isopropanol−0.1% trifluoroacetic acid) resulted in elution of two peaks of inhibitor activity between 31% and 34% isopropanol associated with a major homogeneous protein peak and a minor heterogeneous protein peak. No inhibitor was recovered when an acetonitrile gradient was used instead of isopropanol, indicating that the inhibitor is very hydrophobic. These data suggest that high-performance liquid chromatography offers a simple procedure for the potential purification of IGF inhibitor(s) from normal human serum.     

Partial purification, properties, and subcellulsr distribution of rat liver phosphatidate phosphatase.

Phosphatidate phosphatase (EC 3.1.3.4Y was purified 15- to 20-fold from the soluble fraction of rat liver. The purification procedure involved calcium phosphate gel adsorption and elution, ammonium sulfact precipitation, and molecular-sieve chromatography. For the enzyme assay, and aqueous dispersion of phosphatidate, rather than "membrane-bound" phosphatidate, was used as substrate. The partially purified enzyme depends almost entirely on the presence of Mg2+ for its activity. Morover, the activity of the enzyme is stimulated by phosphatidylcholine. The enzyme exhibits a high substrate specificity for phosphatidate. The apparent Km for phosphatidate is approximately 0.05 mM. The optimum pH is between 7.4 and 7.6. The enzyme is inhibited by fluoride and by p-chloromercuribenzoate. The subcellular distribution of phosphatidate phosphatase in rat liver was studied by assaying the activity of the enzyme in the presence of Mg2+ and phosphatidylcholine. In contrast ot the results of previous studies, most of the enzyme activity was found in the soluble fraction.     

Production and partial characterization of a beta-amylase by Xanthophyllomyces dendrorhous

AIMS: The characterization of a beta-amylase produced by Xanthophyllomyces dendrorhous. METHODS AND RESULTS: Growth in different culture media showed that X. dendrorhous produces an amylase whose synthesis is repressed by the carbon source and induced by starch and maltose. Enzymatic assays using substrates with different degrees of polymerization together with viscosity experiments revealed that the enzyme was beta-amylase. According to the biochemical characterization, the enzyme has a molecular weight of 240 kDa and a Km of 1.35 mg ml-1. The optimum pH and temperature were 5.5 and 50 degrees C, respectively. Using different inhibitors of the enzymatic activity it was shown that cysteine, tryptophan and serine are essential amino acids for catalysis. CONCLUSIONS: Xanthophyllomyces dendrorhous CECT1690 synthesizes and secretes beta-amylase that could be a by-product, in addition to carotenoid pigments, in the fermentation downstream. SIGNIFICANCE AND IMPACT OF THE STUDY: The beta-amylase produced by X. dendrorhous may have certain industrial applications.     

A TRYPSIN‐LIKE PROTEINASE IN THE MIDGUT OF Ectomyelois ceratoniae ZELLER (LEPIDOPTERA: PYRALIDAE): PURIFICATION,CHARACTERIZATION, AND HOST PLANT INHIBITORS

A trypsin‐like proteinase was purified and characterized in the midgut of Ectomyelois ceratoniae. A purification process that used Sepharyl G‐100 and DEAE‐cellulose fast flow chromatographies revealed a proteinase with specific activity of 66.7 μmol/min/mg protein, recovery of 27.04 and purification fold of 23.35. Molecular weight of the purified protein was found to be 35.8 kDa. Optimal pH and temperature were obtained 9 and 20°C for the purified trypsin proteinase, respectively. The purified enzyme was significantly inhibited by PMSF, TLCK, and SBTI as specific inhibitors of trypsins in which TLCK showed the highest inhibitory effect. Trypsin proteinase inhibitors were extracted from four varieties of pomegranate including Brait, Torsh‐Sabz, May‐Khosh, and Shirin by ion exchange chromatography. It was found that fractions 17–20 of Brait; fractions 18 and 21–26 of Torsh‐Sabz; fractions 1–7, 11–17, and 19–21 of May‐Khosh and fraction 8 for Shirin showed presence of trypsin inhibitor in these host. Comparison of their inhibitory effects on the purified trypsin proteinase of E. ceratoniae demonstrated that fractions from May‐khosh variety had the highest effect on the enzyme among other extracted fractions. Characterization of serine proteinases of insects mainly trypsins is one of the promising methods to decrease population and damages via extracting their inhibitors and providing resistant varieties.     

Single step purification of a series of wheat recombinant proteins with expanded bed absorption chromatography

Expanded bed absorption chromatography (EBA) was used to improve and simplify the purification of several wheat recombinant proteins. Binding and elution conditions were set to allow the purification of the over expressed protein in a single step. In comparison with our previous multi step protocol, same purity was obtained while EBA required less time (one day instead of five) and gave a higher yield (63% instead of 10%). This new procedure was then used for the successful purification of five other wheat ns-LTP. Despite their important polymorphism (identity from 44 to 97 %-pHi from 8 to 10), the EBA protocol allowed their purification in a single step.     

Downregulation of a chloroplast-targeted beta-amylase leads to a starch-excess phenotype in leaves

A functional screen in Escherichia coli was established to identify potato genes coding for proteins involved in transitory starch degradation. One clone isolated had a sequence very similar to a recently described chloroplast-targeted beta-amylase of Arabidopsis. Expression of the gene in E. coli showed that the protein product was a functional beta-amylase that could degrade both starch granules and solubilized amylopectin, while import experiments demonstrated that the beta-amylase was imported and processed into pea chloroplasts. To study the function of the protein in transitory starch degradation, transgenic potato plants were generated where its activity was reduced using antisense techniques. Analysis of plants reduced in the presence of this beta-amylase isoform showed that their leaves had a starch-excess phenotype, indicating a defect in starch degradation. In addition, it was shown that the antisense plants degraded only 8-30% of their total starch, in comparison with 50% in the wild type, over the dark period. This is the first time that a physiological role for a beta-amylase in plants has been demonstrated.     

Purification of xanthine oxidase from bovine milk by affinity chromatography with a novel gel

Abstract

A new affinity gel was synthesized for the purification of xanthine oxidase (XO, EC 1.2.3.22) from bovine milk. The gel was prepared on a Sepharose 4B matrix on which a spacer arm based on l-tyrosine was covalently attached via CNBr activation, followed by reaction with the XO inhibitor p-aminobenzamidine. The elution conditions of affinity gel were determined at different pH values and ionic strengths. Maximum elution of XO was achieved at pH 9.0 and ionic strength around 0.4. The overall purification for XO was 1645-fold with 20.49% yield. SDS-PAGE of the enzyme indicates a single band with an apparent MW of 150?kDa. The gel provides a simple, rapid and effective useful for the purification of XO. Heat stability was determined on purified XO activity. Xanthine oxidase was preserved up to 70% with activity exposure of 60?°C and incubated for 60?min. These results indicated that the enzyme was heat stable.     

Purification of beta-glucocerebrosidase by preparative-scale high-performance liquid chromatography: the use of ethylene glycol-containing buffers for chromatography of hydrophobic glycoprotein enzymes

beta-Glucocerebrosidase, partially purified by the method of F. S. Furbish et al. (1977, Proc. Natl. Acad. Sci. USA 74, 3560-3563), was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to contain, in addition to the desired enzyme, variable amounts of a very hydrophobic contaminant (apparent Mr 45,000). Purification of the enzyme was accomplished by gel-permeation HPLC on a TSK 3000 SW column (0.7 X 60 cm). Adsorptive losses of protein on the column were minimized by using buffers containing up to 50% ethylene glycol. We have examined the effects of varying the ethylene glycol concentration on the elution times and recoveries of the two major proteins in this preparation. The high reproducibility of the individual chromatograms permitted the use of an automatic sampler and fraction collector to perform multiple, continuous runs for the purification of milligram quantities of enzyme. Multiple runs of a preparative-scale column, TSK G3000 SWG (2.15 X 60 cm), permitted gram-scale purification of beta-glucocerebrosidase without loss in efficiency of separation. Recovery of enzyme activity is greater than 94% with less than 1% contamination by other proteins. Reaction of enzyme prepared in this fashion with rabbit polyclonal antiserum or mouse monoclonal anti-beta-glucocerebrosidase shows the enzyme to be pure and not immunologically related to the 45,000 Mr contaminant. The specific activity of enzyme prepared by this means is 1.6 X 10(6) nmol/h/mg protein. Inclusion of ethylene glycol in buffers was shown to overcome hydrophobic protein interactions with TSK 3000 SW column matrices for both the soluble human lysosomal enzyme alpha-galactosidase A and the plant toxin ricin.     

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.     

High-performance liquid chromatography of proteins on deformed nonporous agarose beads. Affinity chromatography of dehydrogenases based on cibacron blue-derivatized agarose

Nonporous agarose beads, prepared by shrinkage and cross-linking in organic solvents, were derivatized with Cibacron Blue F3G-A. A compressed bed of these beads was used for purification of dehydrogenases (glucose-6-phosphate dehydrogenase, lactate dehydrogenase and alcohol dehydrogenase). The chromatographic conditions for the purification of glucose-6-phosphate dehydrogenase were optimized by varying the pH of the buffer; the concentrations of eluting agents, i.e. NADP (specific elution) and sodium chloride (nonspecific elution); flow rate; residence time of the protein on the column bed; and protein load. Specific elution with NADP (2 mM in 0.025 M Tris-HCl, pH 8.0) gave the highest recovery (140%) and highest purification factor (200-fold) of the enzyme. The ability of the compressed bed of nonporous agarose beads to tolerate high flow rates was essential, since the recovery of the enzyme activity increased with an increase in flow rate.     

Cloning,Sequencing, and Expression of a β-Amylase Gene from Bacillus cereus var. mycoides and Characterization of Its Products

The cloned gene was composed of 1638 bp for coding plus promoter like and SD-like sequences ahead of it. The deduced amino acid sequence had high similarity with known β-amylases. The N-terminal sequence of the cloned β-amylase seemed to be a signal peptide. The gene was introduced into Bacillus subtilis 1A289 using pHY300PLK as a vector and the expressed protein was recovered from the culture media. The enzyme fraction produced was divided into two components upon the DEAE column chromatography. The amino acid sequence of one fraction (FrI) was the same as the mature enzyme, and the other (FrII) lacked the N-terminal amino acid residue (Ala) of the mature enzyme. The kinetic parameters of the hydrolysis catalyzed by the enzyme component FrI were measured, and the subsite affinities of the enzyme were evaluated. In conclusion, it was shown that the recombinant enzyme was the same as the mature enzyme functionally and proteochemically.     

Purification of Escherichia coli alkaline phosphatase on an ion-exchange high-performance liquid chromatographic column using carboxymethyl dextrans

Carboxymethyl dextrans (CM-Ds) were used on an HPLC ion-exchange column to obtain significantly enriched alkaline phosphatase (EC 3.1.3.1) from a sample of Escherichia coli periplasmic space proteins without significant loss of enzymatic activity. The ability of CM-Ds to separate alkaline phosphatase even when the column was 80-85% saturated with protein demonstrates the potential for high column capacity using CM-Ds. In addition, the fractions containing alkaline phosphatase and CM-Ds were reapplied to the same ion-exchange column under different buffer conditions and purified to homogeneity by salt gradient elution chromatography, thus demonstrating the compatibility of CM-Ds with the latter chromatographic method. The two-step chromatographic procedure yielded enzyme of purity comparable to that of electrophoretically purified E. coli alkaline phosphatase obtained commercially. These studies demonstrate that HPLC displacement chromatography is a mild procedure which allows rapid, quantitative purification of an enzyme. Scaling up with larger columns should allow purification of enzymes of a commercial basis.