Purification of human plasma retinol-binding protein by hydrophobic interaction chromatography

Human plasma retinol-binding protein has been purified to homogeneity by a simple method that requires an ammonium sulfate fractionation, a hydrophobic interaction chromatography on phenyl-Sepharose, which dissociates the complex between retinol-binding protein and its carrier, transthyretin, and a gel filtration on Sephadex G-50. The yield of pure protein is comparable or higher than that obtained with the more complex procedures previously reported.     

Studies of lysozyme binding to histamine as a ligand for hydrophobic charge induction chromatography

Histamine was immobilized on Sepharose CL‐6B (Sepharose) for use as a ligand of hydrophobic charge induction chromatography (HCIC) of proteins. Lysozyme adsorption onto Histamine‐Sepharose (HA‐S) was studied by adsorption equilibrium and calorimetry to uncover the thermodynamic mechanism of the protein binding. In both the experiments, the influence of salt (ammonium sulfate and sodium sulfate) was examined. Adsorption isotherms showed that HA‐S exhibited a high salt tolerance in lysozyme adsorption. This property was well explained by the combined contributions of hydrophobic interaction and aromatic stacking. The isotherms were well fitted to the Langmuir equation, and the equilibrium parameters for lysozyme adsorption were obtained. In addition, thermodynamic parameters (ΔH_ads, ΔS_ads, and ΔG_ads) for the adsorption were obtained by isothermal titration calorimetry by titrating lysozyme solutions into the adsorbent suspension. Furthermore, free histamine was titrated into lysozyme solution in the same salt‐buffers. Compared with the binding of lysozyme to free histamine, lysozyme adsorption onto HA‐S was characterized by a less favorable ΔG_ads and an unfavorable ΔS_ads because histamine was covalently attached to Sepharose via a three‐carbon‐chain spacer. Consequently, the immobilized histamine could only associate with the residues on the protein surface rather than those in the hydrophobic pocket, causing a less favorable orientation between histamine and lysozyme. Further comparison of thermodynamic parameters indicated that the unfavorable ΔS_ads was offset by a favorable ΔH_ads, thus exhibiting typical enthalpy‐entropy compensation. Moreover, thermodynamic analyses indicated the importance of the dehydration of lysozyme molecule and HA‐S during the adsorption and a substantial conformational change of the protein during adsorption. The results have provided clear insights into the adsorption mechanisms of lysozyme onto the new HCIC material. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Proteome analysis of rice root proteins regulated by gibberellin

To gain an enhanced understanding of the mechanism by which gibberellins （GAs） regulate the growth and development of plants, it is necessary to identify proteins regulated by GA. Proteome analysis techniques have been applied as a direct, effective, and reliable tool in differential protein expressions. In previous studies, sixteen proteins showed differences in accumulation levels as a result of treatment with GA3, uniconazole, or abscisic acid （ABA）, and/or the differences between the GA-deficient semi-dwarf mutant, Tan-ginbozu, and normal cultivars. Among these proteins, aldolase increased in roots treated with GA3, was present at low levels in Tan-ginbozu roots, and decreased in roots treated with uniconazole or ABA. In a root elongation assay, the growth of aldolase-antisense transgenic rice was half of that of vector control transgenic rice. These results indicate that increases in aldolase activity stimulate the glycolytic in the GA-induced growth of roots. In among GA, aldolase, and root growth. pathway and may play an important role this review, we discuss the relationship among GA, aldolase, and root growth.     

Purification of cell culture‐derived modified vaccinia ankara virus by pseudo‐affinity membrane adsorbers and hydrophobic interaction chromatography

A purification scheme for cell culture‐derived smallpox vaccines based on an orthogonal downstream process of pseudo‐affinity membrane adsorbers (MA) and hydrophobic interaction chromatography (HIC) was investigated. The applied pseudo‐affinity chromatography, based on reinforced sulfated cellulose and heparin‐MA, was optimized in terms of dynamic binding capacities, virus yield and process productivity. HIC was introduced as a subsequent method to further reduce the DNA content. Therefore, two screens were undertaken. First, several HIC ligands were screened for different adsorption behavior between virus particles and DNA. Second, elution from pseudo‐affinity MA and adsorption of virus particles onto the hydrophobic interaction matrix was explored by a series of buffers using different ammonium sulfate concentrations. Eventually, variations between different cultivation batches and buffer conditions were investigated.The most promising combination, a sulfated cellulose membrane adsorber with subsequent phenyl HIC resulted in overall virus particle recoveries ranging from 76% to 55% depending on the product batch and applied conditions. On average, 61% of the recovered virus particles were infective within all tested purification schemes and conditions. Final DNA content varied from 0.01% to 2.5% of the starting material and the level of contaminating protein was below 0.1%. Biotechnol. Bioeng. 2010;107: 312–320. © 2010 Wiley Periodicals, Inc.     

Analytical characterization of a monoclonal antibody therapeutic reveals a three-light chain species that is efficiently removed using hydrophobic interaction chromatography

Size exclusion high performance liquid chromatography analysis of a human monoclonal antibody (mAb) showed the presence of a new species that eluted with a retention time between the dimeric and monomeric species of the antibody. Extensive characterization of this species, referred to as “shoulder,” indicated that it was a mAb containing an extra light chain and had a molecular weight of approximately 175 kDa. The extra light chain was found to be non-covalently associated with the Fab portion of the protein. The relative amount of shoulder (typically 1−3% of the total mAb present) varied with the Chinese hamster ovary cell line producing the mAb and was not influenced by the growth conditions. Our three-step mAb purification platform using protein A, anion exchange, and cation exchange process steps was successful at removing dimer and higher and lower molecular weight species, but not the shoulder impurity. It was found that hydrophobic interaction chromatography could be used in place of cation exchange to exploit the subtle differences in hydrophobicity between monomer and shoulder. We developed an antibody polishing process using Butyl Sepharose HP resin that is capable of removing the majority of high and low molecular weight impurities yielding 99% pure mAb monomer, virtually devoid of the shoulder species, with a step recovery of about 80%.     

Online fluorescent dye detection method for the characterization of immunoglobulin G aggregation by size exclusion chromatography and asymmetrical flow field flow fractionation

The aim of this study was to develop an online fluorescent dye detection method suitable for high-pressure size exclusion chromatography (HP-SEC) and asymmetrical flow field flow fractionation (AF4). The noncovalent extrinsic fluorescent dye 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (Bis-ANS) was added to the mobile phase or the sample, and the fluorescence emission at 488 nm was recorded on excitation at 385 nm. By combining HP-SEC and AF4 with online dye detection, it was possible to simultaneously detect heat-induced aggregation and structural changes of monomeric and aggregated immunoglobulin G (IgG); an increase in Bis-ANS fluorescence was observed in both the aggregate and monomer fractions. These structural changes of individual fractions, which were not detectable by online UV and multiangle laser light scattering (MALLS) or by stand-alone dynamic light scattering (DLS), intrinsic IgG fluorescence, and far-UV circular dichroism (CD), resulted in progressive aggregation on storage. The developed online fluorescent dye detection for HP-SEC or AF4 with Bis-ANS is a powerful method to detect both aggregation and structural changes of both monomeric and aggregated IgG in heat-stressed formulations.     

Calorimetry for studying the adsorption of proteins in hydrophobic interaction chromatography

Hydrophobic interaction chromatography is a very popular chromatography method for purification of proteins and plasmids in all scales from analytical to industrial manufacturing. Despite this frequent use, the complex interaction mechanism and the thermodynamic aspects of adsorption in hydrophobic interaction chromatography are still not well understood. Calorimetric methods such as isothermal titration calorimetry and flow calorimetry can help to gain a deeper understanding of the adsorption strength, the influence of salt type and temperature. They can be used to study conformational changes of proteins, which are often associated with the adsorption in hydrophobic interaction chromatography. This review offers a detailed introduction into the thermodynamic fundamentals of adsorption in hydrophobic interaction chromatography with a special focus on the potential applications of isothermal titration calorimetry and flow calorimetry for studying specific problems and relationships of the adsorption behavior of proteins and its various influencing factors. Models for characterizing conformational changes upon adsorption are presented together with methods for assessing this problem for different proteins and stationary phases. All of this knowledge can contribute greatly to forming a sound basis for method development, process optimization and finding modelling strategies in hydrophobic interaction chromatography.     

Evaluation of modified chitosan as matrix for hydrophobic interaction chromatography

Chitosan beads were modified with glutaraldehyde and modified chitosan was investigated as matrix for hydrophobic interaction chromatography. The influence of temperature, type of salt and its ionic strength on the adsorption of -galactosidase was studied. -Galactosidase was found to bind in presence of high concentration of ammonium sulphate (3 M, w/v) and 90% of the bound enzyme was eluted with decreasing salt concentration in presence of 10% ethylene glycol. Attempt was made to purify -galactosidase from modified chitosan, -galactosidase showed 1.7-fold purification with 43.96% recovery of enzyme activity. The SDS–PAGE analysis of enzyme showed considerable purification and its molecular weight was found to be 63–64 kDa. Unlike other chromatographic matrices, the prepared chitosan beads were used five times. The results showed that purification and recovery of the enzyme did not change even when column size was increased.     

Studies on the retention of plasmid DNA and Escherichia coli nucleic acids by hydrophobic interaction chromatography

This work presents studies on the interactions of supercoiled plasmid DNA and Escherichia coli genomic DNA (gDNA) and RNA, with an hydrophobic interaction chromatography (HIC) gel, obtained by derivatisation of Sepharose CL-6B with 1,4-butanediol diglycidyl ether. Nucleic acids purified from E. coli were injected separately in the above HIC column and eluted with 1.5 M (NH₄)₂SO₄ in the buffer. The column was able to separate single-stranded from double-stranded nucleic acids. RNA and denatured gDNA were retarded in a different way due to the interactions of the exposed hydrophobic bases with the ligands. Supercoiled plasmid DNA, on the contrary, eluted in the flowthrough. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification of muscle uridine diphosphoglucose pyrophosphorylase by hydrophobic chromatography

Uridine diphosphoglucose pyrophosphorylase was purified 2500-fold from rabbit skeletal muscle with a total recovery of 35% of the initial activity. The present procedure was made possible by an extensive use of hydrophobic chromatography. Purified pyrophosphorylase had a specific activity of 500 mumol/min/mg of protein and was homogeneous by chromatographic and electrophoretic criteria. The enzyme appears to be composed of eight subunits of 53,000 molecular weight each.     

Scale-up of hydrophobic interaction chromatography for the purification of a DNA vaccine against rabies

The purification of a DNA vaccine against rabies by hydrophobic interaction chromatography (HIC) using a Sepharose gel derivatised with 1,4-butanediol diglycidyl ether was scaled up 60 times. The purification profile was not affected by increased loadings (up to 15 mg DNA) and a product with a consistent quality was obtained. Fourteen mg of plasmid with an HPLC purity of 100% were obtained in one run, corresponding to a 95% yield.     

Separation of mAbs molecular variants by analytical hydrophobic interaction chromatography HPLC: overview and applications

Hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) is a powerful analytical method used for the separation of molecular variants of therapeutic proteins. The method has been employed for monitoring various post-translational modifications, including proteolytic fragments and domain misfolding in etanercept (Enbrel®); tryptophan oxidation, aspartic acid isomerization, the formation of cyclic imide, and α amidated carboxy terminus in recombinant therapeutic monoclonal antibodies; and carboxy terminal heterogeneity and serine fucosylation in Fc and Fab fragments. HIC-HPLC is also a powerful analytical technique for the analysis of antibody-drug conjugates. Most current analytical columns, methods, and applications are described, and critical method parameters and suitability for operation in regulated environment are discussed, in this review.     

Bioluminescence assay for estimating the hydrophobic properties of bacteria as revealed by hydrophobic interaction chromatography

The luciferin-luciferase bioluminescence method was used to estimate the number of bacteria retained in neutral and amphiphilic gels and those in the eluate to determine the hydrophobic surface properties of bacteria by using hydrophobic interaction chromatography. Good correlations were found between viable counts and ATP content for Escherichia coli, Pseudomonas fragi, and Listeria monocytogenes. ATP determination was more rapid than viable counts for characterizing the relative hydrophobicity of L. monocytogenes. Quantitative estimations of adsorption of L. monocytogenes on octyl-Sepharose indicate that this microorganism is hydrophilic.     

Proteome analysis of diploid,tetraploid and hexaploid wheat: towards understanding genome interaction in protein expression

Hexaploid wheat (Triticum aestivum L.) is derived from a complex hybridization procedure involving three diploid species carrying the A, B and D genomes. The proteome patterns of diploid, tetraploid and hexaploid wheat were analyzed to explore the genome interaction in protein expression. At least two species from each of the diploid and tetraploid were used to compare their proteome maps with a hexaploid wheat cv. Chinese Spring. The ancestral cultivars were selected based on their history of closeness with the cultivated wheat. Proteins were extracted from seed flour and separated by two-dimensional electrophoresis (2-DE) with isoelectric focusing of pH range from 4-10. 2-DE maps of cultivated and ancestral species were analyzed by computer assisted image analyzer. The region of high molecular weight glutenin subunits of hexaploid wheat showed similarity with those of the diploid donors, BB and DD genomes. The omega gliadin, which is controlled by B genome in common wheat, was assumed to have evolved as a result of interaction between AA and BB genomes. The low molecular weight glutenins and alpha and beta gliadin regions were contributed by the three genomes. This result suggests that the function of donor genomes particularly in the expression of proteins in hexaploid wheat is not totally independent; rather it is the product of interactions among the diploid genomes in the hexaploid nuclear constitutions. The expression of nonstorage proteins was affected substantially due to the removal of the D genome from hexaploid constitution. Location of the structural gene controlling one of the alpha amylase inhibitor proteins in the nonstorage protein region was identified in the short arm of chromosome 3D.     

小峰熊蜂蜂王蛹期发育蛋白质组分析

为了探明小峰熊蜂Bombus hypocrita蜂王蛹期发育蛋白质表达调控方面的特点,揭示其发育的分子机理。采用双向电泳法对小峰熊蜂蜂王蛹期发育进行蛋白质组研究,结果在小峰熊蜂蜂王蛹期的白眼期(A期)、褐眼期(B期)和黑眼期(C期)分别检测到81、80和75个蛋白点,特有蛋白质分别为8个、7个和2个,共有蛋白质为61个,A期到B期有4个蛋白质显著上调,5个显著下调,B期到C期有7个蛋白质显著上调,1个显著下调,A期到C期有10个蛋白质显著上调,有4个显著下调。此外,3个蛋白质是在A、B期表达C期关闭,6个蛋白质A、C期表达,B期关闭,5个蛋白质A期关闭,而B、C期表达。初步表明小峰熊蜂蜂王从蛹期发育到成蜂过程中,不仅需要一些保守蛋白质来调控,而且还需要一些特异蛋白质。     

Separation of mAbs molecular variants by analytical hydrophobic interaction chromatography HPLC: overview and applications

Hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) is a powerful analytical method used for the separation of molecular variants of therapeutic proteins. The method has been employed for monitoring various post-translational modifications, including proteolytic fragments and domain misfolding in etanercept (Enbrel®); tryptophan oxidation, aspartic acid isomerization, the formation of cyclic imide, and α amidated carboxy terminus in recombinant therapeutic monoclonal antibodies; and carboxy terminal heterogeneity and serine fucosylation in Fc and Fab fragments. HIC-HPLC is also a powerful analytical technique for the analysis of antibody-drug conjugates. Most current analytical columns, methods, and applications are described, and critical method parameters and suitability for operation in regulated environment are discussed, in this review.     

Bioluminescence assay for estimating the hydrophobic properties of bacteria as revealed by hydrophobic interaction chromatography.

The luciferin-luciferase bioluminescence method was used to estimate the number of bacteria retained in neutral and amphiphilic gels and those in the eluate to determine the hydrophobic surface properties of bacteria by using hydrophobic interaction chromatography. Good correlations were found between viable counts and ATP content for Escherichia coli, Pseudomonas fragi, and Listeria monocytogenes. ATP determination was more rapid than viable counts for characterizing the relative hydrophobicity of L. monocytogenes. Quantitative estimations of adsorption of L. monocytogenes on octyl-Sepharose indicate that this microorganism is hydrophilic.     

Depletion of highly abundant proteins in blood plasma by hydrophobic interaction chromatography for proteomic analysis

The proteomic analysis of plasma is extremely complex due to the presence of few highly abundant proteins. These proteins have to be depleted in order to detect low abundance proteins, which are likely to be of biomedical interest. In this work it was investigated the applicability of hydrophobic interaction chromatography (HIC) as a plasma fractionation method prior to two-dimensional gel electrophoresis (2DGE). The average hydrophobicity of the 56 main plasma proteins was calculated. Plasma proteins were classified as low, medium and highly hydrophobic through a cluster analysis. The highly abundant proteins showed a medium hydrophobicity, and therefore a HIC step was designed to deplete them from plasma. HIC performance was assessed by 2DGE, and it was compared to that obtained by a commercial immuno-affinity (IA) column for albumin depletion. Both methods showed similar reproducibility. HIC allowed partially depleting α-1-antitrypsin and albumin, and permitted to detect twice the number of spots than IA. Since albumin depletion by HIC was incomplete, it should be further optimized for its use as a complementary or alternative method to IA.