Influence of sodium chloride on hydrophobic adsorption of PEGylated lysozyme

Interaction of macromolecules in aqueous salt‐containing solution with a hydrophobic adsorbent is studied by adsorption equilibrium measurements and by independent isothermal titration calorimetry. The macromolecules are native as well as mono‐, di‐, and tri‐PEGylated lysozyme and four pure PEGs. The hydrophobic adsorbent is Toyopearl PPG‐600M. The salt is sodium chloride. The sodium chloride concentration in the aqueous 25 mM sodium phosphate buffer is varied from 2000 to 4500 mM at pH 7.0 and 25°C. PEGylation of the lysozyme is carried using 5 and 10 kDa PEG chains. The molar enthalpy of adsorption is calculated from the adsorption equilibrium and the calorimetric data. The results show that the adsorption of the PEGylated lysozyme is caused by both the interaction of the lysozyme and the interaction of the PEG chains with the adsorbent, respectively, but the interaction of the lysozyme is stronger than that of PEG. The comparison of the results of the present study on the influence of sodium chloride with a corresponding study on the influence of ammonium sulfate shows that the adsorption mechanism changes upon the variation of the salt. The knowledge of the adsorption mechanisms supports the systematic development of chromatographic purification steps.     

Modeling of protein monomer/aggregate purification and separation using hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) is commonly used to separate protein monomer and aggregate species in the purification of protein therapeutics. Despite being used frequently, the HIC separation mechanism is quite complex and not well understood. In this paper, we examined the separation of a monomer and aggregate protein mixture using Phenyl Sepharose FF. The mechanisms of protein adsorption, desorption, and diffusion of the two species were evaluated using several experimental approaches to determine which processes controlled the separation. A chromatography model, which used homogeneous diffusion (to describe mass transfer) and a competitive Langmuir binary isotherm (to describe protein adsorption and desorption), was formulated and used to predict the separation of the monomer and aggregate species. The experimental studies showed a fraction of the aggregate species bound irreversibly to the adsorbent, which was a major factor governing the separation of the species. The model predictions showed inclusion of irreversible binding in the adsorption mechanism greatly improved the model predictions over a range of operating conditions. The model successfully predicted the separation performance of the adsorbent with the examined feed.     

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 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     

疏水相互作用层析分离重组人干扰素α2b

目的采用疏水相互作用层析分离重组人干扰素α2b,去除干扰素样品中的二聚体,得到高纯度的干扰素用于进一步的研究。方法首先采用阳离子交换层析纯化复性重组人干扰素α2b,去除了大部分的杂蛋白,然后采用疏水相互作用层析纯化重组人干扰素α2b,去除复性过程中产生的错误折叠体和二聚体,并考察盐浓度、pH值、流速和洗脱液中尿素对疏水相互作用层析纯化效果的影响。结果硫酸铵初始浓度1.2 mol/L、缓冲液pH值6.0、流速2.5 mL/min、洗脱液中添加尿素浓度为2 mol/L时疏水相互作用层析纯化效果最佳。最终得到的重组人干扰素α2b非还原型SDS-PAGE电泳均呈单一条带。结论确定了疏水层析纯化重组人干扰素α2b的最优条件,成功提取到具有高活性、高纯度的重组人干扰素α2b纯品。     

On the use of mild hydrophobic interaction chromatography for "method scouting" protein purification strategies in aqueous two-phase systems: a study using model proteins

The behavior of a series of pure proteins partitioned in aqueous two-phase systems is compared with their behavior during mild hydrophobic interaction chromatography (HIC). A simple theoretical rationale for this comparison is presented based upon solvophobic theory. Similarities were found in the behavior of the model proteins in the two forms of partition chromatography. This indicates that HIC may be employed as a rapid instrumental technique for the broad characterization of protein behavior, which may be of benefit in the development of liquid-liquid partitioning strategies. However, it has proved difficult to completely account for this behavior on the basis of the known physical and structural properties of the proteins used. The variety in the detailed partitioning behavior of this small sample of protein types suggests that partition in aqueous two-phase systems is uniquely sensitive to subtle differences in surface properties of complex macromolecules. (c) 1994 John Wiley & Sons, Inc.     

Characterization of denatured proteins by hydrophobic interaction chromatography: a preliminary study

In this preliminary study hydrophobic interaction chromatography (HIC) is proposed as a good tool in order to detect conformational changes induced by chemical denaturants in two globular proteins, cytochrome C (Cyt C) and myoglobin (MYO). Alterations in protein structure were manifested chromatographically by reproducible changes in peak heights, retention time, and appearance of multiple peaks. The HIC behavior of the two model proteins denatured by guanidinium thyocyanate (GdmSCN) was investigated, keeping constant various concentrations of urea in the mobile phase in a TSK-Gel Phenyl-5PW column (TosoBiosep). Suitable elution conditions provide evidence of the simultaneous presence of two denatured forms in the case of MYO, and sequential different denatured states of Cyt C.     

Purification of recombinant aprotinin from transgenic corn germ fraction using ion exchange and hydrophobic interaction chromatography

Plants have attracted interest as hosts for protein expression because of the promise of a large production capacity and a low production cost. However, recovery costs remain a challenge as illustrated for recovery of recombinant aprotinin, a trypsin inhibitor, with removal of native corn trypsin inhibitor from transgenic corn (Azzoni et al. in Biotechnol Bioeng 80:268–276, 2002). When expression is targeted to corn grain fractions, dry milling can separate germ and endosperm fractions. Hence, only the product-containing fraction needs to be extracted, reducing the cost of extraction and the impurity level of the extract. Selective extraction conditions can reduce impurity levels to the point that low-cost adsorbents can result in relatively high purity levels. In this work, we attempted to achieve comparable purity with these lower cost methods. We replaced whole grain extraction and purification of recombinant aprotinin with sequential trypsin affinity and IMAC steps with an alternative of germ fraction extraction and purification with ion exchange and hydrophobic interaction chromatography (HIC). Using germ extraction at acidic pH supplemented with heat precipitation to remove additional host proteins resulted in a higher specific activity feed to the chromatographic steps. The cation exchange step provided 7.6× purification with 76.4% yield and no sodium dodecyl sulfate–polyacrylamide gel electrophoresis detectable native corn trypsin inhibitor. After the HIC step (2.7× step purification with 44.0% yield), the final product had a specific activity that was 75.3% of that of the affinity-purified aprotinin.     

Application of hydrophobic interaction displacement chromatography for an industrial protein purification

Recently it has been established that low molecular weight displacers can be successfully employed for the purification of proteins in hydrophobic interaction chromatography (HIC) systems. This work investigates the utility of this technique for the purification of an industrial protein mixture. The study involved the separation of a mixture of three protein forms, that differed in the C-terminus, from their aggregate impurities while maintaining the same relative ratio of the three protein forms as in the feed. A batch high-throughput screening (HTS) technique was employed in concert with fluorescence spectroscopy for displacer screening in these HIC systems. This methodology was demonstrated to be an effective tool for identifying lead displacer candidates for a particular protein/stationary-phase system. In addition, these results indicate that surfactants can be employed at concentrations above their CMCs as effective displacers. Displacement of the recombinant proteins with PEG-3400 and the surfactant Big Chap was shown to increase the productivity as compared to the existing step-gradient elution process.     

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.     

Mutations for decreasing the immunogenicity and maintaining the function of core streptavidin

The defining property of core streptavidin (cSA) is not only its high binding affinity for biotin but also its pronounced thermal and chemical stability. Although potential applications of these properties including therapeutic methods have prompted much biological research, the high immunogenicity of this bacterial protein is a key obstacle to its clinical use. To this end, we have successfully constructed hypoimmunogenic cSA muteins in a previous report. However, the effects of these mutations on the physicochemical properties of muteins were still unclear. These mutations retained the similar electrostatic charges to those of wild‐type (WT) cSA, and functional moieties with similar hydrogen bond pattern. Herein, we performed isothermal titration calorimetry, differential scanning calorimetry, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis to gain insight into the physicochemical properties and functions of these modified versions of cSA. The results indicated that the hypoimmunogenic muteins retained the biotin‐binding function and the tetramer structure of WT cSA. In addition, we discuss the potential mechanisms underlying the success of these mutations in achieving both immune evasion and retention of function; these mechanisms might be incorporated into a new strategy for constructing hypoimmunogenic proteins.     

Isothermal Titration Calorimetry of Chiral Polymeric Nanoparticles

Chiral polymeric nanoparticles are of prime importance, mainly due to their enantioselective potential, for many applications such as catalysis and chiral separation in chromatography. In this article we report on the preparation of chiral polymeric nanoparticles by miniemulsion polymerization. In addition, we describe the use of isothermal titration calorimetry (ITC) to measure the chiral interactions and the energetics of the adsorption of enantiomers from aqueous solutions onto chiral polymeric nanoparticles. The characterization of chirality in nano‐systems is a very challenging task; here, we demonstrate that ITC can be used to accurately determine the thermodynamic parameters associated with the chiral interactions of nanoparticles. The use of ITC to measure the energetics of chiral interactions and recognition at the surfaces of chiral nanoparticles can be applied to other nanoscale chiral systems and can provide further insight into the chiral discrimination processes of nanomaterials. Chirality 27:613–618, 2015. © 2015 Wiley Periodicals, Inc.     

Influence of tryptophan tags on the purification of cutinase, secreted by a recombinant Saccharomyces cerevisiae, using cationic expanded bed adsorption and hydrophobic interaction chromatography

During cationic bed adsorption (EBA), with cutinase with varying length tryptophan tags (WP)(2)and (WP)(4), 33% and 10% of adsorption capacity and 80% and 32% eluted specific activity were observed in relation to wild type (wt)-cutinase in the conventional process. Therefore, as the hydrophobicity of the protein increases, it is important to integrate the EBA step with a hydrophobic interaction chromatography (HIC) process. As the length of the hydrophobic tag-(WP) increases from n = 2 to n = 4, the purification factor obtained by HIC was 1.8 and 2.2-fold higher than wt-cutinase. However, the recovery yield obtained in HIC decreases substantially as the length of hydrophobic tag increases (97%, 84% and 70% for wt-cutinase, cutinase-(WP)(2) and cutinase-(WP)(4)). The integration of two purification steps, EBA followed by HIC, resulted in the highest overall purity level for cutinase-(WP)(2), and the highest overall recovery yield for wt-cutinase. When optimizing the design of a hydrophobic tag fused to a protein secreted by Saccharomyces cerevisiae it must be considered that the cultivation parameters could impair the downstream process, and consequently the optimum tag is not necessarily the one that presents the highest purification factor in HIC.     

A mild hydrophobic interaction chromatography involving polyethylene glycol immobilized to agarose media refolding recombinant Staphylococcus aureus elongation factor G

Recombinant Staphylococcus aureus elongation factor G (EF-G) is difficult to refold by dilution due to the formation of large amounts of misfolded structures. However, refolding of EF-G by adsorption to a chromatographic column packed with immobilized polyethylene glycol 20,000 (PEG 20 K) followed by pulse elution with 8 M urea resulted in 88% mass recovery and 80% of correctly refolded structure. The PEG 20 K was coupled to brominated allyl group derivatized Sepharose High Performance to construct a mild hydrophobic adsorbent. Various other hydrophobic interaction adsorbents were also attempted to refold EF-G. However, ligands with high hydrophobicity tended to misfold EF-G, resulting in irreversible adsorption. Various solvents, detergents, and low temperature as well as 8 M urea were tried to release bound EF-G. Only pulse elution with 8 M urea was efficient. Urea concentrations favorable for efficiently refolding EF-G were investigated. Low urea concentration produced more misfolded structures.     

Alternative modes of binding of proteins with tandem SH2 domains

The issue of specificity in tyrosine kinase intracellular signaling mediated by src homology 2 (SH2) domains has great importance in the understanding how individual signals maintain their mutual exclusivity and affect downstream responses. Several proteins contain tandem SH2 domains that, on interacting with their ligand, provide a higher level of specificity than can be afforded by the interaction of a single SH2 domain. In this study, we focus on the comparison of two proteins ZAP70 and the p85 subunit of PI 3-kinase, which although distinctly different in function and general structure, possess tandem SH2 domains separated by a linker region and which bind to phosphorylated receptor molecules localized to the cell membrane. Binding studies using isothermal titration calorimetry show that these two proteins interact with peptides mimicking their physiological ligands in very different ways. In the case of the SH2 domains from ZAP70, they interact with a stoichiometry of unity, while p85 is able to make two distinct interactions, one with a stoichiometry of 1:1 and the other with two p85 molecules interacting with one receptor. The observation of two different modes of binding of p85 might be important in providing different cellular responses based on fluctuating intracellular concentration regimes of this protein. Thermodynamic data on both proteins suggest that a conformational change occurs on binding. On investigation of this structural change using a truncated form of p85 (including just the two SH2 domains and the inter-SH2 region), both NMR and circular dichroism spectroscopic studies failed to show significant changes in secondary structure. This suggests that any conformational change associated with binding is small and potentially limited to loop regions of the protein.     

Structural and binding studies of the three-metal center in two mycobacterial PPM Ser/Thr protein phosphatases

Phospho-Ser/Thr protein phosphatases (PPs) are dinuclear metalloenzymes classed into two large families, PPP and PPM, on the basis of sequence similarity and metal ion dependence. The archetype of the PPM family is the α isoform of human PP2C (PP2Cα), which folds into an α/β domain similar to those of PPP enzymes. The recent structural studies of three bacterial PPM phosphatases, Mycobacterium tuberculosis MtPstP, Mycobacterium smegmatis MspP, and Streptococcus agalactiae STP, confirmed the conservation of the overall fold and dinuclear metal center in the family, but surprisingly revealed the presence of a third conserved metal-binding site in the active site. To gain insight into the roles of the three-metal center in bacterial enzymes, we report structural and metal-binding studies of MtPstP and MspP. The structure of MtPstP in a new trigonal crystal form revealed a fully active enzyme with the canonical dinuclear metal center but without the third metal ion bound to the catalytic site. The absence of metal correlates with a partially unstructured flap segment, indicating that the third manganese ion contributes to reposition the flap, but is dispensable for catalysis. Studies of metal binding to MspP using isothermal titration calorimetry revealed that the three Mn²⁺-binding sites display distinct affinities, with dissociation constants in the nano- and micromolar range for the two catalytic metal ions and a significantly lower affinity for the third metal-binding site. In agreement, the structure of inactive MspP at acidic pH was determined at atomic resolution and shown to lack the third metal ion in the active site. Structural comparisons of all bacterial phosphatases revealed positional variations in the third metal-binding site that are correlated with the presence of bound substrate and the conformation of the flap segment, supporting a role of this metal ion in assisting enzyme-substrate interactions.     

Paper-PEG-based membranes for hydrophobic interaction chromatography: purification of monoclonal antibody

This article discusses the preparation of novel Paper-PEG interpenetrating polymer network-based membranes as inexpensive alternative to currently available adsorptive membranes. The Paper-PEG membranes were developed for carrying out hydrophobic interaction membrane chromatography (HIMC). PEG is normally very hydrophilic but can undergo phase separation and become hydrophobic in the presence of high antichaotropic salt concentrations. Two variants of the Paper-PEG membranes, Paper-PEG 1 and Paper-PEG 2 were prepared by grafting different amounts of the polymer on filter paper and these were tested for their hydraulic properties and antibody binding capacity. The better of the two membranes (Paper-PEG 1) was then used for purifying the monoclonal antibody hIgG1-CD4 from simulated mammalian cell culture supernatant. The processing conditions required for purification were systematically optimized. The dynamic antibody binding capacity of the Paper-PEG 1 membrane was about 9 mg/mL of bed volume. A single step membrane chromatographic process using Paper-PEG 1 membrane gave high monoclonal antibody purity and recovery. The hydraulic permeability of the paper-based membrane was high and was maintained even after many runs, indicating that membrane fouling was negligible and the membrane was largely incompressible.     

Non-additivity of Functional Group Contributions in Protein-Ligand Binding: A Comprehensive Study by Crystallography and Isothermal Titration Calorimetry

Additivity of functional group contributions to protein-ligand binding is a very popular concept in medicinal chemistry as the basis of rational design and optimized lead structures. Most of the currently applied scoring functions for docking build on such additivity models. Even though the limitation of this concept is well known, case studies examining in detail why additivity fails at the molecular level are still very scarce. The present study shows, by use of crystal structure analysis and isothermal titration calorimetry for a congeneric series of thrombin inhibitors, that extensive cooperative effects between hydrophobic contacts and hydrogen bond formation are intimately coupled via dynamic properties of the formed complexes. The formation of optimal lipophilic contacts with the surface of the thrombin S3 pocket and the full desolvation of this pocket can conflict with the formation of an optimal hydrogen bond between ligand and protein. The mutual contributions of the competing interactions depend on the size of the ligand hydrophobic substituent and influence the residual mobility of ligand portions at the binding site. Analysis of the individual crystal structures and factorizing the free energy into enthalpy and entropy demonstrates that binding affinity of the ligands results from a mixture of enthalpic contributions from hydrogen bonding and hydrophobic contacts, and entropic considerations involving an increasing loss of residual mobility of the bound ligands. This complex picture of mutually competing and partially compensating enthalpic and entropic effects determines the non-additivity of free energy contributions to ligand binding at the molecular level.     

Modelling and simulation of the adsorption of the lipase from Geotrichum sp. on hydrophobic interaction columns

The Langmuir model fitted well the adsorption isotherms of lipase on the hydrophobic resin. The model parameters, Q _m and k _d, were affected by NaCl concentration: Q _m increased from 31 to 80 U g^–1 resin, and k _d changed from 9.4 to 3 U ml^–1. Column modelling and the simulation data were compared with the experimental data with good agreement. The highest achieved column efficiency was 71%.     

Interaction mechanism of mono-PEGylated proteins in electrostatic interaction chromatography

The retention and binding mechanisms in electrostatic interaction-based chromatography (ion-exchange chromatography) of PEGylated proteins (covalent attachment of polyethylene glycol chains to protein) were investigated using our previously developed model. Lysozyme and bovine serum albumin were chosen as model proteins. The retention volume of PEGylated proteins shifted to lower elution volumes with increasing PEG molecular weight compared with the non-modified (native) protein retention volume. However, PEGylation did not affect the number of binding sites appreciably. The enzyme activity of PEGylated lysozyme measured with a standard insoluble substrate in suspension decreased considerably, whereas the activity with a soluble small-molecule substrate did not drop significantly. These findings indicate that when a protein is mono-PEG-ylated, the binding site is not affected and the elution volume reduces due to the steric hindrance between PEGylated protein and ion-exchange ligand.