Scale-up of recombinant protein purification by hydrophobic interaction chromatography

The scale-up of hydrophobic interaction chromatography is described. Human recombinant superoxide dismutase was used as a model. The scale-up was performed by keeping the height to diameter (H/D) ratio of the column constant. The success of scale-up was evaluated by reversed-phase high-performance liquid chromatography of the eluted material. The wrong H/D ratio causes decreased resolution.     

Effect of surface hydrophobicity distribution on retention of ribonucleases in hydrophobic interaction chromatography

The effect of surface hydrophobicity distribution of proteins on retention in hydrophobic interaction chromatography (HIC) was investigated. Average surface hydrophobicity as well as hydrophobic contact area between protein and matrix were estimated using a classical thermodynamic model. The applicability of the model to predict protein retention in HIC was investigated on ribonucleases with similar average surface hydrophobicity but different surface hydrophobicity distribution. It was shown experimentally that surface hydrophobicity distribution could have an important effect on protein retention in HIC. The parameter "hydrophobic contact area," which comes from the thermodynamic model, was able to represent well the protein retention in HIC with salt gradient elution. Location and size of the hydrophobic patches can therefore have an important effect on protein retention in HIC, and the hydrophobic contact area adequately describes this.     

Mechanism of simultaneously refolding and purification of proteins by hydrophobic interaction chromatographic unit and applications

The hydrophobic amino acid residues of a denatured protein molecule tend to react with the particles of the stationary phase of hydrophobic interaction chromatography (STHIC). These hydrophobic interactions prevent the denatured protein molecules from aggregating with each other. The STHIC can provide high enough energy to a denatured protein molecule to make it dehydration and to refold it into its native or various intermediate states. The outcome not only depends on the specific interactions between amino acids, the structure of STHIC, but also depends on the association between the STHIC and mobile phase. The mechanism of protein refolding and the principle of its quality control by HPHIC were also presented. By appropriate selection of the chromatographic condition, several denatured proteins can be refolded and separated simultaneously in a single chromatographic run. A specially designed unit, with diameter much larger than its length, was designed and employed for both laboratory and preparative     

Rapid purification of detergent-solubilized bovine hormone-sensitive lipase by high performance hydrophobic interaction chromatography

Hormone-sensitive lipase (HSL), the enzyme controlling the rate of adipose tissue lipolysis and also possibly involved in the regulation of steroidogenesis, has been purified from bovine omental adipose tissue. Partially detergent-solubilized, delipidated and purified HSL was obtained through step-elution at conventional DEAE ion-exchange chromatography, followed by concentration on hydroxylapatite. High performance hydrophobic interaction chromatography (HPHIC) on phenylsilica then resulted in an increase of HSL protein purity from 2% to more than 70%. Final purification of the enzyme to apparent homogeneity (greater than 95% protein purity), concentration and removal of most of the detergent was obtained by high performance cation exchange chromatography on Mono S. At least 0.5 mg of highly stable HSL was obtained from 5 kg of bovine omental fat within four working days. The purified lipase had a lower specific activity than previously reported for the corresponding rat enzyme but the preparations have proved very useful for enzyme structure studies and as an antigen.     

Interaction of fibrinogen with nanosilica

Interaction of human plasma fibrinogen (HPF) with fumed nanosilica A-300 in a phosphate buffer solution (PBS) was studied using ¹H NMR spectroscopy with layer-by-layer freezing-out of bulk and interfacial water in the temperature range of 210–273 K, TSDC (90 < T < 265 K), adsorption, FTIR, and UV spectroscopy methods. An increase in concentration of HPF in the PBS leads to a decrease in amounts of structured water (frozen at T < 273 K) because of coagulation of HPF molecules. Addition of nanosilica to the HPF solution strongly reduces the amounts of structured water because of adsorption interaction of HPF molecules with silica nanoparticles, self-association of HPF molecules, formation of denser packed hybrid agglomerates with HPF and silica, and lastly, because of conformational changes of HPF. A monolayer adsorption capacity of A-300 corresponds to 156 mg of HPF per gram of silica. The FTIR and UV spectra show that the HPF adsorption on silica leads to structural changes of the protein molecules. These changes and formation of hybrid HPF/A-300 aggregates can increase the rate of clotting that is of importance on nanosilica application as a component of tourniquet preparations.      

Methods of calculating protein hydrophobicity and their application in developing correlations to predict hydrophobic interaction chromatography retention

Hydrophobic interaction chromatography (HIC) is a key technique for protein separation and purification. Different methodologies to estimate the hydrophobicity of a protein are reviewed, which have been related to the chromatographic behavior of proteins in HIC. These methodologies consider either knowledge of the three-dimensional structure or the amino acid composition of proteins. Despite some restrictions; they have proven to be useful in predicting protein retention time in HIC.     

Self-healing surface hydrophobicity by consecutive release of hydrophobic molecules from mesoporous silica

The paper reports a novel approach to achieve self-healing surface hydrophobicity. Mesoporous silica is used as the reservoir for hydrophobic molecules, i.e., octadecylamine (ODA), that can release and refresh the surface hydrophobicity consecutively. A polymdopamine layer is used to further encapsulate silica-ODA, providing a reactive layer, governing release of the underlying ODA, and improving the dispersivity of silica nanoparticles in bulk resin. The approach arrives at self-healing (super)hydrophobicity without using any fluoro-containing compounds.     

Fluorescence triggered by ligand-protein hydrophobic interaction

Hydrophobic fluorescence:Tan and his colleagues recently introduced a brand new chemotype of environment-sensitive fluorescent turn-on probes to detect the hydrophobic ligand-binding domain by using SBD fluorophore.The design strategy described in this report generalized the environment sensitivity turn-on mechanism to recognize a specific protein,which provides a robust breakthrough for interchanging fluorescence in conventional small-molecule fluorescent imaging.     

高效疏水相互作用色谱法复性与同时纯化重组人Flt3配体的包涵体蛋白质

Flt3配体(FL)是一类具有促进早期造血功能的细胞因子,在促进造血细胞生长发育及造血动员方面具有重要的临床应用价值。为了用基因工程方法获得大量用于临床和研究的重组人FL(rhFL)蛋白质,本文对在大肠杆菌(E. coli)中表达得到的Flt3配体的包涵体进行回收、洗涤,溶解于8 mol/L脲后在高效疏水相互作用色谱(HPHIC)柱上进行rhFL包涵体的复性与同时纯化,并对其保留特征和复性规律进行了研究。结果表明,在连续进样、变性蛋白质质量浓度为8.51 g/L、固定相选用端基为PEG800、流动相添加4 mol/L脲、1.8 mmol/L 还原型谷胱甘肽(GSH)和0.3 mmol/L氧化型谷胱甘肽(GSSH)、pH 7.0的优化条件下,复性与同时纯化rhFL包涵体的质量回收率为36.9%,纯度达94.5%以上。本文仅用一步HPHIC法成功地复性与同时纯化了rhFL蛋白质,为获得高活性的rhFL产品奠定了一定的工作基础。     

Stabilization of peptide fibrils by hydrophobic interaction

Hydrophobic interactions play an important role in assembly processes in aqueous environments. In case of peptide amphiphiles, hydrophobicity is combined with hydrogen bonding to yield well-defined peptide-based aggregates. Here, we report a systematic study after the role of hydrophobic interactions on both stabilization and morphology of a peptide fibrillar assembly. For this purpose, alkyl tails were connected to a known beta-sheet forming peptide with the sequence KTVIIE. The introduction of n-alkyl groups induced thermal stability to the assemblies without affecting the morphology of the peptide aggregates.     

Rapid purification of arrowhead proteinase inhibitors by high performance hydrophobic interaction chromatography on a PEG bonded phase column

A new hydrophobic interaction HPLC column is used for the rapid purification of proteinase inhibitors isolated from arrowhead. The inhibitors, partially purified by DEAE-cellulose column chromatography, are resolved into three components with a mobile phase gradient of decreasing salt concentration from 1.1 M ammonium sulfate in 0.01 M phosphate buffer to phosphate buffer alone. This new HPLC column is found to be very useful for rapid, semipreparative purification of hydrophobic protein and sample loading of up to 1.6 mg of inhibitors can be fully resolved on an analytical column.     

Evaluation of recombinant green fluorescent protein, under various culture conditions and purification with HiTrap hydrophobic interaction chromatography resins

To determine the influence of various culture conditions, transformed cells of Escherichia coli expressing recombinant green fluorescent protein (GFPuv) were grown in nine cultures with four variable conditions (storage of inoculated broth at 4°C prior to incubation, agitation speed, isopropyl-β-d-thiogalactopyranoside [IPTG] concentration, and induction time). The pelleted cells were resuspended in extraction buffer and subjected to the three-phase partitioning (TPP) extraction method. To determine the most appropriate purification resin, protein extracts were eluted through one of four types of HiTrap hydrophobic interaction chromatography (HIC) columns prepacked with methyl, butyl, octyl, or phenyl resins and analyzed further on a 12% sodium dodecylsulfatepolyacrylamidegel. With Coomassie staining, a single band between 27 (standard GFPuv) and 29 kDa (molecular weight standard) was visualized for every HIC column sample. TPP extraction with HIC elution provided about 90% of the GFPuv recovered and eight-fold GFPuv enrichment related to the specific mass. Rotary speed and IPTG concentration showed, respectively, greater negative and positive influences on GFPuv expression at the beginning of the logarithmic phase for the set culture conditions (37°C, 24-h incubation).     

Water interaction with hydrophobic and hydrophilic soot particles

The interaction of water with laboratory soots possessing a range of properties relevant for atmospheric studies is examined by two complementary methods: gravimetrical measurement of water uptake coupled with chemical composition and porosity analysis and HTDMA (humidified tandem differential mobility analyzer) inference of water uptake accompanied by separate TEM (transmission electron microscopy) analysis of single particles. The first method clarifies the mechanism of water uptake for bulk soot and allows the classification of soot with respect to its hygroscopicity. The second method highlights the dependence of the soot aerosol growth factor on relative humidity (RH) for quasi-monodisperse particles. Hydrophobic and hydrophilic soot are qualitatively defined by their water uptake and surface polarity: laboratory soot particles are thus classified from very hydrophobic to very hydrophilic. Thermal soot particles produced from natural gas combustion are classified as hydrophobic with a surface of low polarity since water is found to cover only half of the surface. Graphitized thermal soot particles are proposed for comparison as extremely hydrophobic and of very low surface polarity. Soot particles produced from laboratory flame of TC1 aviation kerosene are less hydrophobic, with their entire surface being available for statistical monolayer water coverage at RH approximately 10%. Porosity measurements suggest that, initially, much of this surface water resides within micropores. Consequently, the growth factor increase of these particles to 1.07 at RH > 80% is attributed to irreversible swelling that accompanies water uptake. Hysteresis of adsorption/desorption cycles strongly supports this conclusion. In contrast, aircraft engine soot, produced from burning TC1 kerosene in a gas turbine engine combustor, has an extremely hydrophilic surface of high polarity. Due to the presence of water soluble organic and inorganic material it can be covered by many water layers even below water saturation conditions. This soot demonstrates a gradual diameter growth factor (D(wet)/D(dry)) increase up to 1.22 at 93% relative humidity, most likely due to the presence of single particles with water soluble material heterogeneously distributed over their surface.     

Purification of labelled antibodies by hydrophobic interaction chromatography

In order to improve the sensitivity of immunometric assays, a chromatographic technique was developed that virtually eliminates components causing non-specific background. Labelled antibodies were applied to a phenyl-Sepharose column in physiological buffer. When labelled anitbodies were purified by this technique, the non-specific background of various time-resolved immunofluorometric assays was reduced 3- to 10-fold and was very close to the instrument background. The assay sensitivity was simultaneously increased by a factor of 2 to 16. This purification method might be used to improve the results of immunometric assays in general.     

Comparison of standard and new generation hydrophobic interaction chromatography resins in the monoclonal antibody purification process

Recent efforts to improve hydrophobic interaction chromatography (HIC) for use in monoclonal antibody (mAb) purification have focused on two approaches: optimization of resin pore size to facilitate mAb mass transport, and use of novel hydrophobic charge induction (HCIC) mixed mode ligands that allow capture of mAbs under low salt conditions. We evaluated standard HIC and new generation HIC and HIC-related chromatography resins for mAb purification process efficiency and product quality both as isolated chromatography steps and in purification process trains. We find that HIC resins with optimized pore size have significantly improved binding capacity which can increase HIC purification unit operation efficiency. The HCIC Mercapto-Ethyl-Pyridine (MEP) resin, which shows a different salt impact trend and impurity resolution pattern from standard HIC resin, can not only capture mAb from crude CHO fermentation supernatant but also substantially enhance mAb purification process flow efficiency when serving as a polishing role.     

Interaction of surfactants with hydrophobic surfaces in nanopores

Surfactant-induced wetting of hydrophobic nanopores is investigated. SDS micelles interact with the C18 layer on the nanopore walls with their hydrophobic tails, creating a charged wall lining with their head groups and inducing a breakthrough of the aqueous solution to wet the pores. The surface coverage of the surfactant molecules is evaluated electrophoretically. A surprising discovery is that pore wetting is achieved with 0.73 μmol/m(2) coverage of SDS surfactant, corresponding to only 18% of a monolayer on the walls of the nanopores. Clearly, the surfactant molecules cannot organize as a compact uninterrupted monolayer. Instead, formation of hemimicelles is thermodynamically favored. Modeling shows that, to be consistent with the experimental observations, the aggregation number of hemimicelles is lower than 25 and the size of hemimicelle is limited to a maximum radius of 11.7 ?. The hydrophobic tails of SDS thus penetrate into and intercalate with the C18 layer. The insight gained in the C18-surfactant interactions is essential in the surfactant-induced solubilization of hydrophobic nanoporous particles. The results have bearing on the understanding of the nature of hydrophobic interactions.     

Microgels formed by electrostatic and hydrophobic interaction of naphthaleneacetic acid with β-cyclodextrin-grafted polyethyleneimine

Microgels were prepared by physically cross-linking β-cyclodextrin-grafted polyethyleneimine (βCD-PEI) using a hydrophobic acidic compound, naphthaleneacetic acid (NAA). Under a strong acidic condition (e.g., pH 3.0), fibrous microgels were observed on a scanning electron microscope (SEM) possibly due to the intermolecular electrostatic interaction of NAA with PEI. In the range of pH 4.0 to pH 8.0, globular microgels were found possibly because an intramolecular electrostatic interaction prevails over an intermolecular interaction. At pH 9.0 and pH 10.0, neither fibrous nor globular microgels were observed due to lack of the electrostatic interaction and the hydrophobic interaction of NAA with βCD-PEI. The release of fluorescein isothiocyanate-dextran (FITC-dextran; M.W., 10,000) from the microgels increased with increasing pH. At pHs higher than pH 3.0, not only the diffusion of the solute, but also the dissolution of the microgels could contribute to the release.     

Enrichment of low-copy-number gene products by hydrophobic interaction chromatography

Enrichment of proteins in solution is the goal of a purification process and often a scientific challenge. We investigated the capacity of hydrophobic interaction chromatography to enrich proteins, potential candidates for novel drug targets. The soluble protein fraction of Haemophilus influenzae was fractionated over a TSK Phenyl column and the proteins resolved were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry. Approximately 150 proteins, bound to the column, were identified, 30 for the first time. Most of the proteins enriched by hydrophobic interaction chromatography were represented by major spots, so that an enrichment of low-copy-number gene products was only partially achieved. The proteins enriched by this chromatographic approach belong to various protein classes, including enzymes, ribosomal proteins and proteins with as yet unknown functions. The results include two-dimensional maps and a list of the proteins enriched by hydrophobic interaction chromatography.