His‐tagged protein purification by metal‐chelate affinity extraction with nickel‐chelate reverse micelles

Di(2‐ethylhexyl) phosphoric acid (HDEHP) was used as a transition metal ion chelator and introduced to the nonionic reverse micellar system composed of equimolar Triton X‐45 and Span 80 at a total concentration of 30 mmol/L. Ni(II) ions were chelated to the HDEHP dimers in the reverse micelles, forming a complex denoted as Ni(II)R₂. The Ni(II)‐chelate reverse micelles were characterized for the purification of recombinant hexahistidine‐tagged enhanced green fluorescent protein (EGFP) expressed in Escherichia coli. The affinity binding of EGFP to Ni(II)R₂ was proved by investigation of the forward and back extraction behaviors of purified EGFP. Then, EGFP was purified with the affinity reverse micelles. It was found that the impurities in the feedstock impeded EGFP transfer to the reverse micelles, though they were little solubilized in the organic phase. The high specificity of the chelated Ni²⁺ ions toward the histidine tag led to the production of electrophoretically pure EGFP, which was similar to that purified by immobilized metal affinity chromatography. A two‐stage purification by the metal‐chelate affinity extraction gave rise to 87% recovery of EGFP. Fluorescence spectrum analysis suggests the preservation of native protein structure after the separation process, indicating the system was promising for protein purification. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Affinity-based reversed micellar protein extraction: II. effect of cosurfactant tail length

The selectivity of protein extraction by reversed micellar solutions can be improved by the addition of affinity cosurfactants bearing ligands which bind strongly to the target protein. The interactions between cosurfactant and protein, as well as the interfacial activity of both the free cosurfactant and the protein-cosurfactant complex, were accounted for in a model of the affinity-partitioning process. The aqueous phase dissociation constant was used to describe the protein-ligand interactions. The interfacial partition coefficient for several cosurfactant families varied with tail length according to the well-established hydrophobic effect. Control studies with alkylated chymotrypsin showed that when longer hydrophobic tails are irreversibly attached to the protein, the protein partitions more strongly to the reversed micellar phase. In contrast, for reversible protein-cosurfactant binding, the model predicts a maximum in protein uptake when the cosurfactant tail length is varied; the decrease at longer tail lengths is due to the lowered aqueous phase concentration of affinity cosurfactant, resulting in the formation of fewer protein-cosurfactant complexes. This behavior was confirmed experimentally. (c) 1993 John Wiley & Sons, Inc.     

Metal chelate affinity precipitation: a new approach to protein purification

Metal chelate affinity precipitation of proteins, a method combining metal–protein interaction and affinity precipitation is being discussed as a selective separation process for proteins. The technique utilizes a flexible soluble–insoluble thermo-responsive polymer with a covalently linked ligand loaded with metal ions. The affinity binding of the target protein varies with different metal ions. Copolymers of N-isopropylacrylamide with 1-vinylimidazole loaded with Cu(II) ions are designed as a potential carriers for affinity purification and proved to be successful for purification of protein inhibitors from a variety of cereals. This revised version was published online in July 2006 with corrections to the Cover Date.     

Single step purification of lactoperoxidase from whey involving reverse micelles‐assisted extraction and its comparison with reverse micellar extraction

The extraction of lactoperoxidase (EC 1.11.1.7) from whey was studied using single step reverse micelles‐assisted extraction and compared with reverse micellar extraction. The reverse micelles‐assisted extraction resulted in extraction of contaminating proteins and recovery of lactoperoxidase in the aqueous phase leading to its purification. Reverse micellar extraction at the optimized condition after forward and backward steps resulted in activity recovery of lactoperoxidase and purification factor of the order of 86.60% and 3.25‐fold, respectively. Whereas reverse micelles‐assisted extraction resulted in higher activity recovery of lactoperoxidase (127.35%) and purification factor (3.39‐fold). The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) profiles also evidenced that higher purification was obtained in reverse micelles‐assisted extraction as compared of reverse micellar extracted lactoperoxidase. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Purification of recombinant protein A by aqueous two-phase extraction integrated with affinity precipitation

Aqueous two-phase extraction incorporated affinity precipitation was examined as a technique for protein purification. An enteric coating polymer, Eudragit S100, was employed as a ligand carrier. Eudragit was specifically partitioned to the top phase in the aqueous two-phase systems. For application of this method to purification of recombinant protein A using human IgG coupled to Eudragit in an aqueous two-phase system, 80% of protein A added was recovered with 81% purity. The purity was enhanced 26-fold by thid method. The IgG-Eudragit could be used repeatedly for the purification process. This seperation method should be applicable to industrial-scale purification as a new purification procedure combining the advantages and compensating for the disadvantages of the aqueous two-phase method and affinity precipitation method. (c) 1992 John Wiley & Sons, Inc.     

Solid‐phase extraction with metal–organic frameworks for the analysis of chiral compounds

Metal–organic frameworks (MOFs) are excellent porous materials with nanoscale cavities and high surface areas, which make them promising as novel adsorbents in solid‐phase extraction (SPE). In this article we report a new application of the chiral MOF [Zn₂(D‐Cam)₂(4,4′‐bpy)]_n in SPE used for the measurement of the enantiomeric excess (ee) of (±)‐1,1′‐bi‐2‐naphthol. Several important experimental parameters that may influence the extraction efficiency were investigated and optimized. Under the optimum conditions, a good linearity (R² > 0.999) was found between the ee value and the reciprocal of the peak areas. When compared with the actual ee measured using chiral HPLC, the SPE‐based assay also showed good accuracy and precision. The results showed that SPE based on chiral MOFs as adsorbents is a simple and effective method for the determination of the ee values of chiral compounds.     

Binding of phosphorylated peptides and inhibition of their interaction with disease‐relevant human proteins by synthetic metal‐chelate receptors

The modulation of biological signal transduction pathways by masking phosphorylated amino acid residues represents a viable route toward pharmacologic protein regulation. Binding of phosphorylated amino acid residues has been achieved with synthetic metal‐chelate receptors. The affinity and selectivity of such receptors can be enhanced if combined with a second binding site. We demonstrate this principle with a series of synthetic ditopic metal‐chelate receptors, which were synthesized and investigated for their binding affinity to phosphorylated short peptides under conditions of physiological pH. The compounds showing highest affinity were subsequently used to inhibit the interaction of the human STAT1 protein to a peptide derived from the interferon‐γ receptor, and between the checkpoint kinase Chk2 and its preferred binding motif. Two of the investigated ditopic synthetic receptors show a significant increase in inhibition activity. The results show that regulation of protein function by binding to phosphorylated amino acids is possible. The introduction of additional binding sites into the synthetic receptors increases their affinity, but the flexibility of the structures investigated so far prohibited stringent amino acid sequence selectivity in peptide binding. Copyright © 2009 John Wiley & Sons, Ltd.     

Exploring additivity effects of double mutations on the binding affinity of protein‐protein complexes

Additivity in binding affinity of protein‐protein complexes refers to the change in free energy of binding (ΔΔG_bind) for double (or multiple) mutations which is approximately equal to the sum of their corresponding single mutation ΔΔG_bind values. In this study, we have explored the additivity effect of double mutants, which shows a linear relationship between the binding affinity of double and sum of single mutants with a correlation of 0.90. However, the comparison of ΔΔG_bind values showed a mean absolute deviation of 0.86 kcal/mol, and 25.6% of the double mutants show a deviation of more than 1 kcal/mol, which are identified as non‐additive. The additivity effects have been analyzed based on the influence of structural features such as accessible surface area, long range order, binding propensity change, surrounding hydrophobicity, flexibility, atomic contacts between the mutations and distance between the 2 mutations. We found that non‐additive mutations tend to be closer to each other and have more contacts. We have also used machine learning methods to discriminate additive and non‐additive mutations using structure‐based features, which showed the accuracies in the range of 0.77–0.92 for protein‐protein complexes belonging to different functions. Further, we have compared the additivity effects of protein stability along with binding affinity and explored the similarities and differences between them. The results obtained in this study provide insights into the effects of various structural features on binding affinity of double mutants, and will aid the development of accurate methods to predict the binding affinity of double mutants.     

Enantioseparation of flurbiprofen enantiomers using chiral ionic liquids by liquid‐liquid extraction

Flurbiprofen is a kind of nonsteroidal anti‐inflammatory drug, which has been widely used in clinic for treatment of rheumatoid arthritis and osteoarthritis. It has been reported that S‐flurbiprofen shows good performance on clinic anti‐inflammatory treatment, while R‐enantiomer almost has no pharmacological activities. It has important practical values to obtain optically pure S‐flurbiprofen. In this work, chiral ionic liquids, which have good structural designability and chiral recognize ability, were selected as the extraction selector by the assistance of quantum chemistry calculations. The distribution behaviors of flurbiprofen enantiomers were investigated in the extraction system, which was composed of organic solvent and aqueous phase containing chiral ionic liquid. The results show that maximum enantioselectivity up to 1.20 was attained at pH 2.0, 25°C using 1,2‐dichloroethane as organic solvent, 1‐butyl‐3‐methylimidazole L‐tryptophan ([Bmim][L‐trp]) as chiral selector. The racemic flurbiprofen initial concentration was 0.2 mmol L^?1, and [Bmim][L‐trp] concentration was 0.02 mol L^?1. Furthermore, the recycle of chiral ionic liquids has been achieved by reverse extraction process of the aqueous phase with chiral selector, which is significant for industrial application of chiral ionic liquids and scale‐up of the extraction process.     

A chelate complex‐enhanced luminol system for selective determination of Co(II), Fe(II) and Cr(III)

A determination method for Co(II), Fe(II) and Cr(III) ions by luminol‐H₂O₂ system using chelating reagents is presented. A metal ion‐chelating ligand complex with a Co(II) ion and a chelating reagent like ethylenediaminetetraacetic acid (EDTA) produced highly enhanced chemiluminescence (CL) intensity as well as longer lifetime in the luminol‐H₂O₂ system compared to metals that exist as free ions. Whereas free Cu(II) and Pb(II) ions had a strong catalytic effect on the luminol‐H₂O₂ system, significantly, the complexes of Cu(II) and Pb(II) with chelating reagents lost their catalytic activity due to the chelating reagents acting as masking agents. Based on the observed phenomenon, it was possible to determine Co(II), Fe(II) and Cr(III) ions with enhanced sensitivity and selectivity using the chelating reagents of the luminol‐H₂O₂ system. The effects of ligand, H₂O₂ concentration, pH, buffer solution and concentrations of chelating reagents on CL intensity of the luminol‐H₂O₂ system were investigated and optimized for the determination of Co(II), Fe(II) and Cr(III) ions. Under optimized conditions, the calibration curve of metal ions was linear over the range of 2.0 × 10^‐8 to 2.0 × 10^‐5 M for Co(II), 1.0 × 10^‐7 to 2.0 × 10^‐5 M for Fe (II) and 2.0 × 10^‐7 to 1.0 × 10^‐4 M for Cr(III). Limits of detection (3σ/s) were 1.2 × 10^‐8, 4.0 × 10^‐8 and 1.2 × 10^‐7 M for Co(II), Fe(II) and Cr(III), respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

Affinity-tagged green fluorescent protein (GFP) extraction from a clarified E. coli cell lysate using a two-phase aqueous micellar system

Green fluorescent protein (GFP) has been proposed as an ideal choice for a protein-based biological indicator for use in the validation of decontamination or disinfection treatments. In this article, we present a potentially scalable and cost-effective way to purify recombinant GFP, produced by fermentation in Escherichia coli, by affinity-enhanced extraction in a two-phase aqueous micellar system. Affinity-enhanced partitioning, which improves the specificity and yield of the target protein by specific bioaffinity interactions, has been demonstrated. A novel affinity tag, family 9 carbohydrate-binding module (CBM9) is fused to GFP, and the resulting fusion protein is affinity-extracted in a decyl beta-D-glucopyranoside (C10G1) two-phase aqueous micellar system. In this system, C10G1 acts as phase forming and as affinity surfactant. We will further demonstrate the implementation of this concept to attain partial recovery of affinity-tagged GFP from a clarified E. coli cell lysate, including the simultaneous removal of other contaminating proteins. The cell lysate was partitioned at three levels of dilution (5x, 10x, and 40x). Irrespective of the dilution level, CBM9-GFP was found to partition preferentially to the micelle-rich phase, with the same partition coefficient value as that found in the absence of the cell lysate. The host cell proteins from the cell lysate were found to partition preferentially to the micelle-poor phase, where they experience less excluded-volume interactions. The demonstration of proof-of-principle of the direct affinity-enhanced extraction of CBM9-GFP from the cell lysate represents an important first step towards developing a cost-effective separation method for GFP, and more generally, for other proteins of interest.     

Heterofunctional Magnetic Metal‐Chelate‐Epoxy Supports for the Purification and Covalent Immobilization of Benzoylformate Decarboxylase From Pseudomonas Putida and Its Carboligation Reactivity

In this study, the combined use of the selectivity of metal chelate affinity chromatography with the capacity of epoxy supports to immobilize poly‐His‐tagged recombinant benzoylformate decarboxylase from Pseudomonas putida (BFD, E.C. 4.1.1.7) via covalent attachment is shown. This was achieved by designing tailor‐made magnetic chelate–epoxy supports. In order to selectively adsorb and then covalently immobilize the poly‐His‐tagged BFD, the epoxy groups (300 µmol epoxy groups/g support) and a very small density of Co²⁺‐chelate groups (38 µmol Co²⁺/g support) was introduced onto magnetic supports. That is, it was possible to accomplish, in a simple manner, the purification and covalent immobilization of a histidine‐tagged recombinant BFD. The magnetically responsive biocatalyst was tested to catalyze the carboligation reactions. The benzoin condensation reactions were performed with this simple and convenient heterogeneous biocatalyst and were comparable to that of a free‐enzyme‐catalyzed reaction. The enantiomeric excess (ee) of (R)‐benzoin was obtained at 99 ± 2% for the free enzyme and 96 ± 3% for the immobilized enzyme. To test the stability of the covalently immobilized enzyme, the immobilized enzyme was reused in five reaction cycles for the formation of chiral 2‐hydroxypropiophenone (2‐HPP) from benzaldehyde and acetaldehyde, and it retained 96% of its original activity after five reaction cycles. Chirality 27:635–642, 2015. © 2015 Wiley Periodicals, Inc.     

AB‐Bind: Antibody binding mutational database for computational affinity predictions

Antibodies (Abs) are a crucial component of the immune system and are often used as diagnostic and therapeutic agents. The need for high‐affinity and high‐specificity antibodies in research and medicine is driving the development of computational tools for accelerating antibody design and discovery. We report a diverse set of antibody binding data with accompanying structures that can be used to evaluate methods for modeling antibody interactions. Our Antibody‐Bind (AB‐Bind) database includes 1101 mutants with experimentally determined changes in binding free energies (ΔΔG) across 32 complexes. Using the AB‐Bind data set, we evaluated the performance of protein scoring potentials in their ability to predict changes in binding free energies upon mutagenesis. Numerical correlations between computed and observed ΔΔG values were low (r = 0.16–0.45), but the potentials exhibited predictive power for classifying variants as improved vs weakened binders. Performance was evaluated using the area under the curve (AUC) for receiver operator characteristic (ROC) curves; the highest AUC values for 527 mutants with |ΔΔG| > 1.0 kcal/mol were 0.81, 0.87, and 0.88 using STATIUM, FoldX, and Discovery Studio scoring potentials, respectively. Some methods could also enrich for variants with improved binding affinity; FoldX and Discovery Studio were able to correctly rank 42% and 30%, respectively, of the 80 most improved binders (those with ΔΔG < −1.0 kcal/mol) in the top 5% of the database. This modest predictive performance has value but demonstrates the continuing need to develop and improve protein energy functions for affinity prediction.     

Optimization of protein extraction from Chlorella Vulgaris via novel sugaring‐out assisted liquid biphasic electric flotation system

Microalgae biomass has been consumed as animal feed, fish feed and in human diet due to its high nutritional value. In this experiment, microalgae specie of Chlorella Vulgaris FSP‐E was utilized for protein extraction via simple sugaring‐out assisted liquid biphasic electric flotation system. The external electric force provided to the two‐phase system assists in disruption of rigid microalgae cell wall and releases the contents of microalgae cell. This experiment manipulates various parameters to optimize the set‐up. The liquid biphasic electric flotation set‐up is compared with a control liquid biphasic flotation experiment without the electric field supply. The optimized separation efficiency of the liquid biphasic electric flotation system was 73.999 ± 0.739% and protein recovery of 69.665 ± 0.862% compared with liquid biphasic flotation, the separation efficiency was 61.584 ± 0.360% and protein recovery was 48.779 ± 0.480%. The separation efficiency and protein recovery for 5 × time scaled‐up system was observed at 52.871 ± 1.236% and 73.294 ± 0.701%. The integration of simultaneous cell‐disruption and protein extraction ensures high yield of protein from microalgae. This integrated method for protein extraction from microalgae demonstrated its potential and further research can lead this technology to commercialization.     

A comprehensive evaluation of imidazole‐zinc reverse stain for current proteomic researches

In this paper, we comprehensively evaluated the capability of imidazole‐zinc reverse stain (ZN) in comparative proteomics. Three commonly used protein gel staining methods, including silver (SN), SYPRO Ruby (SR), and CB stain were investigated alongside for comparison purpose. A transparency scanning procedure, which may deliver more even and contrasting gel images, was found best for documenting ZN stained gels. Our results showed that ZN was more sensitive than SN, SR, and CB. It may reveal as few as 1.8 ng of proteins in a gel. Moreover, ZN was found to provide a linear dynamic range of staining for revealing proteins up to 140 ng, and show an insignificant staining preference. To analyze a ZN stained 2‐D gel image that generally comprises an apparent but even background, the Melanie 4 software was found more suitable than others. Furthermore, ZN demonstrated an equivalent or better MS compatibility than the other three staining methods. Intense and comprehensive MS profiles were frequently observed for ZN stained gel spots. Approximate two‐third of ZN stained gel spots were successfully identified for protein identities. Taken together, our results suggest that the prompt, cost effective and versatile ZN is well suited for current proteomic researches.     

Isolation and characterisation of cDNA encoding a wheat heavy metal‐associated isoprenylated protein involved in stress responses

In cells, metallochaperones are important proteins that safely transport metal ions. Heavy metal‐associated isoprenylated plant proteins (HIPPs) are metallochaperones that contain a metal binding domain and a CaaX isoprenylation motif at the carboxy‐terminal end. To investigate the roles of wheat heavy metal‐associated isoprenylated plant protein (TaHIPP) genes in plant development and in stress responses, we isolated cDNA encoding the wheat TaHIPP1 gene, which contains a heavy metal‐associated domain, nuclear localisation signals and an isoprenylation motif (CaaX motif). Quantitative real‐time PCR analysis indicated that the TaHIPP1 gene was differentially expressed under biotic and abiotic stresses. Specifically, TaHIPP1 expression was up‐regulated by ABA exposure or wounding. Additionally, TaHIPP1 over‐expression in yeast (Schizosaccharomyces pombe) significantly increased the cell growth rate under Cu²⁺ and high salinity stresses. The nuclear localisation of the protein was confirmed with confocal laser scanning microscopy of epidermal onion cells after particle bombardment with chimeric TaHIPP1‐GFP constructs. In addition, TaHIPP1 was shown to enhance the susceptibility of wheat to Pst as determined by virus‐induced gene silencing. These data indicate that TaHIPP1 is an important component in defence signalling pathways and may play a crucial role in the defence response of wheat to biotic and certain abiotic stresses.     

Comparison of different protein precipitation and solid‐phase extraction protocols for the study of the catabolism of peptide drugs by LC‐HRMS

LC‐HRMS‐based identification of the products of peptide catabolism is the key to drive the design of more stable compounds. Because the catabolite of a given peptide can be very different from the parent compound and from other catabolites in terms of physicochemical properties, it can be challenging to develop an analytical method that allows recovery and detection of the parent and all parent‐related catabolites. The aim of this study was to investigate how the recovery and the matrix effect of peptidic drugs and their catabolites are affected by different protein precipitation (PP) and solid‐phase extraction (SPE) protocols. To this purpose, four model peptides representative of different classes (somatostatin, GLP‐2, human insulin and liraglutide) were digested with trypsin and chymotrypsin to simulate proteolytic catabolism. The resulting mixtures of the parent peptides and their proteolytic products covering a wide range of relative hydrophobicity (H_R) and isoelectric points (pI) were spiked in human plasma and underwent different PP and SPE protocols. Recovery and matrix effect were measured for each peptide and its catabolites. PP with three volumes of ACN or EtOH yielded the highest overall recoveries (more than 50% for the four parent peptides and all their catabolites) among all the tested PP and SPE protocols. Mixed‐mode anion exchange (MAX) was the only SPE sorbent among the five tested that allowed to extract all the peptides with recoveries more than 20%. Matrix effect was generally lower with SPE. Overall, it was observed that peptides with either high hydrophilicity (e.g., somatostatin catabolites) or hydrophobicity (GLP‐2 and lipidated liraglutide catabolites) had a much narrower choice of PP solvent or SPE protocol. Simulation of catabolism using recombinant enzymes together with in silico calculation of the H_R and the pI of potential proteolysis products is recommended to select the optimal extraction conditions for the study of peptide catabolism.