Metal-ion Chromatography Research Articles

Study on the transient expression of infectious bronchitis virus spike protein in Nicotiana benthamiana leaves

Infectious bronchitis virus (IBV) is considered as one of the main causes of economic loss in chicken farms worldwide, especially in poultry producing nations. This virus, a member of the Coronaviridae family, is classified into different genotypes based on its surface spike glycoproteins which also play important roles in cell attachment and immune response. Due to continual genetic mutation of IBV, attenuated and inactivated vaccines show decreased effectiveness or lack of cross – protection; thus ongoing studies focus on the development of new generation vaccines to prevent new IBV outbreaks. This paper describes our study on expression of the antigenic region in S1 subunit of the spike in Nicotiana benthamiana. Two expression vectors carrying either S1 or receptor binding domain (RBD) coding gene (pCB301-S1 and pCB301-RBD) were constructed and transformed into tobacco leaves by agroinfiltration method. The RBD showed a clearly higher level of expression compared to the whole S1. Purification of RBD by immobilized metal ion chromatography and size exclusion chromatography represented a mixture of monomer, dimer and trimer glycoprotein with expected sizes in Western blot. In summary, this study demonstrated our primary success in establishing an expression model that could be used to investigate plant-based IBV recombinant vaccine.

Hydrophilic magnetic host-guest Ti-phenolic networks: a promising material for the highly sensitive enrichment of glycopeptides and phosphopeptides.

Protein glycosylation and phosphorylation are the two most important post-translational modifications, which play a vital role in physiological and pathological processes. Before the comprehensive characterization of glycoproteome/phosphoproteome through the mass spectrometry (MS) technique, it is necessary to perform a highly specific enrichment procedure because glycoproteins/phosphoproteins inherently occur in low abundances. Herein, we have reported a novel magnetic β-cyclodextrin-based host-guest Ti-phenolic network material, focusing on the simultaneous enrichment of glycopeptides/phosphopeptides via hydrophilic interaction chromatography and immobilized metal ion chromatography. Ti ions and glutathione-derived adamantine were introduced by metal-phenolic interactions and host-guest interactions. The material possesses biocompatibility, good hydrophilicity, strong magnetic response, metal chelation effect, and demonstrates an excellent enrichment ability towards glycopeptides/phosphopeptides. Combined with MS detection, high sensitivity (0.035/0.01 fmol for IgG/β-casein) and good reusability (6 times) were achieved. In addition, its outstanding specificity was validated in quantities as low as 500 : 1 : 1 for BSA : IgG : β-casein (m/m/m). Benefiting from these merits, the adsorbent material was successfully used for the simultaneous enrichment of phosphopeptides/glycopeptides from human serum and HeLa cell lysate and can be expected to exhibit great applicability for precious and small amounts of biosamples in the study of glycoproteomics/phosphoproteomics.

Producción y caracterización de una esterasa extracelular termoestable de Aspergillus niger

A strain of Aspergillus niger NRRL 337 degrade agro-industrial waste i.e wheat bran in solid state fermentation and produce esterase enzyme. Maximum enzyme activity 15.2 U/ml was produced after 72 h of incubation at 30˚C. The protein was partially purified by performing ammonium sulfate precipitation and immobilized metal ion chromatography (IMAC) technique. SDS-PAGE analysis showed molecular weight of esterase was 70 kDa. Effects of different nitrogrn and carbon sources with different concentrations were also studied. Partially purified enzyme was active at pH of 6.0 for up to 3 h and at temperature of 70˚C for 1 h. Ca2+, Mg2+, Na+1, and K+1 play an important role in increasing enzyme activity. The residual activity of esterase was decreased in the presence of organic solvents. SDS, β-mercaptoethanol, and DMSO are good inhibitors for esterase enzyme. Under these conditions, partially purified esterase with specific activity of 114.20 U/mg and 47.58 purification fold was obtained. The data achieved from this work are important in terms of heat stability and pH of esterase and giving a new enzyme source for industrial applications.

Zinc(II) Chelate Binds to Fibrinogen and Its αC Region

Fibrinogen is the immediate precursor of fibrin blood clots. When fibrinogen is cleaved by thrombin, it assembles into fibrin polymers through intermolecular interactions where calcium and zinc ions may both be involved. Fibrinogen’s αC domains play a major role in fibrin polymer assembly, and they contain a Zn²⁺ binding sites based on peptide studies. However, Zn²⁺ binding to natural αC and fibrin (ogen) remains to be demonstrated. The objective of this study was to directly demonstrate Zn²⁺ interactions with purified fibrinogen and its αC region. Both human and bovine αC regions were investigated due to the significant differences in their protein sequences. To detect Zn²⁺ binding, chelated Zn²⁺ in the form of Zn²⁺nitrilotriacetic acid linked to alkaline phosphatase tracer (ZnTAP) was prepared and used in ELISA and Western blot style methods. Gel electrophoresis was used to monitor changes to fibrinogen after reaction with plasmin. Human and bovine αC regions were obtained by limited plasmin digests of fibrinogen and purified by Con-A and Zn-immobilized metal ion chromatography. The results showed ZnTAP binding to fibrinogen in solution and inhibition of binding to fibrinogen coated microplates by ELISA. Inhibition concentrations (IC50) of 0.58 μM for human fibrinogen and 0.26 μM for bovine fibrinogen were determined. Blot probes revealed ZnTAP binding to fibrinogen with disulfide bonds intact and to the Aα and Bβ chains with disulfide bonds reduced. Reduced Aα chain reaction was less than that of intact fibrinogen and the reduced Bβ chain was only weakly reactive. With plasmin treatment, ZnTAP binding decreased by ELISA in parallel with the degradation of αC region by gel electrophoresis analysis. Purified αC bound ZnTAP in ELISA and on Western blots. In this study we demonstrated that Zn²⁺ binds to fibrinogen and its Aα and Bβ chains, and to the αC region of the Aα chain. The results support a role for protein conformation in Zn²⁺ binding and demonstrate the utility of the ZnTAP complex with tracer for fibrinogen binding interactions.

A versatile tool in controlling aggregation and Ag nanoparticles assisted in vitro folding of thermally denatured zDHFR

BackgroundProteins have tendency to form inactive aggregates at higher temperatures due to thermal instability. Maintenance of thermal stability is essential to gain the protein in sufficient quantity and biologically active form during their commercial production. MethodsBL21-DE3 Rosetta E. coli cells which contains plasmid pET43.1a vector was used for producing zDHFR protein commercially. The purification of N-terminal Histidine tagged zDHFR was performed by Immobilized Metal Ion chromatography (IMAC). Investigations were performed in existence and non existence of Silver nanoparticles (AgNPs). The inactivation kinetics of zDHFR in existence and non existence of AgNPs were monitored over a range of 40–80 °C as monitored by UV–Visible absorption spectroscopy. ResultsThe protein completely lost its activity at 55 °C. Kinetics of inactivated zDHFR follows first order model in presence and absence of AgNPs. Decrease in rate constant (k) values at respective temperatures depicts that AgNPs contribute in the thermostability of the protein. AgNPs also assists in regaining the activity of zDHFR protein. ConclusionsAgNPs helps in maintaining thermostability and reducing the aggregation propensity of zDHFR protein. General significanceResult explains that AgNPs are recommended as a valuable system in enhancing the industrial production of biologically active zDHFR protein which is an important component in folate cycle and essential for survival of cells and prevents the protein from being aggregated.

Recombinant N-glycosylation isoforms of Legume lectins: Production and purification from Nicotiana benthamiana leaves following RuBisCO depletion

An efficient purification of recombinant proteins often requires a high ratio of recombinant to host proteins. In plants, Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant leaf protein, thus strongly impacting purification yield. Here, we describe a simple and robust purification procedure for recombinant proteins based on a differential precipitation of RuBisCO. In this context, four Legume lectin domains of Arabidopsis thaliana which belong to receptor-like kinases and cell wall proteins were produced from Nicotiana benthamiana leaves. The recombinant proteins exhibit a unique lectin domain consisting of around 250 amino acid residues with several predicted N-glycosylation sites and a six His-tag at the N-terminus. After ammonium sulphate precipitation of total soluble proteins, depletion of RuBisCO was obtained using citrate and succinate buffers during the salting-in step: this depletion was pH-dependent and the presence of di- or tri-carboxylic acids was required. The depleted protein extracts were then subjected to two chromatographic steps which were used in the negative mode to submit a protein fraction enriched as much as possible in recombinant lectin domains to a third chromatographic step (immobilized metal-ion chromatography). Three of the Legume lectin domains were purified near to homogeneity and revealed multiple N-glycosylation isoforms, particularly those from receptor-like kinases, which were characterised using specific lectins and deglycosylation enzymes. The production and purification of recombinant lectin domains will facilitate their biochemical characterisation in the context of cell-to-cell signalling and cell wall organisation.

Characterization and Expression Profiling of Recombinant Parathyroid Hormone (rhPTH) Analog 1–34 in Escherichia coli, Precise with Enhanced Biological Activity

Human truncated parathyroid hormone [hPTH (1–34)], a peptide hormone which accelerated the research interest towards the theraputical applications. This study outlines the effective expression of [hPTH (1–34)] in Escherichia coli and the recuperation of highly soluble truncated PTH from the fusion protein by proteolytic digestion. Successful expression of glutathione S-transferase (GST) fusion protein was achieved by incorporating truncated PTH with an enzymatic cleavage site into the “N” terminal GST of pGEX-4T-3 expression vector. Under the optimized condition, we achieved more than 120 mg of pure hPTH (1–34) per liter of bacterial culture, with an overall yield of 39%. Purification process was carried out through immobilized metal ion chromatography and membrane filter to produce maximum purity. Physical characterization using western blot analysis showed that the extracted truncated PTH is intact and reacts with anti-PTH antibodies. In vitro analysis of PTH stimulated adenylyl cyclase activation in UMR 106 cells confirmed biological activity for purified protein. We believe this vector and production methodology represents a generally applicable tool for the generation of recombinant peptides.

Detection of histidine-rich glycoprotein and fibrinogen with nickel-enzyme conjugates: Purification of rabbit HRG

Nickel-bound alkaline phosphatase and peroxidase enzymes were used to investigate nickel binding to plasma proteins. Rabbit plasma dilutions to 25,000 were positive by ELISA, while Western blot analysis showed a prominent reaction with histidine-rich glycoprotein (HRG)1 and lower reaction with fibrinogen (Fgn). To confirm their identities, purified HRG and Fgn were demonstrated to react with the nickel-bound enzymes by Western analysis. With disulfide bonds reduced, HRG and Fgn α-chain reactions were demonstrated. HRG reactions were shown in other species, including human, bovine, chicken and guinea pig, demonstrating general applicability of the detection method. To enhance the purification of rabbit HRG, ammonium sulfate fractionation, immobilized metal ion chromatography and ion-exchange chromatography were optimized. Purified HRG contained trace components larger than HRG that reacted with nickel-enzymes and also with an antibody to HRG by Western analysis, confirming the trace components are related to HRG. These results demonstrate the utility of nickel-enzymes together with antibodies to detect HRG.

Transduction of Recombinant M3-p53-R12 Protein Enhances Human Leukemia Cell Apoptosis.

Tumor suppressor protein p53 plays important roles in initiating cell cycle arrest and promoting tumor cell apoptosis. Previous studies have shown that p53 is either mutated or defective in approximately 50% of human cancers; therefore restoring normal p53 activity in cancer cells might be an effective anticancer therapeutic approach. Herein, we designed a chimeric p53 protein flanked with the MyoD N-terminal transcriptional activation domain (amino acids 1-62, called M3) and a poly-arginine (R12) cell penetrating signal in its N-and C-termini respectively. This chimeric protein, M3-p53-R12, can be expressed in E. coli and purified using immobilized metal ion chromatography followed by serial refolding dialysis. The purified M3-p53-R12 protein retains DNA-binding activity and gains of cell penetrating ability. Using MTT assay, we demonstrated that M3-p53-R12 inhibited the growth of K562, Jurkat as well as HL-60 leukemia cells carrying mutant p53 genes. Results from FACS analysis also demonstrated that transduction of M3-p53-R12 protein induced cell cycle arrest of these leukemia cells. Of special note, M3-p53-R12 has no apoptotic effect on normal mesenchymal stem cells (MSC) and leukocytes, highlighting its differential effects on normal and tumor cells. To sum up, our results reveal that purified recombinant M3-p53-R12 protein has functions of suppressing the leukemia cell lines' proliferation and launching cell apoptosis, suggesting the feasibility of using M3-p53-R12 protein as an anticancer drug. In the future we will test whether this chimeric protein can preferentially trigger the death of malignant cancer cells without affecting normal cells in animals carrying endogenous or xenographic tumors.

Deciphering ligand specificity of a Clostridium thermocellum family 35 carbohydrate binding module (CtCBM35) for gluco- and galacto- substituted mannans and its calcium induced stability.

This study investigated the role of CBM35 from Clostridium thermocellum (CtCBM35) in polysaccharide recognition. CtCBM35 was cloned into pET28a (+) vector with an engineered His6 tag and expressed in Escherichia coli BL21 (DE3) cells. A homogenous 15 kDa protein was purified by immobilized metal ion chromatography (IMAC). Ligand binding analysis of CtCBM35 was carried out by affinity electrophoresis using various soluble ligands. CtCBM35 showed a manno-configured ligand specific binding displaying significant association with konjac glucomannan (K a = 14.3×104 M−1), carob galactomannan (K a = 12.4×104 M−1) and negligible association (K a = 12 µM−1) with insoluble mannan. Binding of CtCBM35 with polysaccharides which was calcium dependent exhibited two fold higher association in presence of 10 mM Ca2+ ion with konjac glucomannan (K a = 41×104 M−1) and carob galactomannan (K a = 30×104 M−1). The polysaccharide binding was further investigated by fluorescence spectrophotometric studies. On binding with carob galactomannan and konjac glucomannan the conformation of CtCBM35 changed significantly with regular 21 nm peak shifts towards lower quantum yield. The degree of association (K a) with konjac glucomannan and carob galactomannan, 14.3×104 M−1 and 11.4×104 M−1, respectively, corroborated the findings from affinity electrophoresis. The association of CtCBM35with konjac glucomannan led to higher free energy of binding (ΔG) −25 kJ mole−1 as compared to carob galactomannan (ΔG) −22 kJ mole−1. On binding CtCBM35 with konjac glucomannan and carob galactomannan the hydrodynamic radius (RH) as analysed by dynamic light scattering (DLS) study, increased to 8 nm and 6 nm, respectively, from 4.25 nm in absence of ligand. The presence of 10 mM Ca2+ ions imparted stiffer orientation of CtCBM35 particles with increased RH of 4.52 nm. Due to such stiffer orientation CtCBM35 became more thermostable and its melting temperature was shifted to 70°C from initial 50°C.

Lactadherin C2 Domain Exhibits Ptd-L-Ser Specificity and Anticoagulant Properties Distinct From Homologous Factor VIII C2 Domain and Full-Length Lactadherin

AbstractAbstract 1105 Background:Lactadherin (aka mfg-e8) is a milk-fat globule membrane protein with a domain structure of EGF1-EGF2-C1-C2, where the lectin-like C1 and C2 domains are homologous to the membrane binding domains of factor VIII and factor V. Like factor VIII and factor V, lactadherin exhibits calcium-independent membrane binding that is selective for phosphatidyl-L-serine (Ptd-L-Ser). Lactadherin also binds preferentially to convex membranes, competes efficiently for binding sites of factor VIII and factor V, and can function as an anticoagulant via competition for these binding sites. On stressed endothelial cells lactadherin binds to filopodia and the cell margins, identifying sites that have exposed Ptd-L-Ser and support assembly of the prothrombinase complex.The crystallographic structure of the lactadherin C2 domain (Lact-C2) and structure-function studies have shown that membrane binding is mediated by a longer β-hairpin turn, with different residues than fVIII-C2 and fV-C2. Further, we have shown that Lact-C2 maintains specificity for phosphatidylserine, in contrast to fVIII-C2, and that fluorescent fusion proteins containing Lact-C2 can be used as intracellular phosphatidylserine probes (Yeung et al. Science 2008;319:210). However, the extent to which Lact-C2 retains the membrane binding and anticoagulant properties of full-length lactadherin, has not been studied. Methods:Lact-C2 was produced in E. coliand purified by metal ion chromatography followed by gel filtration. Competition experiments were performed by flow cytometry using phospholipid bilayers supported by glass microspheres to determine Lact-C2's ability to block binding sites of FITC-labeled lactadherin, or fluorescein-labeled factor VIII and factor V. Lact-C2 was also labeled with FITC to measure its binding to sonicated or 100 nm diameter, extruded vesicles with varying PS content in order to assess Ptd-L-Ser selectivity and membrane curvature sensitivity. Two-step amidolytic factor Xase and prothrombinase assays were used to assess the ability of Lact-C2 to block activity. Fluorescence microscopy experiments were used to compare the binding of Alexa 647-labeled Lact-C2 vs. FITC-labeled lactadherin on staurosphorine-treated HeLa cells. Results:Lact-C2 showed stereospecific binding to Ptd-L-Ser vs. Ptd-D-Ser in vesicles of 4% and 10% PS. Lact-C2 was sensitive to vesicle curvature, detecting as little as 1% Ptd-L-Ser on sonicated vesicles but requiring 4% Ptd-L-Ser on extruded vesicles. Lact-C2 competed for 89% of lactadherin binding sites and 84% of factor VIII binding sites. Inhibition of factor Xase activity plateaued at 89% reduction vs. >99% reduction for lactadherin. Lact-C2 also competed for 61% of factor V binding sites corresponding to an 82% reduction in prothrombinase activity. We are currently comparing the distribution of binding sites for Lact-C2 vs. lactadherin on stressed HeLa cells, with preliminary data showing distinct, but overlapping, binding site distribution. Discussion:Lact-C2 exhibits stereospecific Ptd-L-Ser binding and convex curvature preference similar to full-length lactadherin. Lact-C2 contrasts with fVIII-C2 in Ptd-L-Ser specificity and capacity to compete with factor VIII and inhibit factor Xase activity and prothrombinase activity.These results provide a framework for interpreting experiments in which Lact-C2 is used as an anticoagulant or as a calcium-independent probe for exposed membrane Ptd-L-Ser. Lact-C2 is able to bind to only a subset of lactadherin binding sites, highlighting the importance of the lactadherin C1 domain for high affinity binding and underscoring the largely unappreciated complexity of phospholipid membrane binding sites. Disclosures:Shi:Brigham and Women’s Hospital: Use of Lactadherin to detect phosphataidylserine, Use of Lactadherin to detect phosphataidylserine Patents & Royalties.

Recombinant Production, Isotope Labeling and Purification of ENOD40B: A Plant Peptide Hormone

The plant peptide hormone ENOD40B was produced in a protein production strain of Escherichia coli harboring an induction controller plasmid (Rosetta(DE3)pLysS) as a His6-tagged ubiquitin fusion protein. The fusion protein product was denatured and refolded as part of the isolation procedure and purified by immobilized metal ion chromatography. The peptide hormone was released from its fusion partner by adding yeast ubiquitin hydrolase (YUH) and subsequently purified by reversed phase chromatography. The purity of the resulting peptide fragment was assayed by MALDITOF mass spectrometry and NMR spectroscopy. The final yields of the target peptide were 7.0 mg per liter of LB medium and 3.4 mg per liter of minimal medium.

Hierarchical Fe-ZSM-5 zeolite monolithic column for enhanced phosphorylated protein immobilization and identification

A hierarchical Fe-ZSM-5 zeolite monolithic column was fabricated for immobilization of low abundance phosphoprotein through specific coordination between heteroatomic Fe in the framework of the zeolite with the phosphate group in the micro-environment of the protein. Compared to traditionally immobilized metal ion chromatography, this method could be directly manipulated and handily adapted for phosphoproteomics identification.

A proteomic approach to identification of plutonium-binding proteins in mammalian cells

Plutonium can enter the body through different routes and remains there for decades; however its specific biochemical interactions are poorly defined. We, for the first time, have studied plutonium-binding proteins using a metalloproteomic approach with rat PC12 cells. A combination of immobilized metal ion chromatography, 2D gel electrophoresis, and mass spectrometry was employed to analyze potential plutonium-binding proteins. Our results show that several proteins from PC12 cells show affinity towards Pu(4+)-NTA (plutonium bound to nitrilotriacetic acid). Proteins from seven different spots in the 2D gel were identified. In contrast to the previously known plutonium-binding proteins transferrin and ferritin, which bind ferric ions, most identified proteins in our experiment are known to bind calcium, magnesium, or divalent transition metal ions. The identified plutonium interacting proteins also have functional roles in downregulation of apoptosis and other pro-proliferative processes. MetaCore™ analysis based on this group of proteins produced a pathway with a statistically significant association with development of neoplastic diseases.

Large-scale phosphotyrosine proteomic profiling of rat renal collecting duct epithelium reveals predominance of proteins involved in cell polarity determination

Although extensive phosphoproteomic information is available for renal epithelial cells, previous emphasis has been on phosphorylation of serines and threonines with little focus on tyrosine phosphorylation. Here we have carried out large-scale identification of phosphotyrosine sites in pervanadate-treated native inner medullary collecting ducts of rat, with a view towards identification of physiological processes in epithelial cells that are potentially regulated by tyrosine phosphorylation. The method combined antibody-based affinity purification of tyrosine phosphorylated peptides coupled with immobilized metal ion chromatography to enrich tyrosine phosphopeptides, which were identified by LC-MS/MS. A total of 418 unique tyrosine phosphorylation sites in 273 proteins were identified. A large fraction of these sites have not been previously reported on standard phosphoproteomic databases. All results are accessible via an online database: http://helixweb.nih.gov/ESBL/Database/iPY/. Analysis of surrounding sequences revealed four overrepresented motifs: [D/E]xxY*, Y*xxP, DY*, and Y*E, where the asterisk symbol indicates the site of phosphorylation. These motifs plus contextual information, integrated using the NetworKIN tool, suggest that the protein tyrosine kinases involved include members of the insulin- and ephrin-receptor kinase families. Analysis of the gene ontology (GO) terms and KEGG pathways whose protein elements are overrepresented in our data set point to structures involved in epithelial cell-cell and cell-matrix interactions ("adherens junction," "tight junction," and "focal adhesion") and to components of the actin cytoskeleton as major sites of tyrosine phosphorylation in these cells. In general, these findings mesh well with evidence that tyrosine phosphorylation plays a key role in epithelial polarity determination.

Recombinant Surface Proteomics as a Tool to Analyze Humoral Immune Responses in Bovines Infected by Mycoplasma mycoides Subsp. mycoides Small Colony Type

A systematic approach to characterize the surface proteome of Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC), the causative agent of contagious bovine pleuropneumonia (CBPP) in cattle, is presented. Humoral immune responses in 242 CBPP-affected cattle and controls were monitored against one-third of the surface proteins of M. mycoides SC in a high throughput magnetic bead-based assay. Initially, 64 surface proteins were selected from the genome sequence of M. mycoides SC and expressed as recombinant proteins in Escherichia coli. Binding of antibodies to each individual protein could then be analyzed simultaneously in minute sample volumes with the Luminex suspension array technology. The assay was optimized on Namibian CBPP-positive sera and Swedish negative controls to allow detection and 20-fold mean signal separation between CBPP-positive and -negative sera. Signals were proven to be protein-specific by inhibition experiments, and results agreed with Western blot experiments. The potential of the assay to monitor IgG, IgM, and IgA responses over time was shown in a proof-of-concept study with 116 sera from eight animals in a CBPP vaccine study. In conclusion, a toolbox with recombinant proteins and a flexible suspension array assay that allows multiplex analysis of humoral immune responses to M. mycoides SC has been created.

Production of recombinant proteins in Escherichia coli using an N-terminal tag derived from sortase

The use of Escherichia coli protein expression systems has many benefits, including the ease of propagation, amounts of protein that can be generated and cost. However, this host has some drawbacks due to difficulties in the production of soluble foreign proteins with their alternate codon usage bias, reductive cytoplasmic environment and lack of complex post-translational modifications. We have designed a novel fusion protein tag derived from the sequence of sortase (SrtA) which we have named Solubility 'eNhancing'Ubiquitous Tag (SNUT). Here we demonstrate its application and effectiveness as an N-terminal fusion tag for the expression and purification of proteins that could not be effectively produced with other tags. We show this tag can be utilized for the purification of proteins through both native and refolding immobilized metal ion chromatography and in combination with an anti-SNUT monoclonal antibody, can also be used as a detection tag. This tag may prove useful in circumventing expression and purification issues with the production of proteins in bacterial expression hosts.

Surface energetics to assess biomass attachment onto immobilized metal-ion chromatography adsorbents in expanded beds

Cell-to-support interaction and cell-to-cell agglomeration phenomena have been studied in a model system composed of intact yeast cells and Chelating-Streamline™ adsorbents. Biomass components and beaded adsorbents were mainly characterized by contact angle determinations with three diagnostic liquids. Complementarily, zeta potential measurements were performed. These experimental values were employed to calculate free energy of interaction versus distance profiles in aqueous media. The effect of immobilized metal-ion type and buffer pH on the interaction energy was evaluated. Calculations indicated that moderate interaction between cell particles and adsorbent beads can develop due to the presence of Cu2+ ions onto the solid phase. The strength of interaction increased with buffer pH, within the range 6.0 to 8.3 e.g. secondary energy pockets increased from |15| to |60| kT. Cell-to-cell secondary energy minimum was ≥ |14| kT showing low-to-moderate tendencies to aggregate, particularly at pH ≥ 8. Extended DLVO predictions were generally confirmed by biomass deposition experiments. However, an exception was found when working with immobilized Cu2+ at pH 8 since yeast cells were able to sequestrate such immobilized ions. Therefore, lower-than-expected values for the depositions coefficient (α) were observed. Understanding biomass attachment onto Chelating supports can help in better design and operate expanded bed adsorption of bioproducts.

Thiol protection in membrane protein purifications: A study with phage holins

The lambda holin, or S105, is a small cytoplasmic membrane protein that controls the timing of host lysis. Using thiol-specific reagents, we determined that the single cysteine residue within S105 was heterogeneously modified during membrane extraction and subsequent immobilized metal ion chromatography. Here we describe the use of a specific and reversible thiol reagent, 2,2′-dithiodipyridine, to generate purified protein with its cysteine residues in the native thiol state. The 2,2′-dithiodipyridine protection protocol was also successfully used for another unrelated holin, S 2168, and should be generally useful for the purification of membrane proteins.

Analytical and Separation Chemistry by Taking Advantage of Organic Photochromism Combined with Macrocyclic Chemistry

Photochromic crown compounds, such as monomeric and polymeric crown compounds incorporating spirobenzopyran, spironaphthooxazine, spirobenzothiapyran, Malachite Green, and Crystal Violet moieties, were synthesized for applications in the fields of analytical and separation chemistry. The photochromic crown compounds, especially crowned spirobenzopyran derivatives, were applied to metal ion extraction, extraction spectrophotometry, membrane transport, and ion chromatography, the sensitivity and selectivity of which were controlled and enhanced photochemically. Also, micelles and vesicles containing lipophilic crowned spirobenzopyrans and Malachite Greens were tested for their usefulness in spectrophotometric and fluorometric metal-ion determination and photocontrol of organic substance in aqueous systems. The polymeric crowned spirobenzopyrans were tried to use as a photochemical valve based on photochemical rheology changes. Moreover, attempts were made to directly observe photoinduced aggregation of the spirobenzopyran moiety and photoresponsive rheology changes of the polymeric crowned spirobenzopyran, by using chemical force microscopy.