Ni NTA Research Articles

Secretory expression of lacticin Q fused with SUMO in Bacillus subtilis

Lacticin Q is a 53-amino acid Class II bacteriocin produced by Lactococcus lactis QU 5. It shows antibacterial activity comparable to that of nisin A in terms of both spectrum and intensity. Moreover, it remains stable at alkaline pH values, while nisin A was inactivated. It may possibly be employed as an alternative to or in combination with nisin A. The objective of this study was to express lacticin Q extracellularly with Small ubiquitin-related modifier (SUMO) fusion technology in Bacillus subtilis. Secretory SUMO-lacticin Q fusion protein was efficiently produced in B. subtilis WB600 transformed with the recombinant expression plasmid and accounted for 19% of the culture supernatant proteins. Fusion SUMO-lacticin Q was purified by nickel nitrilotriacetate (Ni–NTA) affinity chromatography and digested with SUMO protease to release lacticin Q. Lacticin Q was further purified by Ni–NTA chromatography to yield about 2.5mg of lacticin Q with more than 93% purity from 1L of supernatant of fermentation culture. An activity assay indicated that the recombinant bacteriocin exhibited excellent antimicrobial activity against indicator strains. The results obtained suggest that the secretory lacticin Q was efficiently expressed using SUMO fusion technology in B. subtilis. The expression and purification system could promote the application of lacticin Q in food and medicine.

Chitinase A from Stenotrophomonas maltophilia shows transglycosylation and antifungal activities

Stenotrophomonas maltophilia chitinase (StmChiA and StmChiB) genes were cloned and expressed as soluble proteins of 70.5 and 41.6kDa in Escherichia coli. Ni–NTA affinity purified StmChiA and StmChiB were optimally active at pH 5.0 and 7.0, respectively and exhibited broad range pH activity. StmChiA and StmChiB had an optimum temperature of 40°C and are stable up to 50 and 40°C, respectively. Hydrolytic activity on chitooligosaccharides indicated that StmChiA was an endo-acting enzyme releasing chitobiose and StmChiB was both exo/endo-acting enzyme with the release of GlcNAc as the final product. StmChiA showed higher preference to β-chitin and exhibited transglycosylation on even chain length tetra- and hexameric substrates. StmChiA, and not StmChiB, was active on chitinous polymers and showed antifungal activity against Fusarium oxysporum.

Pitfalls in screening streptococci for retrieving superior streptokinase (SK) genes: no activity correlation for streptococcal culture supernatant and recombinant SK

Streptokinase (SK), the heterogeneous protein family secreted by some groups of β-hemolytic streptococci (βHS), is a plasminogen activator and well-known drug for thrombolytic therapy. Differences in plasminogen activation property of streptococcal culture supernatants (SCS) have been traditionally used to identify superior producer strains and SK genes (skc) for recombinant SK (rSK) production. However, the role of SK heterogeneity and whether SK activities in SCS correlate with that of their corresponding rSK is a matter of debate. To address these concerns, SCS of nine group C streptococci (GCS) screened among 252 βHS clinical isolates were compared for plasminogen activation using S-2251 chromogenic assay. The GCS (Streptococcus equisimilis) showing the highest (GCS-S87) and lowest (GCS-S131) activities were selected for PCR-based isolation of skc, cloning and rSK production in Escherichia coli. The 6×His-tagged rSK proteins were purified by NI-NTA chromatography, analyzed by SDS-PAGE and Western blotting and their activities were determined. While SCS of GCS-S87 and GCS-S131 showed different plasminogen activations (95 and 35 %, respectively) compared to that of the reference strain (GCS-9542), but interestingly rSK of all three strains showed close specific activities (1.33, 1.70, and 1.55 × 10(4) IU mg(-1)). Accordingly, SKS87 and SKS131 had more than 90 % sequence identity at the amino acids level compared to SK9542. Therefore, SK heterogeneity by itself may not contribute to the differences in plasminogen activation properties of SCS and evaluation of this activity in SCS might not be a proper assay for screening superior skc.

Distortion of the amide-I and -II bands of an α-helical membrane protein, pharaonis halorhodopsin, depends on thickness of gold films utilized for surface-enhanced infrared absorption spectroscopy

Surface-enhanced infrared absorption with attenuated total reflection (ATR-SEIRA) is a powerful tool for exploring molecular mechanisms of membrane proteins at the monolayer level. However, the band intensity, position, and direction can be largely influenced by the presence of a thin gold film, as observed for small molecules existing in close proximity to the surface. Here we investigated influence on the band shapes of an α-helical membrane protein, pharaonis halorhodopsin (pHR), attached on the gold surface through a complex formation between a six-histidines tag and a Ni–nitrilotriacetic acid (Ni–NTA) linker. Normal, bipolar, and inverted shapes of amide-I and -II bands were observed with an increase in film thickness, although pHR molecules would locate relatively far from the surface. The physical origin of this interesting phenomenon has been identified by changing incident angle, polarization, and film deposition rate. We find that the observed absorption anomalies are due to the influence of perpendicularly polarized light. Furthermore, it is shown that the band shapes are normal below the percolation threshold, and bipolar ones occur when an anomalous absorption by the films is strong, while the inverted ones develop with films in which surface scattering is predominant.

Recombinant VP1 protein expressed in Pichia pastoris induces protective immune responses against EV71 in mice

Human enterovirus 71 (EV71) is one of the major causative agents of hand, foot and mouth disease and is also associated with serious neurological diseases in children. Currently, there are no effective antiviral drugs or vaccines against EV71 infection. VP1, one of the major immunogenic capsid proteins of EV71, is widely considered to be the candidate antigen for an EV71 vaccine. In this study, VP1 of EV71 was expressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast Pichia pastoris, and purified by Ni–NTA affinity chromatography. Immunogenicity and vaccine efficacy of the recombinant VP1 were assessed in mouse models. The results showed that the recombinant VP1 could efficiently induce anti-VP1 antibodies in BALB/c mice, which were able to neutralize EV71 viruses in an in vitro neutralization assay. Passive protection of neonatal mice further confirmed the prophylactic efficacy of the antisera from VP1 vaccinated mice. Furthermore, VP1 vaccination induced strong lymphoproliferative and Th1 cytokine responses. Taken together, our study demonstrated that the yeast-expressed VP1 protein retained good immunogenicity and was a potent EV71 vaccine candidate.

Chemical modification of Nitzschia panduriformis's frustules for protein and viral nanoparticle adsorption

The frustule of diatoms, through appropriate chemical modification, can be developed for a high adsorption level of recombinant proteins and viral nanoparticles. Field emission scanning electron microscopy (FE-SEM) analysis of clean frustules revealed a 3D loculate areolae structure (valvar phase porous pattern of the siliceous cell wall). Isocyanatopropyl triethoxysilane (IPS) and iminodiacetic acid (IDA) were used to immobilize Cu2+ ions (an average Cu2+ adsorption capacity about 190μmol of Cu2+/ml of the Cu2+-coupled biosilica reached). FE-SEM, energy dispersion X-ray spectroscopy (EDS) and Fourier transform infrared (FT-IR) were used to confirm the chemical modification of the Cu2+-coupled biosilica. Protein adsorption was confirmed with the detection of a recombinant (His)6-tagged green fluorescent protein binding using fluorescent microscopy. Infectious bursal disease virus VP2-441 subviral particles (SVPs) were found to bind to the Cu2+-coupled biosilica (approximately 3×10−9mol of VP2-441 SVPs/ml of modified frustules), a level higher than the previously obtained 9×10−10mol/ml for SVP binding using a commercial Ni–NTA resin. These give diatom frustules the potential to be developed into a material useful in viral nanoparticle purification systems or as a biosensor for the detection of viruses.

Efficient expression and purification of tag-free Epstein–Barr virus EBNA1 protein in Escherichia coli by auto-induction

Epstein–Barr nuclear antigen 1 (EBNA1) is the essential Epstein–Barr virus (EBV) protein at the interface between the EBV genome and the host chromatin. It is EBNA1’s task to guarantee replication and segregation of the multicopy closed circular viral genome in infected cells. While EBNA1’s functions are relatively well understood, little is known about the molecular mechanisms of EBNA1 mediating chromatin tethering and DNA replication. To characterize those, purified EBNA1 would be a very useful tool in many different biochemical assays. For long, it was not possible to overexpress sufficient quantities of EBNA1 in Escherichia coli (E. coli) due to its rare codon usage, especially in the N-terminal part of the protein. Recently, some groups succeeded in purifying EBNA1 from bacteria using advanced inducible E. coli cells [1–3]. However, all purification procedures ended in a His-tagged version of EBNA1, which might influence EBNA1’s function in biological assays. Therefore, we inserted a tobacco etch virus (TEV)-cleavage site between the N-terminal His-tag and the following open reading frame of EBNA1. Using sequential Ni–NTA and gel filtration columns and TEV protease-mediated cleavage upon autoinduction, we were able to purify functional EBNA1 protein featuring just a single additional, artificial N-terminal glycine residue. Following our simple and fast purification scheme we were able to synthesize 2mg of highly pure EBNA1 protein per liter culture.

Facilitated Substrate Channeling in a Self‐Assembled Trifunctional Enzyme Complex

Most cascade enzymes in metabolic pathways are spatially held together by noncovalent protein–protein interactions. The formation of a cascade enzyme complex often allows the product of one enzyme to be transferred to an adjacent enzyme where it acts as the substrate, thereby resulting in an enhanced reaction rate, because reaching equilibrium in the cytoplasm is not required; this mechanism is called substrate channeling. In nature, most intracellular enzyme complexes are dynamic so that they may be dissociated or associated, thereby resulting in forestallment of substrate competition among different pathways, regulation of metabolic fluxes, mitigation of metabolite inhibition, and circumvention of unfavorable equilibrium and kinetics. The simplest way to facilitate substrate channeling between cascade enzymes is the construction of fusion proteins, but substrate channeling in fusion proteins might not take place. The assembly of numerous enzymes and/or co-enzymes in vitro is called cascade enzyme biocatalysis and has been proposed for the implementation of complicated bioconversion that microbes and chemical catalysts cannot do, such as hydrogen production from cellulosic materials and water with high yield. Inspired by natural enzyme complexes (e.g., metabolons, which are complexes of sequential enzymes of a metabolic pathway), the construction of static rather than dynamic enzyme complexes could be an important approach to accelerating reaction rates among cascade enzymes and to avoiding the regulation of enzyme–enzyme interactions. For example, Wilner et al. linked glucose oxidase and horseradish peroxidase by DNA scaffolds of different lengths, resulting in reaction rates that were enhanced by 20–30-fold. However, DNA scaffolds may be too costly for scale-up as compared to protein scaffolds. Minteer and co-workers demonstrated that chemical cross-linking of proteins within the mitochondria of Saccharomyces cerevisiae resulted in significant increases of the power output in enzymatic fuel cells. But chemical covalent linking often impairs enzyme activity so that it may not be applied to most intracellular enzymes. Herein we demonstrate a general approach for constructing a static self-assembled enzyme complex by using the highaffinity interaction between cohesin and dockerin modules, which occur in natural extracellular complexed cellulase systems, called cellulosomes. Cohesin domains are part of the natural scaffoldin protein of the cellulosome, which is crucial to the construction of the cellulase complex by binding to enzymes carrying dockerin domains. Bayer et al. proposed to construct designed enzyme complexes by utilizing speciesspecific dockerins and cohesins, which can bind tightly in these complexes at a molar ratio of 1:1. Later, several synthetic mini-cellulosomes containing various extracellular glycoside hydrolases were constructed. However, no one attempted to construct an enzyme complex containing cascade enzymes from a metabolic pathway by using dockerins and cohesins and investigated its potential applications in cascade enzyme biocatalysis. Triosephosphate isomerase (TIM, EC 5.3.1.1), aldolase (ALD, EC 4.1.2.13), and fructose 1,6-bisphosphatase (FBP, EC3.1.3.11) are cascade enzymes in the glycolysis and gluconeogenesis pathways. TIM catalyzes the reversible conversion of glycer-aldehyde-3-phosphate (G3P) to dihydroxy-acetone phosphate (DHAP). ALD catalyzes the reversible aldol condensation of G3P and DHAP to fructose-1,6bisphosphate (F16P). FBP catalyzes the irreversible conversion of F16P to fructose-6-phosphate (F6P; Scheme 1). Previous studies reported that substrate channeling existed in dynamic metabolons of enzymes such as TIM, ALD, or FBP. Three dockerin-free proteins: Thermus thermophilus HB27 TIM (TTC0581) as well as the Thermotoga maritima ALD (TM0273) and FBP (TM1415) were expressed in E. coli and purified to homogeneity by using nickel–nitrilotriacetate (Ni–NTA) resin or a self-cleaving intein. However, a mixture of these three enzymes did not form a putative enzyme complex, as examined by affinity electrophoresis (data not shown). The synthetic static three-enzyme complex was assembled in vitro through a synthetic trifunctional scaffoldin containing a family 3 cellulose-binding module (CBM3) at the N terminus followed by three different types of cohesins from the Clostridium thermocellum ATCC 27405 CipA, Clostridium cellulovorans ATCC 35296 CbpA, and Ruminococcus flavefaciens ScaB (cohesins CTCoh, CCCoh, and RFCoh, [*] Dr. C. You, S. Myung, Y.-H. P. Zhang Biological Systems Engineering Department Virginia Tech, 304 Seitz Hall Blacksburg, VA 24061 (USA) E-mail: ypzhang@vt.edu Homepage: http://www.sugarcar.com

Cloning analysis of ferritin and the cisplatin-subunit for cancer cell apoptosis in Aplysia juliana hepatopancreas

Ferritin, an iron storage protein, plays a key role in iron metabolism in vivo. Here, we have cloned an inducible ferritin cDNA with 519bp within the open reading frame fragment from the hepatopancreas of Aplysia juliana (AJ). The subunit sequence of the ferritin was predicted to be a polypeptide of 172 amino acids with a molecular mass of 19.8291kDa and an isoelectric point of 5.01. The cDNA sequence of hepatopancreas ferritin in AJ was constructed into a pET-32a system for expressing its relative protein efficiently in E. coli strain BL21, under isopropyl-β-d-thiogalactoside induction. The recombinant ferritin, which was further purified on a Ni–NTA resin column and digested with enterokinase, was detected as a single subunit of approximately 20kDa mass using both SDS-PAGE and mass spectrometry. The secondary structure and phosphorylation sites of the deduced amino acids were predicted using both ExPASy proteomic tools and the NetPhos 2.0 server, and the subunit space structure of the recombinant AJ ferritin (rAjFer) was built using a molecular operating environment software system. The result of in-gel digestion and identification using MALDI-TOF MS/MS showed that the recombinant protein was AjFer. ICP-MS results indicated that the rAjFer subunit could directly bind to cisplatin[cis-Diaminedichloroplatinum(CDDP)], giving approximately 17.6 CDDP/ferritin subunits and forming a novel CDDP-subunit. This suggests that a nanometer CDDP core-ferritin was constructed, which could be developed as a new anti-cancer drug. The flow cytometry results indicated that CDDP–rAjFer could induce Hela cell apoptosis. Results of the real-time PCR and Western blotting showed that the expression of AjFer mRNA was up-regulated in AJ under Cd2+ stress. The recombinant AjFer protein should prove to be useful for further study of the structure and function of ferritin in Aplysia.

Metal ion affinity purification of proteins by genetically incorporating metal-chelating amino acids

Affinity tags are efficient tools for protein purification. They allow simple one-step purification of proteins to high purity. However, in some cases the tags cause structural and functional changes in a protein, and need to be removed. Therefore, affinity tags that are readily introduced into proteins with minimal perturbation and have specific affinity for purification are desired. Herein, two metal-chelating amino acids derived from 2,2′-bipyridine and 8-hydroxyquinoline were genetically incorporated into glutathione S-transferase (GST) and the mutant proteins were purified by using the metal ion affinity of the unnatural amino acids. The purification of the GST mutants containing 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA) showed that the proteins could be efficiently enriched in Ni–NTA by the metal ion affinity of the unnatural amino acid and purified to excellent purity. This method should be very useful for general protein affinity purification, especially for proteins whose structure or function is affected by affinity tags fused to N- or C-terminals.

Molecular cloning, expression, and characterization of a Ca2+-dependent nuclease of Arabidopsis thaliana

A gene encoding a Ca2+-dependent nuclease (AtCaN2) consisting of 226 amino acids from Arabidopsis thaliana was isolated. The AtCaN2 gene was expressed in Escherichia coli as a His fusion protein. The His–AtCaN2 fusion protein was purified by affinity chromatography using a Ni–NTA column. Western blot analysis using an anti-His antibody showed that the His–AtCaN2 fusion protein was not degraded. The nuclease activity of the His–AtCaN2 fusion protein was highest at 37°C, required a neutral or weakly-alkaline environment, and was stimulated by Ca2+ and Mg2+, but inhibited by Zn2+ and high concentration of Mn2+.

Eliciting cross-neutralizing antibodies in mice challenged with a dengue virus envelope domain III expressed inEscherichia coli

Dengue viruses (DENVs) are mosquito-borne infectious pathogens that pose a serious global public health threat, and at present, no therapy or effective vaccines are available. Choosing suitable units as candidates is fundamental for the development of a dengue subunit vaccine. Domain III of the DENV-2 E protein (EDIII) was chosen in the present study and expressed in Escherichia coli by N-terminal fusion to a bacterial leader (pelB), and C-terminal fusion with a 6×His tag based on the functions of DENV structure proteins, especially the neutralizing epitopes on the envelope E protein. After two-step purification using Ni-NTA affinity and cation-exchange chromatography, the His-tagged EDIII was purified up to 98% homogenicity. This recombinant EDIII was able to trigger high levels of neutralizing antibodies in both BALB/c and C57BL/6 mice. Both the recombinant EDIII and its murine antibodies protected Vero cells from DENV-2 infection. Interestingly, the recombinant EDIII provides at least partial cross-protection against DENV-1 infection. In addition, the EDIII antibodies were able to protect suckling mice from virus challenge in vivo. These data suggest that a candidate molecule based on the small EDIII protein, which has neutralizing epitopes conserved among all 4 DENV serotypes, has important implications.

Cloning and constitutive expression of His-tagged xylanase GH 11 from Penicillium occitanis Pol6 in Pichia pastoris X33: Purification and characterization

High-level constitutive expression of xylanase GH11 from Penicillium occitanis Pol6 termed PoXyn2 was achieved using the methylotrophic yeast Pichia pastoris. The PoXyn2 cDNA encoding for a mature xylanase of 320 amino acids was subcloned into the pGAPZαA vector, to construct recombinant xylanse with six histidine residues at the N-terminal and further integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAP) constitutive promoter. Activity assay and SDS–PAGE demonstrate that the His-tagged xylanase was extracellularly expressed in P. pastoris and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography (Ni–NTA resin). The purified PoXyn2 showed a single band on SDS–PAGE with an apparent molecular weight of 30kDa. The xylanase activity was optimal at pH 3.0 and 50°C. The specific activity measured for Oat Spelt Xylan was 8549.85Umg−1. The apparent The KM and Vmax values were 8.33±0.7mgml−1and 58.82±0.9μmolmin−1ml−1, respectively, as measured on Oat Spelt Xylan. This is the first report demonstrating the possibility of mass production of P. occitanis xylanase using P. pastoris.

Purification, characterization and reconstitution into membranes of the oligomeric c-subunit ring of thermophilic FoF1-ATP synthase expressed in Escherichia coli

FoF1-ATP synthase catalyzes ATP synthesis coupled with proton-translocation across the membrane. The membrane-embedded Fo portion is responsible for the H+ translocation coupled with rotation of the oligomeric c-subunit ring, which induces rotation of the γ subunit of F1. For solid-state NMR measurements, FoF1 of thermophilic Bacillus PS3 (TFoF1) was overexpressed in Escherichia coli and the intact c-subunit ring (TFoc-ring) was isolated by new procedures. One of the key improvement in this purification was the introduction of a His residue to each c-subunit that acts as a virtual His10-tag of the c-ring. After solubilization from membranes by sodium deoxycholate, the c-ring was purified by Ni–NTA affinity chromatography, followed by anion-exchange chromatography. The intactness of the isolated c-ring was confirmed by high-resolution clear native PAGE, sedimentation analysis, and H+-translocation activity. The isotope-labeled intact TFoc-ring was successfully purified in such an amount as enough for solid-state NMR measurements. The isolated TFoc-rings were reconstituted into lipid membranes. A solid-state NMR spectrum at a high quality was obtained with this membrane sample, revealing that this purification procedure was suitable for the investigation by solid-state NMR. The purification method developed here can also be used for other physicochemical investigations.

Expression and purification of the active soluble form of Bacillus sphaericus binary toxin for structural analysis

The binary toxin produced from Bacillus sphaericus is highly toxic against larvae of Culex and Anopheles mosquitoes. The two major components of the binary toxin are 42-kDa BinA and 51-kDa BinB, which are produced as crystalline inclusions during sporulation. Currently, there is no detailed knowledge of the molecular mechanism of the binary toxin, mainly due to the lack of structural information. Herein, we describe an expression protocol with modified conditions allowing production of soluble, biologically active BinA and BinB for further structural analysis. The binA and binB genes from B. sphaericus 2297 strain were independently cloned and fused with a polyhistidine tag at their N-termini. Both (His)6-tagged BinA and (His)6-tagged BinB were expressed as soluble forms at low temperature. Highly pure proteins were obtained after two-step purification by Ni–NTA affinity and size exclusion chromatography. In vitro activation by trypsin digestion generated a resistant fragment, of 40kDa for BinA, and of 45kDa for BinB, and an oligomeric complex of BinA and BinB in solution was observed after proteolytic activation. Their functional and structural properties were confirmed by a biological assay and far-UV circular dichroism, respectively. The mixture of BinA and BinB, either as a protoxin or as a trypsin-activated form, exhibited high mosquito-larvicidal activity against Culex quinquefasciatus larvae with LC50 of about 10ng/ml, while no toxicity was observed from the single binary toxin component. Results from far-UV circular dichroism of BinA and BinB suggest the presence of mainly β-structure. The expression and purification protocols reported here will be useful for the production of the active and homogeneous binary toxin to allow further detailed structural investigation.

Ubiquitin-intein and SUMO2-intein fusion systems for enhanced protein production and purification

Although most commonly used for protein production, expression of soluble and functional recombinant protein in Escherichia coli is still a major challenge. The development and application of fusion tags that can facilitate protein expression and solubility partly solve this problem, however, under most circumstance, the fusion tags have to be removed by proteases in order to use the proteins. Because the tag removal using proteases increases cost and introduces extra purification steps, it remains a significant problem that must be resolved before being widely used in industry production. Ubiquitin and SUMO have been successfully used to enhance protein expression and solubility. In the last decades, intein has also been widely used in protein production for its self-cleavage property, which could help to remove the fusion tag without any protease. Here, we take the advantages of ubiquitin, SUMO2 and intein in protein expression. We constructed tandem ubiquitin-intein and SUMO2-intein fusion tags, and chose human MMP13 (amino acid 104–274) and eGFP as the passenger proteins that fused to the C-terminus of the tags. These constructs were expressed in E. coli and both MMP13 and eGFP expression and solubility were evaluated. Both tags showed the ability to enhance the solubility of MMP13 and eGFP and improve the expression of eGFP, and the SUMO2-intein having a more significant effect. Both ubiquitin-intein-eGFP and SUMO2-intein-eGFP were purified using Ni–NTA column chromatography and self-cleavaged by changing pH. The recombinant un-tagged eGFP were released and eluted with high homogeneity. In summary, ubiquitin-intein and SUMO2-intein are convenient and useful fusion tags that can enhance the expression, solubility and improve the purification process of the model heterologous protein and these tags may have a good prospect in protein production.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of a major group 7 allergen, Der f 7, from the dust mite Dermatophagoides farinae

Der f 7 is a major group 7 allergen from the dust mite Dermatophagoides farinae that shows 86% sequence identity to the homologous allergen Der p 7 from D. pteronyssinus. Der f 7 was successfully overexpressed in an Escherichia coli expression system and purified to homogeneity using Ni-NTA affinity and size-exclusion column chromatography. SeMet-labelled Der f 7 was crystallized by the hanging-drop vapour-diffusion method using a reservoir solution consisting of 0.1 M bis-tris pH 7.4 and 28% polyethylene glycol monomethyl ether 2000 at 293 K. X-ray diffraction data were collected to 2.24 Å resolution using synchrotron radiation. The crystals belonged to the orthorhombic system, space group P2(1)2(1)2(1), with unit-cell parameters a = 50.19, b = 58.67, c = 123.81 Å. Based on the estimated Matthews coefficient (2.16 Å(3) Da(-1)), two molecules of Der f 7 could be present in the asymmetric unit of the crystal lattice.

Development of a Histidine-Targeted Spectrophotometric Sensor Using Ni(II)NTA-Functionalized Au and Ag Nanoparticles

An antibody-free diagnostic reagent has been developed based on the aggregation-induced colorimetric change of Ni(II)NTA-functionalized colloidal gold and silver nanoparticles. This diagnostic strategy utilizes the high binding affinity of histidine-rich proteins with Ni(II)NTA to capture and cross-link the histidine-rich protein mimics with the silver and gold nanoparticles. In model studies, the aggregation behavior of the Ni(II)NTA nanoparticles was tested against synthetic targets including charged poly(amino acid)s (histidine, lysine, arginine, and aspartic acid) and mimics of Plasmodium falciparum histidine-rich protein 2 (pfHRP-II). Aggregation of the nanoparticle sensor was induced by all of the basic poly(amino acid)s including poly(l-histidine) within the pH range (5.5-9.0) tested, which is likely caused by the coordination between the multivalent polymer target and Ni(II)NTA groups on multiple particles. The peptide mimics induced aggregation of the nanoparticles only near their pK(a)'s with higher limits of detection. In addition, monomeric amino acids do not show any aggregation behavior, suggesting that multiple target binding sites are necessary for aggregation. Long-term stability studies showed that gold but not silver nanoparticles remained stable and exhibited similar aggregation behavior after 1 month of storage at room temperature and 37 °C. These results suggest that Ni(II)NTA gold nanoparticles could be further investigated for use as a sensor to detect histidine-rich proteins in biological samples.

Application of displacement principle for detecting heavy metal ions and EDTA using microcantilevers

A microcantilever sensor based on a reversible displacement reaction was developed. The demonstration was performed with the Ni–NTA–EDTA sample system. Ni ions, a typical heavy metal, were injected and adsorbed onto a NTA-functionalized AFM cantilever in a fluid cell. This adsorption resulted in tensile stress which was detected by monitoring the corresponding deflection of the microcantilever. In the next step, EDTA was injected into the cell to release the adsorbed Ni ions from the surface of the cantilever. Since EDTA molecules have a stronger affinity for Ni ions compared to NTA, they compete with and eventually replace the NTA molecules and take over the binding positions on Ni. The displacement of Ni ions from the cantilever by EDTA, followed by water flow removes the tensile stress and restores the cantilever deflection to its original value. In this way, both heavy metal ions and EDTA can be specifically recognized. From the cantilever deflection, the concentration of Ni ions and EDTA can be quantified. The detection limits are presently at the micromolar level. Another weaker Ni ion binder, imidazole, was unable to dissociate Ni ions from NTA and produce similar effects. The demonstrated principle can be used for microcantilever sensors which are capable of regeneration for multiple uses. The method presented can be generalized to detect other reactants.

Reconstitution and Organization of Photosynthetic Antenna Protein Complex Bearing Functional Hydrophilic Domains

Abstract Bacterial photosynthetic antenna polypeptide (LH1-α) was synthesized as a water-soluble fusion protein with maltose-binding protein (MBP) and a His-tag portion (MBP–rubα-YH) using an E. coli expression system. Reconstitution experiments indicated that LH1-type complexes with pigments were successfully formed, regardless of the presence of a large hydrophilic MBP portion. The reconstituted complex was immobilized onto a Au electrode via a His-tag–Ni–NTA interaction. When the complex was incorporated into a planar lipid bilayer, protruding MBP portions were clearly observed by AFM.