Sandwich Antibody Arrays Using Recombinant Antibody-Binding Protein L

Antibody arrays are a useful for detecting antigens and other antibodies. This technique typically requires a uniform and well-defined orientation of antibodies attached to a surface for optimal performance. A uniform orientation can be achieved by modification of antibodies to include a single site for attachment. Thus, uniformly oriented antibody arrays require a bioengineered modification for the antibodies directly immobilization on the solid surface. In this study, we describe a “sandwich-type” antibody array where unmodified antibodies are oriented through binding with regioselectively immobilized recombinant antibody-binding protein L. Recombinant proL-CVIA bearing C-terminal CVIA motif is post-translationally modified with an alkyne group by protein farnesyltransferase (PFTase) at the cysteine residue in the CVIA sequence to give proL-CVIApf, which is covalently attached to an azido-modified glass slide by a Huisgen [3 + 2] cycloaddition reaction. Slides bearing antibodies bound to slides coated with regioselectively immobilized proL-CVIApf gave stronger fluorescence outputs and those where the antibody-binding protein was immobilized in random orientations on an epoxy-modified slide. Properly selected capture and detection antibodies did not cross-react with immobilized proL-CVIApf in sandwich arrays, and the proL-CVIApf slides can be used for multiple cycles of detected over a period of several months

Kinetic and Binding Studies of <i>Streptococcus pneumoniae</i> Type 2 Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase

Type 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) converts isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP), the two fundamental building blocks of isoprenoid molecules. IDI-2 is found in many species of bacteria and is a potential antibacterial target since this isoform is non-homologous to the type 1 enzyme in <i>Homo sapiens</i>. IDI-2 requires a reduced flavin mononucleotide to form the catalytically active ternary complex, IDI-2·FMNH₂·IPP. For IDI-2 from the pathogenic bacterium <i>Streptococcus pneumoniae</i>, the flavin can be treated kinetically as a dissociable cosubstrate in incubations with IPP and excess NADH. Under these conditions, the enzyme follows a modified sequential ordered mechanism where FMN adds before IPP. Interestingly, the enzyme shows sigmoidal behavior when incubated with IPP and NADH with varied concentrations of FMN in aerobic conditions. In contrast, sigmoidal behavior is not seen in incubations under anaerobic conditions where FMN is reduced to FMNH₂ before the reaction is initiated by addition of IPP. Stopped-flow experiments revealed that FMN, whether bound to IDI-2 or without enzyme in solution, is slowly reduced in a pseudo-first-order reaction upon addition of excess NADH (<i>k</i>_red^FMN = 5.7 × 10^–3 s^–1 and <i>k</i>_red^IDI‑2·FMN = 2.8 × 10^–3 s^–1), while reduction of the flavin is rapid upon addition of NADH to a mixture of IDI-2·FMN, and IPP (<i>k</i>_red^{IDI‑2·FMN·IPP} = 8.9 s^–1). Similar experiments with dithionite as the reductant gave <i>k</i>_red^FMN = 221 s^–1 and <i>k</i>_red^IDI‑2·FMN = 411 s^–1. Dithionite reduction of FMN in the IDI-2·FMN and IPP mixture was biphasic with <i>k</i>_red^{IDI‑2·FMN·IPP (fast)} = 326 s^–1 and <i>k</i>_red^{IDI‑2·FMN·IPP (slow)} = 6.9 s^–1 The pseudo-first-order rate constant for the slow component was similar to those for NADH reduction of the flavin in the IDI-2·FMN and IPP mixture and may reflect a rate-limiting conformational change in the enzyme

Site-Selective Synthesis of ¹⁵N- and ¹³C‑Enriched Flavin Mononucleotide Coenzyme Isotopologues

Flavin mononucleotide (FMN) is a coenzyme for numerous proteins involved in key cellular and physiological processes. Isotopically labeled flavin is a powerful tool for studying the structure and mechanism of flavoenzyme-catalyzed reactions by a variety of techniques, including NMR, IR, Raman, and mass spectrometry. In this report, we describe the preparation of labeled FMN isotopologues enriched with ¹⁵N and ¹³C isotopes at various sites in the pyrazine and pyrimidine rings of the isoalloxazine core of the cofactor from readily available precursors by a five-step chemo-enzymatic synthesis

Tyrosine <i>O</i>‑Prenyltransferase SirD Catalyzes <i>S</i>‑, <i>C</i>‑, and <i>N</i>‑Prenylations on Tyrosine and Tryptophan Derivatives

The tyrosine <i>O</i>-prenyltransferase SirD in <i>Leptosphaeria maculans</i> catalyzes normal prenylation of the hydroxyl group in tyrosine as the first committed step in the biosynthesis of the phytotoxin sirodesmin PL. SirD also catalyzes normal <i>N</i>-prenylation of 4-aminophenylalanine and normal <i>C</i>-prenylation at C7 of tryptophan. In this study, we found that 4-mercaptophenylalanine and several derivatives of tryptophan are also substrates for prenylation by dimethylallyl diphosphate. Incubation of SirD with 4-mercaptophenylalanine gave normal <i>S</i>-prenylated mercaptophenylalanine. We found that incubation of the enzyme with tryptophan gave reverse prenylation at N1 in addition to the previously reported normal prenylation at C7. 4-Methyltryptophan also gave normal prenylation at C7 and reverse prenylation at N1, whereas 4-methoxytryptophan gave normal and reverse prenylation at C7, and 7-methyltryptophan gave normal prenylation at C6 and reverse prenylation at N1. The ability of SirD to prenylate at three different sites on the indole nucleus, with normal and reverse prenylation at one of the sites, is similar to behavior seen for dimethylallyltryptophan synthase. The multiple products produced by SirD suggests it and dimethylallyltryptophan synthase use a dissociative electrophilic mechanism for alkylation of amino acid substrates

Regioselective Covalent Immobilization of Catalytically Active Glutathione S‑Transferase on Glass Slides

The high selectivity of protein farnesyltransferase was used to regioselectively append farnesyl analogues bearing bioorthogonal alkyne and azide functional groups to recombinant Schistosoma japonicum glutathione S-transferase (GSTase) and the active modified protein was covalently attached to glass surfaces. The cysteine residue in a C-terminal CVIA sequence appended to N-terminally His₆-tagged glutathione S-transferase (His₆-GSTase-CVIA) was post-translationally modified by incubation of purified protein or cell-free homogenates from E. coli M15/pQE-His₆-GSTase-CVIA with yeast protein farnesyltransferase (PFTase) and analogues of farnesyl diphosphate (FPP) containing ω-azide and alkyne moieties. The modified proteins were added to wells on silicone-matted glass slides whose surfaces were modified with PEG units containing complementary ω-alkyne and azide moieties and covalently attached to the surface by a Cu­(I)-catalyzed Huisgen [3 + 2] cycloaddition. The wells were washed and assayed for GSTase activity by monitoring the increase in <i>A</i>₃₄₀ upon addition of 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione (GT). GSTase activity was substantially higher in the wells spotted with alkyne (His₆-GSTase-CVIA-PE) or azide (His₆-GSTase-CVIA-AZ) modified glutathione-S-transferase than in control wells spotted with farnesyl-modified enzyme (His₆-GSTase-CVIA-F)

Absolute Configuration of Hydroxysqualene. An Intermediate in Bacterial Hopanoid Biosynthesis

Squalene (SQ) is a key intermediate in hopanoid biosynthesis. Many bacteria synthesize SQ from farnesyl diphosphate (FPP) in three steps: FPP to (1<i>R</i>,2<i>R</i>,3<i>R</i>)-presqualene diphosphate (PSPP), (1<i>R</i>,2<i>R</i>,3<i>R</i>)-PSPP to hydroxysqualene (HSQ), and HSQ to SQ. Chemical, biochemical, and spectroscopic methods were used to establish that HSQ synthase synthesizes (<i>S</i>)-HSQ. In contrast, eukaryotic squalene synthase catalyzes solvolysis of (1<i>R</i>,2<i>R</i>,3<i>R</i>)-PSPP to give (<i>R</i>)-HSQ. The bacterial enzyme that reduces HSQ to SQ does not accept (<i>R</i>)-HSQ as a substrate

Barringtonia racemosa Blume

原著和名: サガリバナ科名: サガリバナ科 = Lecythidaceae採集地: 台湾 恒春熱帯植物園 (台湾省 恒春熱帯植物園)採集日: 1968/8/9採集者: 萩庭丈壽整理番号: JH007629国立科学博物館整理番号: TNS-VS-95762

Regioselective Covalent Immobilization of Recombinant Antibody-Binding Proteins A, G, and L for Construction of Antibody Arrays

Immobilized antibodies are useful for the detection of antigens in highly sensitive microarray diagnostic applications. Arrays with the antibodies attached regioselectively in a uniform orientation are typically more sensitive than those with random orientations. Direct regioselective immobilization of antibodies on a solid support typically requires a modified form of the protein. We now report a general approach for the regioselective attachment of antibodies to a surface using truncated forms of antibody-binding proteins A, G, and L that retain the structural motifs required for antibody binding. The recombinant proteins have a C-terminal CVIX protein farnesyltransferase recognition motif that allows us to append a bioorthogonal azide or alkyne moiety and use the Cu­(I)-catalyzed Huisgen cycloaddition to attach the binding proteins to a suitably modified glass surface. This approach offers several advantages. The recombinant antibody-binding proteins are produced in <i>Escherichia coli</i>, chemoselectively modified posttranslationally in the cell-free homogenate, and directly attached to the glass surface without the need for purification at any stage of the process. Complexes between immobilized recombinant proteins A, G, and L and their respective strongly bound antibodies were stable to repeated washing with PBST buffer at pH 7.2. However, the antibodies could be stripped from the slides by treatment with 0.1 M glycine·HCl buffer, pH 2.6, for 30 min and regenerated by shaking with PBS buffer, pH 7.2, at 4 °C overnight. The recombinant forms of proteins A, G, and L can be used separately or in combination to give glass surfaces capable of binding a wide variety of antibodies

The iGen algorithm.

<p>(a) Schematic overview. Red modules can be run in parallel on multiple computer cores. (b) Reaction types applied to the carbocations, obtained from mechanistic studies of terpenoid synthases.</p

All monoterpene skeletons identified by iGen.

<p>Red skeletons have products associated with EC numbers.</p