Development of a scintillation proximity assay for beta-ketoacyl-acyl carrier protein synthase III

Assays of beta-ketoacyl-acyl carrier protein synthases III (KASIII; FabH), a key enzyme initiating bacterial type II fatty acid biosynthesis, usually involve incubation of radiolabeled acetyl-coenzyme A and malonyl-acyl carrier protein (MACP). The radiolabeled acetoacetyl-ACP product is precipitated and separated from the substrate before quantitation. We have developed a scintillation proximity assay (SPA) where use of biotinylated MACP (BMACP) allows the generation of a biotinylated acetoacetyl-ACP. This product, when captured by the streptavidin-coated scintillant-impregnated microspheres, generates an SPA signal. A BMACP K(m) of 7.1 microM was determined using this SPA with the Streptomyces glaucescens FabH. A similar MACP K(m) (6 microM) was determined in a precipitation assay, demonstrating that BMACP is an effective substrate for FabH. IC(50) values of 15.2 microM (SPA) and 24.8 microM were obtained with iodoacetamide and the S. glaucescens FabH. Comparable IC(50) values of 160 microM (SPA) and 125 microM were also obtained with the antibiotic thiolactomycin and the Escherichia coli FabH. These observations demonstrate that FabH inhibitors can be readily detected using a SPA with BMACP and that the effectiveness of inhibitors in the SPA is comparable to that obtained using MACP and a standard TCA precipitation assay. A FabH SPA adaptable to high-throughput screening should facilitate the discovery of potential novel antibiotics.     

Secretion of recombinant proteins into the culture medium by Escherichia coli and Staphylococcus aureus

The ability of staphylococcal protein A (SPA) to bind to the Fc part of IgG has been used for the purification of a number of heterologous gene products as fusion proteins. Both the SPA promoter and signal sequence function in Escherichia coli, as well as in a number of Gram-positive bacteria, which facilitates comparisons of the expressed specific products in different hosts. The expression system developed for E. coli yields excretion of the fusion protein to the growth medium, which makes E. coli a competitive alternative to Gram-positive bacteria for the expression of secreted products. The human peptide hormones insulin-like growth factors (IGF) I and II were expressed using the protein A system in E. coli and Staphylococcus aureus. Despite a high degree of structural homology, large differences in the yields were observed in the two hosts. This underlines the importance of investigating different bacterial hosts for a particular protein product.     

Sequential Peptide Affinity (SPA) system for the identification of mammalian and bacterial protein complexes

A vector system is described that combines reliable, very low level, regulated protein expression in human cells with two affinity purification tags (Sequential Peptide Affinity, or SPA, system). By avoiding overproduction of the target protein, this system allows for the efficient purification of natural protein complexes and their identification by mass spectrometry. We also present an adaptation of the SPA system for the efficient purification and identification of protein complexes in E. coli and, potentially, other bacteria.     

基于免疫纳米颗粒强化的压电免疫传感器检测大肠杆菌O157︰H7

摘要：【目的】结合纳米技术建立检测大肠杆菌（Escherichia coli）O157︰H7高灵敏检测技术。【方法】采用化学共沉淀法制备出核心粒径约为10 nm的免疫纳米磁颗粒,柠檬酸钠还原法制备粒径约为20 nm的免疫胶体金。压电免疫传感器通过金黄色葡萄球菌蛋白A（Protein A from Staphylococcus aureus SPA）法将抗体固定于石英晶振上,两种免疫纳米颗粒借助不同的抗体连接于传感器上对检测频率信号进行放大。【结果】SPA在石英晶振上的最佳固定浓度和时间为1.2 mg/mL和40 min,抗体的最佳固定浓度和时间为1.0 mg/mL和60 min。压电免疫传感器通过两种免疫纳米颗粒的放大作用,使其对大肠杆菌O157︰H7的检测限从104 cfu/mL提高到101 cfu/mL。【结论】免疫纳米颗粒强化对压电免疫传感器的检测频率信号具有很好的放大效应,可以明显提高其检测灵敏度。     

Renewable reagentless hydrogen peroxide sensor based on direct electron transfer of horseradish peroxidase immobilized on colloidal gold-modified electrode

The polC gene from Streptococcus pyogenes (S. pyogenes, strain SF370) has been cloned and expressed in Escherichia coli (E. coli) as a fusion protein containing an N-terminal histidine tag. The purified recombinant enzyme showed an apparent molecular mass of 160 kDa on SDS-PAGE and a specific activity of 3.5 nmol/min/mg when assayed in the presence of calf thymus DNA and the four deoxyribonucleoside triphosphates. This activity was inhibited by TMAU, a specific inhibitor of PolC. To facilitate kinetic studies, and high-throughput assays, a double-stranded oligo DNA primer/template was used as a substrate. The minimum requirement for the length of the substrate was a 20-base oligo primer annealed to a 35-base template. PolC activity was detected either by a filter-binding format or by a novel homogeneous scintillation proximity assay (SPA). Sensitivity to inhibition by anilinouracil analogs was improved by incorporating three deoxycytidines in the template strand as the first 3 bases to be copied by the polymerase. Inhibition of PolC activity by trimethyleneanilinouracil by the filtration and SPA methods gave comparable results, but the SPA assay uses less radioactive label, is less time-consuming, and is amenable to high-throughput formatting.     

Homogenous assays for Escherichia coli DnaB-stimulated DnaG primase and DnaB helicase and their use in screening for chemical inhibitors

Escherichia coli DnaG primase is a single-stranded DNA-dependent RNA polymerase. Primase catalyzes the synthesis of a short RNA primer to initiate DNA replication at the origin and to initiate Okazaki fragment synthesis for synthesis of the lagging strand. Primase activity is greatly stimulated through its interaction with DnaB helicase. Here we report a 96-well homogeneous scintillation proximity assay (SPA) for the study of DnaB-stimulated E. coli primase activity and the identification of E. coli primase inhibitors. The assay uses an adaptation of the general priming reaction by employing DnaG primase, DnaB helicase, and ribonucleotidetriphosphates (incorporation of [(3)H]CTP) for in vitro primer synthesis on single-stranded oligonucleotide and M13mp18 DNA templates. The primase product is captured by polyvinyl toluene-polyethyleneimine-coated SPA beads and quantified by counting by beta-scintography. In the absence of helicase as a cofactor, primer synthesis is reduced by 85%. The primase assay was used for screening libraries of compounds previously identified as possessing antimicrobial activities. Primase inhibitory compounds were then classified as direct primase inhibitors or mixed primase/helicase inhibitors by further evaluation in a specific assay for DnaB helicase activity. By this approach, specific primase inhibitors could be identified.     

Nucleotide sequence analysis of the gene for protein A from Staphylococcus aureus Cowan 1 (NCTC8530) and its enhanced expression in Escherichia coli

Nucleotide sequence analysis of the gene (spa) for staphylococcal protein A (SPA) from Staphylococcus aureus strain Cowan 1 (NCTC8530) shows that the sequence differs from previously reported SPA nucleotide sequences, especially in the number of repeat units in the cell-wall-binding region of the gene. Dot matrix comparison with streptococcal protein G and the macrophage receptor for the constant fragment (Fc) of immunoglobulins shows a limited but significant homology. The homology to the latter probably identifies the Fc-binding region in the immunoglobulin-binding domains of SPA. Enhanced production of SPA in Escherichia coli was achieved using the lac promoter immediately upstream from the spa gene.     

Voltage-gated K+ channels at an early stage of chronic hypoxia-induced pulmonary hypertension in newborn piglets

Our purpose was to determine whether smooth muscle cell membrane properties are altered in small pulmonary arteries (SPA) of piglets at an early stage of pulmonary hypertension. Piglets were raised in either room air (control) or hypoxia for 3 days. A microelectrode technique was used to measure smooth muscle cell membrane potential (E(m)) in cannulated, pressurized SPA (100- to 300-microm diameter). SPA responses to the voltage-gated K(+) (K(V)) channel antagonist 4-aminopyridine (4-AP) and the K(V)1 family channel antagonist correolide were measured. Other SPA were used to assess amounts of K(V)1.2, K(V)1.5, and K(V)2.1 (immunoblot technique). E(m) was more positive in SPA of chronically hypoxic piglets than in SPA of comparable-age control piglets. The magnitude of constriction elicited by either 4-AP or correolide was diminished in SPA from hypoxic piglets. Abundances of K(V)1.2 were reduced, whereas abundances of both K(V)1.5 and K(V)2.1 were unaltered, in SPA from hypoxic piglets. At least partly because of reduced amounts of K(V)1.2, smooth muscle cell membrane properties are altered such that E(m) is depolarized and K(V) channel family function is impaired in SPA of piglets at an early stage of chronic hypoxia-induced pulmonary hypertension.     

Anti-idiotypic protein domains selected from protein A-based affibody libraries

Three pairs of small protein domains showing binding behavior in analogy with anti-idiotypic antibodies have been selected using phage display technology. From an affibody protein library constructed by combinatorial variegation of the Fc binding surface of the 58 residue staphylococcal protein A (SPA)-derived domain Z, affibody variants have been selected to the parental SPA scaffold and to two earlier identified SPA-derived affibodies. One selected affibody (Z(SPA-1)) was shown to recognize each of the five domains of wild-type SPA with dissociation constants (K(D)) in the micromolar range. The binding of the Z(SPA-1) affibody to its parental structure was shown to involve the Fc binding site of SPA, while the Fab-binding site was not involved. Similarly, affibodies showing anti-idiotypic binding characteristics were also obtained when affibodies previously selected for binding to Taq DNA polymerase and human IgA, respectively, were used as targets for selections. The potential applications for these types of affinity pairs were exemplified by one-step protein recovery using affinity chromatography employing the specific interactions between the respective protein pair members. These experiments included the purification of the Z(SPA-1) affibody from a total Escherichia coli cell lysate using protein A-Sepharose, suggesting that this protein A/antiprotein A affinity pair could provide a basis for novel affinity gene fusion systems. The use of this type of small, robust, and easily expressed anti-idiotypic affibody pair for affinity technology applications, including self-assembled protein networks, is discussed.     

Light-induced degradation of SPA2 via its N-terminal kinase domain is required for photomorphogenesis

Arabidopsis (Arabidopsis thaliana) CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) and members of the SUPPRESSOR OF PHYTOCHROMEA-105 (SPA) protein family form an E3 ubiquitin ligase that suppresses light signaling in darkness by polyubiquitinating positive regulators of the light response. COP1/SPA is inactivated by light to allow photomorphogenesis to proceed. Mechanisms of inactivation include light-induced degradation of SPA1 and, in particular, SPA2, corresponding to a particularly efficient inactivation of COP1/SPA2 by light. Here, we show that SPA3 and SPA4 proteins are stable in the light, indicating that light-induced destabilization is specific to SPA1 and SPA2, possibly related to the predominant function of SPA1 and SPA2 in dark-grown etiolating seedlings. SPA2 degradation involves cullin and the COP10-DEETIOLATED-DAMAGED-DNA BINDING PROTEIN (DDB1) CDD complex, besides COP1. Consistent with this finding, light-induced SPA2 degradation required the DDB1-interacting Trp-Asp (WD)-repeat domain of SPA2. Deletion of the N-terminus of SPA2 containing the kinase domain led to strong stabilization of SPA2 in darkness and fully abolished light-induced degradation of SPA2. This prevented seedling de-etiolation even in very strong far-red and blue light and reduced de-etiolation in red light, indicating destabilization of SPA2 through its N-terminal domain is essential for light response. SPA2 is exclusively destabilized by phytochrome A in far-red and blue light. However, deletion of the N-terminal domain of SPA2 did not abolish SPA2-phytochrome A interaction in yeast nor in vivo. Our domain mapping suggests there are two SPA2-phytochrome A interacting domains, the N-terminal domain and the WD-repeat domain. Conferring a light-induced SPA2-phyA interaction only via the WD-repeat domain may thus not lead to COP1/SPA2 inactivation.

Light inactivates the COP1/SPA2 repressor of photomorphogenesis through cullin- and CDD-mediated degradation of SPA2, whereas the family members SPA3 and SPA4 are stable in the light.     

Light exposure of Arabidopsis seedlings causes rapid de-stabilization as well as selective post-translational inactivation of the repressor of photomorphogenesis SPA2

The COP1/SPA complex acts as an E3 ubiquitin ligase to repress photomorphogenesis by targeting activators of the light response for degradation. Genetic analysis has shown that the four members of the SPA gene family (SPA1-SPA4) have overlapping but distinct functions. In particular, SPA1 and SPA2 differ in that SPA1 encodes a potent repressor in light- and dark-grown seedlings, but SPA2 fully loses its function when seedlings are exposed to light, indicating that SPA2 function is hyper-inactivated by light. Here, we have used chimeric SPA1/SPA2 constructs to show that the distinct functions of SPA1 and SPA2 genes in light-grown seedlings are due to the SPA protein sequences and independent of the SPA promoter sequences. Biochemical analysis of SPA1 and SPA2 protein levels shows that light exposure leads to rapid proteasomal degradation of SPA2, and, more weakly, of SPA1, but not of COP1. This suggests that light inactivates the COP1/SPA complex partly by reducing SPA protein levels. Although SPA2 was more strongly degraded than SPA1, this was not the sole reason for the lack of SPA2 function in the light. We found that the SPA2 protein is inherently incapable of repressing photomorphogenesis in light-grown seedlings. The data therefore indicate that light inactivates the function of SPA2 through a post-translational mechanism that eliminates the activity of the remaining SPA2 protein in the cell.     

QCM immunosensor detection of Escherichia coli O157:H7 based on beacon immunomagnetic nanoparticles and catalytic growth of colloidal gold

In this paper, we describe a novel method for detecting Escherichia coli (E. coli) O157:H7 by using a quartz crystal microbalance (QCM) immunosensor based on beacon immunomagnetic nanoparticles (BIMPs), streptavidin-gold, and growth solution. E. coli O157-BIMPs were magnetic nanoparticles loaded with polyclonal anti-E. coli O157:H7 antibody (target antibody, T-Ab) and biotin-IgG (beacon antibody, B-Ab) at an optimized ratio of 1:60 (T-Ab:B-Ab). E. coli O157:H7 was captured and separated by E. coli O157-BIMPs in a sample, and the streptavidin-gold was subsequently conjugated to E. coli O157-BIMPs by using a biotin-avidin system. Finally, the gold particles on E. coli O157-BIMPs were enlarged in growth solution, and the compounds containing E. coli O157:H7, E. coli O157-BIMPs, and enlarged gold particles were collected using a magnetic plate. The QCM immunosensor was fabricated with protein A from Staphylococcus aureus and monoclonal anti-E. coli O157:H7 antibody. The compounds decreased the immunosensor's resonant frequency. E. coli O157-BIMPs and enlarged gold particles were used as "mass enhancers" to amplify the frequency change. The frequency shift was correlated to the bacterial concentration. The detection limit was 23 CFU/ml in phosphate-buffered saline and 53 CFU/ml in milk. This method could successfully detect E. coli O157:H7 with high specificity and stability. The entire procedure for the detection of E. coli O157:H7 took only 4 h.     

Expression of Pseudomonas fluorescens D-galactose dehydrogenase in E. coli

To investigate the heterologous expression of Pseudomonas genes in Escherichia coli we have cloned P. fluorescens DNA in an E. coli [cosmid] system. A colony bank representing the whole P. fluorescens chromosome was screened immunologically using a modification of the method described by Broome and Gilbert (1978). Radioactive labelling of the antibodies was replaced by conjugation with horseradish peroxidase. Among 523 E. coli colonies one was D-galactose dehydrogenase-positive. The expression of this enzyme in primary clones was lower than in the uninduced Pseudomonas. Subcloning of the D-galactose dehydrogenase gene, in vitro mutagenesis of the DNA, and coupling to a strong E. coli promoter yielded an E. coli strain that produces 90 times more of the enzyme than the induced P. fluorescens.     

Molecules incorporating a benzothiazole core scaffold inhibit the N-myristoyltransferase of Plasmodium falciparum

Recombinant N-myristoyltransferase of Plasmodium falciparum (termed PfNMT) has been used in the development of a SPA (scintillation proximity assay) suitable for automation and high-throughput screening of inhibitors against this enzyme. The ability to use the SPA has been facilitated by development of an expression and purification system which yields considerably improved quantities of soluble active recombinant PfNMT compared with previous studies. Specifically, yields of pure protein have been increased from 12 microg x l(-1) to >400 microg x l(-1) by use of a synthetic gene with codon usage optimized for expression in an Escherichia coli host. Preliminary small-scale 'piggyback' inhibitor studies using the SPA have identified a family of related molecules containing a core benzothiazole scaffold with IC50 values <50 microM, which demonstrate selectivity over human NMT1. Two of these compounds, when tested against cultured parasites in vitro, reduced parasitaemia by >80% at a concentration of 10 microM.     

The expression of biologically active cholera toxin in Escherichia coli.

Chromosomal DNA from Vibrio cholerae El Tor strain 1621 was digested with Hind III and the products fractionated by centrifugation through a sucrose gradient. A 15kb fragment containing the toxin gene of V. cholerae was identified by its homology with the heat labile toxin (LT) gene of toxigenic E. coli. This fragment was cloned in E. coli using pAT153 and subsequently characterised by digestion with different restriction endonucleases. Sequences homologous to the LT gene were identified by hybridisation and then sub-cloned using either pAT153 or pACYC184. Expression of the cloned CT gene in E. coli was detected using both cell culture and ELISA assays. One recombinant plasmid coded for the synthesis of an immunologically active but biologically inactive derivative of CT.     

Staphylococcal protein A as a fusion partner directs secretion of the e1alpha and e1beta subunits of pea mitochondrial pyruvate dehydrogenase by Bacillus subtilis

Staphylococcal protein A (SPA)-based vectors were constructed to direct secretion of the E1alpha and E1beta subunits of Pisum sativum mitochondrial pyruvate dehydrogenase from Bacillus subtilis. These proteins were not exported when the signal peptide from levansucrase (SacBSP) was fused to their N-termini. Both SacBSP-E1alpha and SacBSP-E1beta fusion proteins were insoluble in the cytoplasm. However, when the SPA open-reading frame was inserted between SacBSP and E1alpha or E1beta, corresponding fusion proteins were secreted from the cells. The first (E) IgG-binding domain of SPA was sufficient to direct low level secretion of both fusion proteins (SacBSP-E-E1alpha and SacBSP-E-E1beta). Adding the second (D) IgG-binding domain improved extracellular protein yields 3- to 4-fold over E alone, but was not as efficient as secretion of the full-length (EDABC) SPA-fusion proteins. All constructs were based on the pUB110-derived multicopy plasmid pWB705. Separate B. subtilis strains transformed with SacBSP-E-E1alpha-His(6) or SacBSP-E1beta were cocultivated in the presence of Ni-NTA agarose. The native pyruvate dehydrogenase alpha2beta2 structure was bound to the affinity matrix, demonstrating assembly after secretion. The use of SPA as a fusion partner during expression of heterologous proteins by B. subtilis provides the basis of a versatile system that can be used to study both secretion and protein:protein interactions.     

人早胚发育相关蛋白ZNF268的抗体制备

目的:获得纯化抗原用于制备ZNF268的多克隆抗体。方法:PCR扩增目的基因片Spacer区序列,亚克隆入融合蛋白表达载体pET28a+,构建了重组质粒pET28a+/SPA。然后将该重组质粒转化E.coliDE3(Rosseta),IPTG诱导表达,SDS-PAGE分离,获纯化融合蛋白6His-SPA。用6His-SPA免疫家兔,颈动脉取血,分离血清,从血清中获得ZNF268特异的多克隆抗体。结论:通过构建融合蛋白重组表达质粒pET28a+/SPA,用获得的初步纯化融合蛋白6His-SPA,制备了特异的ZNF268的多克隆抗体6His-SPA Ab。     

Feasibility of using GFP-expressing Escherichia coli, coupled with fluorimetry, to determine protozoan ingestion rates

The feasibility of using a live Escherichia coli population, which had been engineered to express the green fluorescent protein (GFP), coupled with fluorimetry, was tested as a means for determining protozoan ingestion rates. Its potential use was based on evidence that once cells are acidified, e.g. in a food vacuole, the fluorescence is lost. Of the 29 protozoa tested, over 85% ingested the GFP-expressing E. coli and a detailed experiment with the ciliate Tetrahymena pyriformis was carried out, principally to assess the performance of the live bacterium against two commonly used surrogate prey, i.e. fluorescently labelled bacteria (FLB) and fluorescently labelled microspheres (FLMs). A decrease in GFP-expressing E. coli fluorescence and, hence, concentration, was recorded by fluorimetry and epifluorescence microscopy, with calculated ingestion rates being equivalent. A higher ingestion rate was determined by counting the number of fluorescent E. coli within the ciliate over 120 s, but this was equivalent to that obtained for the stained E. coli using the same direct method of analysis. However, the ciliate was shown to process the stained and unstained E. coli cells differently, with only the latter resulting in an increase in ciliate abundance.