Characterization of a monomeric heat-labile classical alkaline phosphatase from Anabaena sp. PCC7120.

Alkaline phosphatases (APs), known inducible enzymes of the Pho regulon and poorly characterized in cyanobacteria, hydrolyze phosphomonoesters to produce inorganic phosphate (P(i)) during P(i) starvation. In this study, two predicted alkaline phosphatase genes in the genome of Anabaena sp. PCC 7120, all2843 and alr5291, were apparently induced during P(i) starvation. Sequence analysis showed that alr5291 encodes a protein that is an atypical alkaline phosphatase like other cyanobacteria PhoAs, but the protein encoded by all2843 is very similar to the classical PhoAs, such as Escherichia coli alkaline phosphatase (EAP). To date, there have been no reports about classical phoA in cyanobacterial genomes. The alkaline phosphatase AP(A), coded by all2843, is characterized as a metalloenzyme containing Mg2+ and Zn2+ with molar ratio of 1 : 2. Site-directed mutagenesis analysis indicated that, though the active center of AP(A) is highly conserved in comparison with EAP, differences do exist between AP(A) and EAP in metal ion coordination. Besides, biochemical analysis revealed that AP(A) is a monomeric protein and inactivated rapidly at 50 degrees C. These results suggest that AP(A) is the first monomeric heat-labile classical PhoA found in cyanobacteria.

Biochemical characterization and mutational improvement of a thermophilic esterase from Sulfolobus solfataricus P2.

A thermophilic esterase, SsoPEst, from Sulfolobus solfataricus P2 was cloned and expressed in E. coli AD494 (DE3). Gene sequencing indicated the encoded 353 amino acids had less than 32% identity with reported esterases. The recombinant enzyme hydrolyzed p-nitrophenyl esters but not tributyrin or tricaprylin, exhibiting the highest specific activity (1.1 U/mg) with p-nitrophenyl caprylate. The enzyme was optimally active at pH 5.5 and 80 degrees C. It retained 50% activity after 1 h incubation at 80 degrees C. Activity was significantly inhibited by PMSF. Five SsoPEst mutants were generated by site-directed mutagenesis. One mutant had a higher specific activity of 2.8 U/mg at 37 degrees C and 14 U/mg at 80 degrees C than the wild-type enzyme which exhibited 0.7 U/mg at 37 degrees C and 3.8 U/mg at 80 degrees C against p-nitrophenyl butyrate.

Seeding-induced self-assembling protein nanowires dramatically increase the sensitivity of immunoassays.

Aiming to build a supersensitive and easily operable immunoassay, bifunctional protein nanowires were generated by seeding-induced self-assembling of the yeast amyloid protein Sup35p that genetically fused with protein G and an enzyme (methyl-parathion hydrolase, MPH), respectively. The protein nanowires possessed a high ratio of enzyme molecules to protein G, allowing a dramatic increase of the enzymatic signal when protein G was bound to an antibody target. As a result, a 100-fold enhancement of the sensitivity was obtained when applied in the detection of the Yersinia pestis F1 antigen.

Characterization of glycerol dehydratase expressed by fusing its alpha- and beta-subunits.

The gdh and gdhr genes, encoding B(12)-dependent glycerol dehydratase (GDH) and glycerol dehydratase reactivase (GDHR), respectively, in Klebsiella pneumoniae, were cloned and expressed in E. coli. Part of the beta-subunit was lost during GDH purification when co-expressing alpha, beta and gamma subunit. This was overcome by fusing the beta-subunit to alpha- or gamma-subunit with/without the insertion of a linker peptide between the fusion moieties. The kinetic properties of the fusion enzymes were characterized and compared with wild type enzyme. The results demonstrated that the fusion protein GDHALB/C, constructed by linking the N-terminal of beta-subunit to the C-terminal of alpha subunit through a (Gly(4)Ser)(4) linker peptide, had the greatest catalytic activity. Similar to the wild-type enzyme, GDHALB/C underwent mechanism-based inactivation by glycerol during catalysis and could be reactivated by GDHR.

Anti-allergic effects of ethanol extracts from brown seaweeds.

Ethanol extracts of brown seaweeds from Pakistan and China were isolated and compared for their antiallergenic activities. They included Sargassum tennerimum (ST) and Sargassum cervicorne (SC) from Pakistan, and Sargassum graminifolium turn (SG), Sargassum thunbergii (STH), and Laminaria japonica (LJ) from China. The ethanol extracts of these brown seaweeds were optimized at 85% (v/v) ethanol for the maximum yield of phlorotannin, an inhibitor against hyaluronidase. Total phlorotannins contained in the crude extracts were measured as 1.71% (SG), 0.74% (STH), 0.97% (LJ), 3.30% (SC), and 5.06% (ST). The 50% inhibitory concentrations (IC(50)) of Pakistani SC and ST were 109.5 and 21 microg/ml, respectively, lower than those of Chinese SG, STH, and LJ (134, 269, and 148 microg/ml, respectively). An antiallergic drug, disodium cromoglycate (DSCG), had an IC(50)=39 microg/ml, and a natural inhibitor of hyaluronidase, catechin, had an IC(50)=20 microg/ml. The IC(50) of ST extract was found similar to that of catechin (21 vs 20 microg/ml) and lower than that of DSCG (21 vs 39 microg/ml). This suggests that ST is a potent inhibitor of hyaluronidase, indicating a promising future development of natural antiallergic medicines or functional foods.

Phage display mediated immuno-PCR.

Immuno-PCR (IPCR) is a powerful detection technology in immunological study and clinical diagnosis due to its ultrasensitivity. Here we introduce a new strategy termed phage display mediated immuno-PCR (PD-IPCR). Instead of utilization of monoclonal antibody (mAb) and chemically bond DNA that required in the conventional IPCR, a recombinant phage particle is applied as a ready reagent for IPCR experiment. The surface displayed single chain variable fragment (scFv) and phage DNA themselves can directly serve as detection antibody and PCR template, respectively. The aim of the design is to overcome shortcoming of low detection sensitivity of scFv so as to largely facilitate the real application of scFv in immunoassay. The idea has been demonstrated by applying hantaan virus nucleocapsid protein (NP) and prion protein (PrP) as detection targets in three experimental protocols (indirect, sandwich and real-time PD-IPCR assays). The detection sensitivity was increased 1000- to 10,000-folds compared with conventional enzyme-linked immunosorbent assays (ELISAs). This proof-of-concept study may serve as a new model to develop an easy to operate, low cost and ultrasensitive immunoassay method for broad applications.

An auto-biotinylated bifunctional protein nanowire for ultra-sensitive molecular biosensing.

In order to obtain an ultra-sensitive molecular biosensor, we designed an auto-biotinylated bifunctional protein nanowire (bFPNw) based on the self-assembly of a yeast amyloid protein, Sup35, to which protein G and a biotin acceptor peptide (BAP) were genetically fused. These auto-biotinylated bFPNws can transfer hundreds of commercially available diagnostic enzymes to an antigen-antibody complex via the biotin-avidin system, greatly enhancing the sensitivity of immune-biosensing. Compared to our previously reported seeding-induced bFPNws (Men et al., 2009), these auto-biotinylated bFPNws gave greater signal amplification, reduced non-specific binding and improved stability. The auto-biotinylated self-assembled bFPNw molecular biosensors were applied to detect Yersinia pestis (Y. pestis) F1 antigen and showed a 2000- to 4000-fold increase in sensitivity compared to traditional immunoassays, demonstrating the potential use of these self-assembling protein nanowires in biosensing.

[Investigation on the relationship among histamine, tryptase and beta-hexosaminidase in the process of mast cell degranulation.].

AIM: To investigate the relationship among histamine, tryptase and beta-hexosaminidase in the process of mast cell release and search for the suitable index to describe mast cell degranulation. METHODS: Mast cell degranulation was performed in vitro, in which histamine, tryptase and beta-hexosaminidase are detected by spectrophotometry. RESULTS: The concordance was found to exit among the three mediums, furthermore the study discloses that more similarity was found between tryptase and beta-hexosaminidase. Histamine was difficult and inconstant to be detected. CONCLUSION: Tryptase and beta-hexosaminidase are more suitable and accurately to reflect mast cell degranulation in vitro than histamine. Tryptase is the excellent index to detect mast cell degranulation in vitro.

On-chip ligation of multiplexing probe-pairs for identifying point mutations out of dense SNP loci.

Detailed analyses of dense single nucleotide polymorphism (SNP) loci within rifampin-resistance determining region (RRDR) are very important for the early assessment of drug resistance of Mycobacterium tuberculosis. A strategy was developed here to specifically identify point mutations out of dense SNP loci by on-chip ligation of multiplexing probe-pairs (MPPs). A probe-pair combines a common probe with a discriminating probe which is covalently attached to a DNA chip. The common probe hybridizes to the discriminating probe via a unique "zip-code complement". The allele-specific part on the 3'-end of the discriminating probe becomes covalently ligated to the adjacent part on the 5'-end of the common probe if and only if a mutation is present. Thus upon zip-code recognition, the process of identifying a mutation of interest is entirely located into corresponding well on the chip. As a consequence, cross-reactions and biased competitive attachments to targets, both of which result from the presence of various multiplexing probes, are greatly minimized. Mutation detection was performed by direct visualization using enzyme-linked assay. The method was demonstrated with an initial set of 24 probe-pairs targeting 22 clinically meaningful mutations within an 81-bp RRDR. 130-bp fragments of the rpoB gene from 15 clinical isolates were identified and were in 100% agreement with results from independent sequencing.

Loop-mediated isothermal amplification for rapid detection of Bacillus anthracis spores.

A loop-mediated isothermal amplification (LAMP) assay system was employed for detecting Bacillus anthracis spores in pure cultures as well as in various simulated powder samples. The specificity of the designed LAMP primer sets was validated by assaying 13 B. anthracis strains and 33 non-B. anthracis species. The detection limits of the LAMP assay were 10 spores/tube for pure cultures and 100 spores/2 mg powder for simulated powder samples. The results show that the LAMP protocol is a promising method for detecting B. anthracis.

Construction of single chain variable fragment (ScFv) and BiscFv-alkaline phosphatase fusion protein for detection of Bacillus anthracis.

This paper describes an attempt for convenient and sensitive detection of Bacillus anthracis with single chain variable fragment (scFv)-based protein chip. Phage display technology was employed to generate scFv by using the protective antigen (PA) of B. anthracis for immunization. V(H) and V(L) genes of the scFv were amplified separately by reverse transcriptase-PCR from mRNA of immunized mice and then assembled into scFv gene with a linker DNA sequence. The scFv gene was inserted into a phagemid vector pCANTAB-5E and then transformed into Escherichia coli TG1 to yield recombinant phages after infection with helper phage M13KO7. After six rounds of panning with PA, the phage clones displaying scFv fragments of the antibody were selected by ELISA. One phage clone scFv-6w10 showing the strongest positive signal in ELISA was selected. To enhance the affinity of the scFv-6w10, a recombinant bivalent single-chain Fv antibody (biscFv-6w10) directed against PA was constructed and tested in functional assays. The affinity of the biscFv-6w10 was much higher than that of scFv-6w10 and reached 6.5 x 10(9) M(-1). An expression system was constructed for the production of E. coli alkaline phosphatase (EAP) labeled biscFv-6w10 (biscFv-6w10-EAP) in E. coli cells. The expressed fusion protein retained both antigen-specific binding and enzymatic activity and thus directly served as an enzyme-labeled antibody. Detections of PA and bacterial cells of B. anthracis using biscFv-6w10-EAP and Cy3-labeled biscFv-6w10 were performed on a protein chip. The fusion protein (biscFv-6w10-EAP) chip could detect 10 pg of PA and 500-1000 bacterial cells in approximately 2 h, while the sensitivity of Cy3-labeled protein chip reached 1 pg of PA and 50-100 cells within 2 h.