Ultrasensitive immuno-detection using viral nanoparticles with modular assembly using genetically-directed biotinylation

We report a novel, modular approach to immuno-detection based on antibody recognition and PCR read-out that employs antibody-conjugated bacteriophage and easily-manipulated non-pathogenic viruses as affinity agents. Our platform employs phage genetically tagged for in vivo biotinylation during phage maturation that can easily be linked, through avidin, to any biotinylated affinity agent, including full-length antibodies, peptides, lectins or aptamers. The presence of analyte is reported with high sensitivity through real-time PCR. This approach avoids the need to clone antibody-encoding DNA fragments, allows the use of full-length, high affinity antibodies and, by having DNA reporters naturally encapsulated inside the bacteriophage, greatly reduces nonspecific binding of DNA. We validate the efficacy of this new approach through the detection of Vascular Endothelial Growth Factor, a known angiogenic cancer biomarker protein, at attomolar concentrations in bronchoalveolar lavage fluid.     

Orotidine-5′-monophosphate decarboxylase fromPseudomonas aeruginosa PAO1: Cloning,overexpression, and enzyme characterization

Orotidine-5-monophosphate decarboxylase (OMPdecase) catalyzes the final step in pyrimidine biosynthesis, the conversion of orotidine-5-monophosphate (OMP) to uridine-5-monophosphate. ThepyrF gene, encoding OMPdecase, was isolated from a chromosomal library ofPseudomonas aeruginosa PAO1 by screening for complementation of anEscherichia coli and aP. aeruginosa pyrF mutant. The nucleotide sequence of a 2510-bp chromosomal DNA fragment, complementing both strains, was determined (EMBL accession number X65613). On this a 696-bp open reading frame capable of encoding the 24 kDa OMPdecase was identified. Despite a generally good correspondence to other OMPdecase sequences, theP. aeruginosa gene was unique in that it did not constitute part of an operon. ThepyrF gene was amplified by polymerase chain reaction, overexpressed in the pT7-7/E. coli BL21(DE3) system and purified to near electrophoretic homogeneity by anion exchange chromatography. Characterization of the purified enzyme revealed the following data, aK_m value for OMP of 9.91 M and an isoelectric point of 6.65. No major decrease in enzyme activity was observed in a pH range between 7.8 and 10.2. Gel electrophoresis under nondenaturing conditions suggested that the native form of OMPdecase is the dimer.     

The alanine racemase of Mycobacterium smegmatis is essential for growth in the absence of D-alanine

Alanine racemase, encoded by the gene alr, is an important enzyme in the synthesis of d-alanine for peptidoglycan biosynthesis. Strains of Mycobacterium smegmatis with a deletion mutation of the alr gene were found to require d-alanine for growth in both rich and minimal media. This indicates that alanine racemase is the only source of d-alanine for cell wall biosynthesis in M. smegmatis and confirms alanine racemase as a viable target gene for antimycobacterial drug development.     

DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in <Emphasis Type=Italic>Escherichia coli</Emphasis>

Background  

Manufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis.     

Characterization of the Alanine Racemases from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1

Alanine racemases are ubiquitous, almost uniquely prokaryotic enzymes catalyzing the racemization between l- and d-alanine. The requirement for d-alanine as a necessary component of the bacterial cell wall makes this class of enzymes a logical target for the development of novel antibiotics. In an effort to better understand the structure and mechanism of these enzymes, we have cloned the two independent alanine racemases from Pseudomonas aeruginosa, an important opportunistic bacterial pathogen of humans and animals. The dadX(PA) and alr(PA) genes have been sequenced, overexpressed, and their activity was demonstrated by complementing d-alanine auxotrophs of Escherichia coli. Both gene products were purified to electrophoretic homogeneity, the enzymes were characterized biochemically, and preliminary crystals were obtained.     

Hemocyanins in spiders, VI[1]. Comparison of the polypeptide chains of Eurypelma californicum hemocyanin.

The subunits of the hemocyanin from the tarantula, Eurypelma californicum, were isolated, following dissociation at pH 9.6, by a sequence of chromatographic and electrophoretic steps. Fraction 2 (containing two chains, a and c2) and the constituent polypeptide chains of the dimeric subunit 4D (b and c4) were resolved by anion exchange chromatography at pH 8.9 and 6.5, respectively. Since c2 and c4 have different electrophoretic mobilities in polyacrylamide gradient gels, the total number of different polypeptide chains is seven. The amino acid compositions of the seven chains are reported. There are major differences for at least half of the amino acids, while more consistent proportions become evident, if the amino acids are grouped by types of side chains. The N-terminal amino acid is proline in the case of chains b and e,, while no end group called be detected in any of the other chains by different methods. The C-terminal end group was found to be valine in both chains d and e. Cleavage by 70% formic acid, and by cyanogen bromide in formic acid results in fragmentation patterns distinct for each chain. After cyanogen bromide cleavage, the two largest peptides of each chain are of molecular weight near 2400. Tryptic fingerprints also reveal significant differences between all chains. Subunit heterogeneity of Eurypelma hemocyanin is clearly not the consequence of secondary modifications, but resides in major differences of the amino acid sequences.     

Purification and preliminary crystallization of alanine racemase from Streptococcus pneumoniae

Background

Over the past fifteen years, antibiotic resistance in the Gram-positive opportunistic human pathogen Streptococcus pneumoniae has significantly increased. Clinical isolates from patients with community-acquired pneumonia or otitis media often display resistance to two or more antibiotics. Given the need for new therapeutics, we intend to investigate enzymes of cell wall biosynthesis as novel drug targets. Alanine racemase, a ubiquitous enzyme among bacteria and absent in humans, provides the essential cell wall precursor, D-alanine, which forms part of the tetrapeptide crosslinking the peptidoglycan layer.

Results

The alanine racemases gene from S. pneumoniae (alr _SP ) was amplified by PCR and cloned and expressed in Escherichia coli. The 367 amino acid, 39854 Da dimeric enzyme was purified to electrophoretic homogeneity and preliminary crystals were obtained. Racemic activity was demonstrated through complementation of an alr auxotroph of E. coli growing on L-alanine. In an alanine racemases photometric assay, specific activities of 87.0 and 84.8 U mg^-1 were determined for the conversion of D- to L-alanine and L- to D-alanine, respectively.

Conclusion

We have isolated and characterized the alanine racemase gene from the opportunistic human pathogen S. pneumoniae. The enzyme shows sufficient homology with other alanine racemases to allow its integration into our ongoing structure-based drug design project.     

Rare target enrichment for ultrasensitive PCR detection using cot-rehybridization and duplex-specific nuclease

Nucleic acid detection by polymerase chain reaction (PCR) is invaluable for the detection of dilute and rare sequences, including pathogens and infrequent species in complex clinical and environmental backgrounds. The presence of excess complex background nucleic acid can reduce sensitivity and specificity. This is because mispriming can cause failure of the amplification reaction. Here we describe a new approach to ultrasensitive PCR detection, using enrichment of rare target nucleic acid from abundant background by combining the classic technique of cot-rehybridization to convert the abundant background to double-stranded form, with the use of a newly described, highly processive duplex-specific crab nuclease. We show that trace sequences in a vast excess of background DNA can be undetectable by PCR, independent of the amount of the mixture added to the PCR, and that these sequences can be made detectable by background suppression using this method.     

Crystal structure at 1.45 A resolution of alanine racemase from a pathogenic bacterium, Pseudomonas aeruginosa, contains both internal and external aldimine forms

The structure of the catabolic alanine racemase, DadX, from the pathogenic bacterium Pseudomonas aeruginosa, reported here at 1.45 A resolution, is a dimer in which each monomer is comprised of two domains, an eight-stranded alpha/beta barrel containing the PLP cofactor and a second domain primarily composed of beta-strands. The geometry of each domain is very similar to that of Bacillus stearothermophilus alanine racemase, but the rotation between domains differs by about 15 degrees. This change does not alter the structure of the active site in which almost all residues superimpose well with a low rms difference of 0.86 A. Unexpectedly, the active site of DadX contains a guest substrate that is located where acetate and propionate have been observed in the Bacillus structures. It is modeled as d-lysine and oriented such that its terminal NZ atom makes a covalent bond with C4' of PLP. Since the internal aldimine bond between the protein lysine, Lys33, and C4' of PLP is also unambiguously observed, there appears to be an equilibrium between both internally and externally reacted forms. The PLP cofactor adopts two partially occupied conformational states that resemble previously reported internal and external aldimine complexes.