I. G. Shemyakin

Purification of filamentous bacteriophage for phage display using size-exclusion chromatography

Phage display has proved itself as a powerful and convenient approach for rapid sampling of billions of protein-aceous structures against a ligand of interest. The most widespread system uses display of proteins and peptides as fusions to coat proteins III or VIII of the M13/fd phage. Efficient screening of phage display library requires high purity of the input phage particles. Double precipitation by polyeth-ylene glycol (PEG) with an average molecular weight of 8000 Da (PEG-8000) is a standard technique for purification of filamentous bacterio-phages (1,2). Certain contaminants could still remain in the PEG-purified phage preparation. Although efficient removal of contaminating RNases from bacteriophage preparation using treatment by ionic detergent followed by ultracentrifugation in CsCl gradient was reported (3), this method is costly, time-consuming, and is not applicable if the native state of the phage-displayed protein or peptide is affected by ionic detergents and high salt. Size-exclusion chromatography (SEC) has been used in preparation of pure eukaryotic viruses (4,5) and bacteriophage λ (6). We assumed that SEC can be the method of choice for efficient purification of filamentous phages under mild conditions. In this work we describe a rapid and simple technique for chromatographic purifi-cation of filamentous phage particles in Sephacryl™ S-500 resin (Amersham Biosciences, Piscataway, NJ, USA).The purity of phage preparations obtained after double PEG precipi-tation was assayed by electrophoresis in Tris-tricine (7). Phage particles displayed the single-chain variable fragment (scFv) of the MRL-4 anti-DNA monoclonal antibody (MAb) (8) fused to the C-terminal portion of the pIII minor phage coat protein (δpIII). Production of the scFv-δpIII fusion cloned into the pTScDisp phagemid (GenBank

Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches

Lethal factor (LF), a zinc-dependent protease of high specificity produced by Bacillus anthracis, is the effector component of the binary toxin that causes death in anthrax. New therapeutics targeting the toxin are required to reduce systemic anthrax-related fatalities. In particular, new insights into the LF catalytic mechanism will be useful for the development of LF inhibitors. We evaluated the minimal length required for formation of bona fide LF substrates using substrate phage display. Phage-based selection yielded a substrate that is cleaved seven times more efficiently by LF than the peptide targeted in the protein kinase MKK6. Site-directed mutagenesis within the metal-binding site in the LF active center and within phage-selected substrates revealed a complex pattern of LF-substrate interactions. The elementary steps of LF-mediated proteolysis were resolved by the stopped-flow technique. Pre-steady-state kinetics of LF proteolysis followed a four-step mechanism as follows: initial substrate binding, rearrangement of the enzyme-substrate complex, a rate-limiting cleavage step, and product release. Examination of LF interactions with metal ions revealed an unexpected activation of the protease by Ca2+ and Mn2+. Based on the available structural and kinetic data, we propose a model for LF-substrate interaction. Resolution of the kinetic and structural parameters governing LF activity may be exploited to design new LF inhibitors.

Role of Structure-Based Changes due to Somatic Mutation in Highly Homologous DNA-Binding and DNA-Hydrolyzing Autoantibodies Exemplified by A23P Substitution in the VH Domain.

Anti-DNA autoantibodies are responsible for tissue injury in lupus. A subset of DNA-specific antibodies capable of DNA cleavage can be even more harmful after entering the living cells by destroying nuclear DNA. Origins of anti-DNA autoantibodies are not fully understood, and the mechanism of induction of DNA-cleaving activity remains speculative. The autoantibody BV04-01 derived from lupus-prone mouse is the only DNA-hydrolyzing immunoglobulin with known 3D structure. Identification and analysis of antibodies homologous to BV04-01 may help to understand molecular bases and origins of DNA-cleaving activity of autoantibodies. BLAST search identified murine anti-DNA autoantibody MRL-4 with sequences of variable region genes highly homologous to those of autoantibody BV04-01. Despite significant homology to BV04-01, not only MRL-4 had no DNA-cleaving activity, but also reversion of its unusual P23 mutation to the germline alanine resulted in a dramatic loss of affinity to DNA. Contrary to this effect, transfer of the P23 mutation to the BV04-01 has resulted in a significant drop in DNA binding and almost complete loss of catalytic activity. In the present paper we analyzed the properties of two homologous autoantibodies and mutants thereof and discussed the implications of unusual somatic mutations for the development of autoantibodies with DNA-binding and DNA-hydrolyzing activity.

Molecular Characteristics of the Mycobacterium tuberculosis LAM-RUS Family Prevalent in Central Russia

ABSTRACT We analyzed IS6110-associated polymorphisms in the phospholipase C genes of 107 isolates of Mycobacterium tuberculosis selected to be representative of isolates circulating in central Russia. We found that the majority of Latin American-Mediterranean family strains contained an insertion in a unique position in the plcA gene, suggesting a common ancestor. This insertion can serve as a specific genetic marker for this group, which we designate the LAM-RUS family.

The Prospects for Using Aptamers in Diagnosing Bacterial Infections

Nucleic acid-based aptamers are widely accepted as promising tools for development of a plethora of diagnostic and therapeutic preparations, as well as means of environmental monitoring. The properties of aptamers permit them to be regarded as fully synthetic analogs of antibodies. Moreover, certain properties of aptamers render them superior to antibodies in terms of the development of new diagnostic and monitoring systems that combine high sensitivity and specificity with high reproducibility and inexpensive manufacture. In particular, aptamer-based technologies for detection of biomolecules and whole microorganisms can be employed in solving the problems of highly sensitive express diagnostics of bacterial pathogens. The present review summarizes the current state of the techniques developed for aptamer-based detection of bacteria and their components and discusses the potential of their practical application.

Ultrasensitive detection of protease activity of anthrax and botulinum toxins by a new PCR-based assay

Anthrax and botulism are dangerous infectious diseases that can be fatal unless detected and treated quickly. Fatalities from these diseases are primarily due to endopeptidase toxins secreted by the pathogens. Rapid and sensitive detection of the presence of active toxins is the key element for protection from natural outbreaks of anthrax and botulism, as well as from the threat of bioterrorism. We describe an ultrasensitive polymerase chain reaction (PCR)-based assay for detecting proteolytic activity of anthrax and botulinum toxins using composite probes consisting of covalent peptide-DNA conjugate for the detection of anthrax, and noncovalent protein-aptamer assembly to assay botulinum toxin activity. Probes immobilized on the solid-phase support are cleaved by toxins to release DNA, which is detected by real-time PCR. Both assays can detect subpicogram quantities of active toxins isolated from composite matrices. Special procedures were developed to isolate intact toxins from the matrices under mild conditions. The assay is rapid, uses proven technologies, and can be modified to detect other proteolytic and biopolymer-degrading enzymes.

Special features of immune response to the lethal toxin of Bacillus anthracis

We and other authors have recently shown that the pattern of the immune response to components of anthrax, the Bacillus anthracis lethal toxin, is complex. In addition to the neutralizing antibodies, the antitoxin antibody pool contains antibodies enhancing the toxin lethal action. We mapped the epitopes in the protective antigen that are responsible for the induction of both antibody types. In this study, we obtained new data on the cytotoxicity of the B. anthracis lethal toxin toward the J774 A.1 cell line in the presence of monoclonal antibodies to various domains of the protective antigen and the lethal factor. The role of the Fc fragment of immunoglobulins in enhancing the lethal toxin action was shown. These results may serve as a basis for the development of a new generation vaccine for anthrax.

Immune response to Vi-antigen of Salmonella typhi is dependent on introduction of positive charge and shape of charged group into polysaccharide

A model of a controlled conversion of polysaccharide Vi-antigen of S. typhi into zwitterionic antigen is proposed. The immunological properties of modifications of this antigen conjugated to a protein support were studied.

Antibiotic-dependent selection of E. coli clones with increased chaperone activity for highly efficient production of full-length soluble new delhi metallo-beta-lactamase

New Delhi metallo-beta-lactamase (NDM-1) is a plasmid-borne enzyme conferring bacterial resistance to any known beta-lactam antibiotics. There is urgent need to develop efficient NDM-1 inhibitors and approaches for early diagnostics of NDM-1 that necessitates structural studies of the enzyme and analysis of the secretion pathways and localization of the protein. Recombinant full-length NDM-1 is produced in E. coli in active form and mostly accumulates in inclusion bodies. We developed a new system that uses antibiotic pressure to select E. coli producing increased quantities of the soluble NDM-1 and showed that the efficiency of production of the soluble NDM-1 depends on the level of chaperone activity in a bacterial cell.

Dominant genotypes of Mycobacterium tuberculosis strains isolated from prison inmates in Ozerki

A correlation was observed between the Beijing/W and LAM families of Mycobacterium tuberculosis and drug resistance, and the transmissiveness of multidrug-resistant strains belonging to these two families was also shown.