Showing papers on "Lambda phage published in 2013"

Oligopeptide M13 Phage Display in Pathogen Research

Abstract: Phage display has become an established, widely used method for selection of peptides, antibodies or alternative scaffolds. The use of phage display for the selection of antigens from genomic or cDNA libraries of pathogens which is an alternative to the classical way of identifying immunogenic proteins is not well-known. In recent years several new applications for oligopeptide phage display in disease related fields have been developed which has led to the identification of various new antigens. These novel identified immunogenic proteins provide new insights into host pathogen interactions and can be used for the development of new diagnostic tests and vaccines. In this review we focus on the M13 oligopeptide phage display system for pathogen research but will also give examples for lambda phage display and for applications in other disease related fields. In addition, a detailed technical work flow for the identification of immunogenic oligopeptides using the pHORF system is given. The described identification of immunogenic proteins of pathogens using oligopeptide phage display can be linked to antibody phage display resulting in a vaccine pipeline.

Simultaneous display of two large proteins on the head and tail of bacteriophage lambda

Abstract: Background Consistent progress in the development of bacteriophage lambda display platform as an alternative to filamentous phage display system was achieved in the recent years. The lambda phage has been engineered to display efficiently multiple copies of peptides or even large protein domains providing a powerful tool for screening libraries of peptides, proteins and cDNA.

In Vivo cII, gpt, and Spi− Gene Mutation Assays in Transgenic Mice and Rats

Abstract: Transgenic mutation assays are used to identify and characterize genotoxic hazards and for determining the mode of action for carcinogens. The three most popular transgenic mutational models are Big Blue® (rats or mice), Muta™ mouse (mice), and gpt-delta (rats or mice). The Big Blue® and Muta™ mouse models use the cII gene as a reporter of mutation whereas gpt-delta rodents use the gpt gene and the red/gam genes (Spi⁻ selection) as mutation reporter genes. Here we describe methodology for conducting mutation assays with these transgenes. Transgenes recovered from tissue DNA are packaged into infectious lambda phage, bacteria are infected with the phage, and cII-mutant and Spi⁻ plaques and gpt-mutant colonies are isolated using selective conditions and quantified. Selected mutants can be further analyzed for identification of small sequence alterations in the cII and gpt genes and large deletions at the Spi⁻ locus.

Escherichia coli as Host and Pathogen

Abstract: Enterohemorrhagic E. coli (EHEC) are highly infectious food-borne pathogens that cause severe diarrhoea in both, industrialised and developing countries all over the world. Their pathogenicity factors involve shiga-like toxins and a type III secretion system along with so called effector proteins, which are translocated directly into the cytoplasm of their host cells, usually enterocytes. Most of these proteins are encoded in pathogenicity islands within the bacterial genome that are framed by sequences of lambdoid phages. Some of these phages are still able to produce infectious particles after UV induction. In this study I generated two protein-protein interactomes, namely EHEC-host and phage lambda-E. coli. For the EHEC-host interactome, 34 effector proteins that had been previously shown to be secreted into human host cells were cloned and screened against pooled human cDNA and ORF libraries via yeast two-hybrid screening. This resulted in 35 reproducible interactions of 15 EHEC effectors with 34 human proteins, of which only four had been published previously. Inclusion of secondary human protein interactors retrieved from the BioGRID database revealed that EHEC effectors are interconnected in the human cell. The translocated intimin receptor (TIR) that was found to interact with eight human proteins was compared to its homologue in enteropathogenic E. coli (EPEC). This revealed that five of the eight EHEC TIR interactors also interact with EPEC TIR. Another interaction discovered in this study involves the EHEC effector NleF, previously a protein of unknown function, and human caspase-9. LUMIER assays against other human caspases identified caspase-4 and -8 as additional binding partners of NleF. Tests with purified enzymes revealed that NleF can potentially inhibit all three caspases. The effector decreased caspase activity significantly in HeLa cell lysate and impaired apoptosis induction in HeLa and Caco-2 cells. A collaboration partner solved the crystal structure of the NleF/caspase-9 complex, which suggested a dominant role of the carboxy-terminal four amino acids in caspase-9 binding and inhibition. I was able to confirm these findings by constructing NleF versions with mutagenized carboxy-termini. NleF versions that lacked the last four amino acids or comprised an additional carboxy-terminal alanine were unable to bind any of the three caspases or impair apoptosis. Apoptosis inhibition is a strategy often applied by viral and bacterial pathogens. Even though NleF is not the only effector protein capable of inhibiting apoptosis in human cells, direct inhibition of caspases by bacterial effectors has not been reported to date. The phage lambda-E. coli.interactome was generated during my research stay at the J. Craig Venter Institute in Rockville (USA). I screened 68 phage lambda proteins against the E. coli W3110 ORF library via yeast two-hybrid screening using two different vector systems. This resulted in 144 reproducible interacting pairs. The phage lambda and E. coli proteins involved in interactions were categorized in functional groups and analysed for interactions between phage and host groups.

PCR Analysis: Detection Of Bacteriophage Contamination In Biotechnology Used To Produce Recombinant drugs

Abstract: Contamination is a major drawback of the world in biotechnology field and it has cost of losing important biological products like recombinant drugs or valuable research. The causative agents are different chemicals, invertebrates, bacteriophages, bacteria, fungi, parasites, viral species and even other cell lines. In this study, E. coli BL-21 DE3 (DE3 has λ - bacteriophage genome) which is cloned with therapeutic drug were studied during 2 years (2008-2010) to detect their contaminations and the causative organisms. Samples were examined for bacteriophage contamination using conventional molecular biology techniques. Using polymerase chain reaction (PCR) technology, primers were specifically designed for lambda phage DE3 genome in E. coli BL-21 (DE3) and bacteriophage was detected in positive control. No bacteriophage was detected in any of the negative control samples. PCR results were confirmed by virus isolation experiments performed with PCR-positive and negative samples. This indicates λDE3 from E . coli BL-21 (DE3) is expressed and this led to cell lysis. The results obtained in this study furnish evidence suggesting that the employed assay system constitutes an effective tool for the detection of contamination in culture systems. To sum up, PCR represents a sensitive, specific, accurate, inexpensive, and quick bacteriophage detection assay that is suitable for the routine screening of cultures.