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1	Towards Development of an Immunoassay Utilizing Circularly Permutated Proteins to Detect Environmental Contaminants Zunnoon Khan, Sara 29 August 2013 (has links) A fusion protein composed of antibody fragments and β-lactamase was earlier created by Kojima et al. (2011), with antigen specificities against a bone disease marker and a pesticide. The enzyme was circularly permutated and fused to the variable heavy and light chain antibody fragments, thereby ensuring inactivity until binding of the target antigen triggered enzyme activation. Upon activation, the β-lactamase produced a colorimetric signal, which indicated antigen presence. In this work, a similar strategy was used to create two novel fusion proteins composed of circularly permuted β-lactamase and superfolder green fluorescent protein with anti-benzo[a]pyrene variable antibody fragments. The fusion proteins were designed and expressed in E. coli for the development of a single-step visual immunoassay. It was hypothesized that the cp reporter proteins would be activated once the binding of B[a]P to the variable antibody fragments occurred, and this interaction was expected to produce a detectable colorimetric or fluorescent signal. Although positive results were obtained in one instance, substantial supportive evidence in favour of the hypothesis could not be obtained. / SENTINEL Bioactive Paper Network, Natural Sciences and Engineering Research Council of Canada (NSERC), Canada Research Chairs Program. immunoassay B[a]P benzo[a]pyrene circular permutation circularly permutated environmental contaminant beta-lactamase superfolder GFP green fluorescent protein fluorescence nitrocefin cross-reactivity imidazole carcinogen detection method fusion protein colorimetric reaction scFv anti-B[a]P scFv cp
2	Orthogonality and Codon Preference of the Pyrrolysyl-tRNA Synthetase-tRNAPyl pair in Escherichia coli for the Genetic Code Expansion Odoi, Keturah 2012 May 1900 (has links) Systematic studies of basal nonsense suppression, orthogonality of tRNAPyl variants, and cross recognition between codons and tRNA anticodons are reported. E. coli displays detectable basal amber and opal suppression but shows a negligible ochre suppression. Although detectable, basal amber suppression is fully inhibited when a pyrrolysyl-tRNA synthetase (PylRS)-tRNAPyl_CUA pair is genetically encoded. trnaPyl_CUA is aminoacylated by an E. coli aminoacyl-tRNA synthetase at a low level, however, this misaminoacylation is fully inhibited when both PylRS and its substrate are present. Besides that it is fully orthogonal in E. coli and can be coupled with PylRS to genetically incorporate a NAA at an ochre codon, tRNAPyl_UUA is not able to recognize an UAG codon to induce amber suppression. This observation is in direct conflict with the wobble base pair hypothesis and enables using an evolved M. jannaschii tyrosyl-tRNA synthetase-tRNAPyl_UUA pair and the wild type or evolved PylRS-tRNAPyl_UUA pair to genetically incorporate two different NAAs at amber and ochre codons. tRNAPyl_UCA is charged by E. coli tryptophanyl-tRNA synthetase, thus not orthogonal in E. coli. Mutagenic studies of trnaPyl_UCA led to the discovery of its G73U form which shows a higher orthogonality. Mutating trnaPyl_CUA to trnaPyl_UCCU not only leads to the loss of the relative orthogonality of tRNAPyl in E. coli but also abolishes its aminoacylation by PylRS. Pyl, pyrrolysine PylRS, pyrrolysyl-tRNA synthetase NAA, noncanonical amino acid aaRS, aminoacyl-tRNA synthetase T4 PNK, T4 polynucleotide kinase AzF, para-azido-L-phenylalanine PCR, polymerase chain reaction RF, release factor TrpRS, tryptophanyl-tRNA synthetase ArgRS, arginyl-tRNA synthetase Trp, tryptophan Lys, lysine Glu, glutamate Gln, glutamine Phe, phenylalanine Arg, arginine.

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Towards Development of an Immunoassay Utilizing Circularly Permutated Proteins to Detect Environmental Contaminants

Orthogonality and Codon Preference of the Pyrrolysyl-tRNA Synthetase-tRNAPyl pair in Escherichia coli for the Genetic Code Expansion