Browsing by keyword "Bacteriophages"
Now showing items 1-4 of 4
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A new TAG-72 cancer marker peptide identified by phage displayRadiolabeled peptides as markers of cancer targets have demonstrated their value in diagnostic imaging and radiotherapy. The 16 mer f88-4/Cys6 phage display library was applied to affinity purified TAG-72 and three consensus peptides were identified: VHHSCTKLTHCCQNWH (A2-13), GGVSCMQTSPVCENNL (A2-6) and TKRDCSAQNYGCQKAI (A2-11). The A2-13 and A2-6 phages showed the highest percent binding to LS-174T cells by flow cytometry and were 3-fold higher than a control phage, while fluorescence microscopy showed that both A2-6 and A2-13 phages bound to the LS-174T cell membrane. However, only the A2-6 phage demonstrated specificity by low binding to the TAG-72 negative cell HT-29. Furthermore, the synthesized free A2-6 peptide demonstrated specific binding to LS-174T cells by flow cytometry and by immunohistochemical staining of xenograft tumor compared to normal colon. These data indicate that the A2-6 peptide is specific for the TAG-72 cancer target.
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DNA methylation alters the pattern of spontaneous mutation in Escherichia coli cells (mutD) defective in DNA polymerase III proofreadingWe have shown previously that dam mutants of Escherichia coli have a weak mutator phenotype which generates mostly transition mutations in the P22 mnt gene. In contrast, in mutD5 cells, which have a strong mutator phenotype, transversion mutations were the most prevalent. A dam-16 mutD5 strain, defective in both DNA polymerase III associated-proofreading and Dam-directed mismatch repair exhibits a strong mutator phenotype but, surprisingly, its mutation spectrum is similar to that of the dam rather than the mutD parent. The most likely explanation is that Dam-directed mismatch repair in the mutD5 strain corrects most of the potential transition mutations (therefore yielding transversions) in the newly synthesised strand. When the dam-16 allele is present together with mutD5 a reduced efficiency of repair as well as loss of strand discrimination and misdirected repair results in the appearance of transition mutations at high frequency.
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Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2CRISPR-Cas adaptive immune systems function to protect bacteria from invasion by foreign genetic elements. The CRISPR-Cas9 system has been widely adopted as a powerful genome-editing tool, and phage-encoded inhibitors, known as anti-CRISPRs, offer a means of regulating its activity. Here, we report the crystal structures of anti-CRISPR protein AcrIIC2Nme alone and in complex with Nme1Cas9. We demonstrate that AcrIIC2Nme inhibits Cas9 through interactions with the positively charged bridge helix, thereby preventing sgRNA loading. In vivo phage plaque assays and in vitro DNA cleavage assays show that AcrIIC2Nme mediates its activity through a large electronegative surface. This work shows that anti-CRISPR activity can be mediated through the inhibition of Cas9 complex assembly.
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Isolation of a monoclonal antibody from a phage display library binding the rhesus macaque MHC class I allomorph Mamu-A1*001Monoclonal antibodies that bind to human leukocyte antigen (HLA) are useful tools for HLA-typing, tracking donor-recipient chimerisms after bone marrow transplants, and characterizing specific major histocompatibility complexes (MHC) on cell surfaces. Unfortunately, equivalent reagents are not available for rhesus macaques, which are commonly used animal as models in organ transplant and infectious disease research. To address this deficiency, we isolated an antibody that recognizes the common Indian rhesus macaque MHC class I molecule, Mamu-A1*001. We induced Mamu-A1*001-binding antibodies by alloimmunizing a female Mamu-A1*001-negative rhesus macaque with peripheral blood mononuclear cells (PBMC) from a male Mamu-A1*001-positive donor. A Fab phage display library was constructed with PBMC from the alloimmunized macaque and panned to isolate an antibody that binds to Mamu-A1*001 but not to other common rhesus macaque MHC class I molecules. The isolated antibody distinguishes PBMC from Mamu-A1*001-positive and -negative macaques. Additionally, the Mamu-A1*001-specific antibody binds the cynomolgus macaque MHC class I ortholog Mafa-A1*001:01 but not variants Mafa-A1*001:02/03, indicating a high degree of binding specificity. The Mamu-A1*001-specific antibody will be useful for identifying Mamu-A1*001-positive rhesus macaques, for detecting Mamu-A1*001-positive cells in populations of Mamu-A1*001-negative cells, and for examining disease processes that alter expression of Mamu-A1*001 on cell surfaces. Moreover, the alloimmunization process we describe will be useful for isolating additional MHC allomorph-specific monoclonal antibodies or antibodies against other polymorphic host proteins which are difficult to isolate with traditional technologies.
