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Wild-type Molecule (wild-type + molecule)
Selected AbstractsFunctional allelic loss detected at the protein level in archival human tumours using allele-specific E-cadherin monoclonal antibodiesTHE JOURNAL OF PATHOLOGY, Issue 5 2002Karl-Friedrich Becker Abstract Immunohistochemical analysis has been used to show that expression of the homophilic cell-to-cell adhesion molecule, E-cadherin, is frequently altered in human cancers, including gastric and breast carcinoma. Besides genetic down-regulation, structural mutations such as in-frame deletions of exon 8 and exon 9 were frequently found; these may affect the binding of monoclonal antibodies used for immunohistochemical analysis. In this study it was found that antibodies HECD-1 and E9, two monoclonal antibodies often used in E-cadherin immunoanalysis, react with epitopes present at least in part in exon 8 and exon 9, respectively. This study generated and characterized a mutation-specific monoclonal antibody, E-cad delta 8-1, reacting with the mutant protein lacking exon 8 but not with the wild-type molecule. By using E-cad delta 8-1 and HECD-1, it was possible separately to analyse the immunoreactivity of mutant and normal E-cadherin proteins, respectively, in an allele-specific manner in archival material. A similar analysis was performed using E9 and the previously characterized mutation-specific antibody E-cad delta 9-1. Typically, in gastric and breast cancer harbouring E-cadherin splice site gene mutations, the mutant proteins were expressed but the wild-type protein was not detected in malignant tissues. These results indicate that variant-specific monoclonal antibodies can be used to identify differentially expressed E-cadherin proteins. For immunohistochemical analysis of E-cadherin, at least two different monoclonal antibodies should be used to exclude alterations of the epitopes resulting in failure to detect a mutant protein. Copyright © 2002 John Wiley & Sons, Ltd. [source] Phage display selection of hairpin loop soyacystatin variants that mediate high affinity inhibition of a cysteine proteinaseTHE PLANT JOURNAL, Issue 5 2001Hisashi Koiwa Summary Two hairpin-loop domains in cystatin family proteinase inhibitors form an interface surface region that slots into the active site cleft of papain-like cysteine proteinases, and determine binding affinity. The slot region surface architecture of the soybean cysteine proteinase inhibitor (soyacystatin N, scN) was engineered using techniques of in vitro molecular evolution to define residues that facilitate interaction with the proteinase cleft and modulate inhibitor affinity and function. Combinatorial phage display libraries of scN variants that contain mutations in the essential motifs of the first (QVVAG) and second (EW) hairpin-loop regions were constructed. Approximately 1010,1011 phages expressing recombinant scN proteins were subjected to biopanning selection based on binding affinity to immobilized papain. The QVVAG motif in the first hairpin loop was invariant in all functional scN proteins. All selected variants (30) had W79 in the second hairpin-loop motif, but there was diversity for hydrophobic and basic amino acids in residue 78. Kinetic analysis of isolated scN variants identified a novel scN isoform scN(LW) with higher papain affinity than the wild-type molecule. The variant contained an E78L substitution and had a twofold lower Ki (2.1 pm) than parental scN, due to its increased association rate constant (2.6 ± 0.09 × 107 m,1sec,1). These results define residues in the first and second hairpin-loop regions which are essential for optimal interaction between phytocystatins and papain, a prototypical cysteine proteinase. Furthermore, the isolated variants are a biochemical platform for further integration of mutations to optimize cystatin affinity for specific biological targets. [source] Production of native and modified recombinant Der p 1 molecules in tobacco plantsCLINICAL & EXPERIMENTAL ALLERGY, Issue 5 2009D. Burtin Summary Background As a complex molecule requiring post-translational processing, it has been difficult to produce the Der p 1 major allergen from the Dermatophagoides pteronyssinus house dust mite in a recombinant form. Objective Here, we tested whether transgenic tobacco plants are suitable to express Der p 1, either as a wild-type molecule or as variants lacking N -glycosylation sites (Gly,) and/or cysteine protease activity (Enz,). Methods Using Agrobacterium tumefaciens -based transformation, pro Der p 1 molecules bearing mutations within either the N -glycosylation sites (N34Q, N150Q) and/or the cysteine protease-active site (C132V) were expressed in tobacco plants. After purification by ion exchange chromatography, allergens were characterized using immunoblotting, circular dichroism (CD), as well as basophil and T lymphocyte stimulation assays. Results Four forms of recombinant Der p 1 (i.e. wild-type Gly+/Enz+, as well as Gly,/Enz+, Gly+/Enz, or Gly,/Enz, variants) were successfully expressed in tobacco leaves as pro Der p 1 molecules. Spontaneous cleavage of the pro-peptide was observed in tobacco leaf extracts for all forms of recombinant Der p 1 (r Der p 1). CD confirmed that all r Der p 1 molecules, with the exception of the Gly,/Enz, variant, exhibited secondary structures comparable to the natural protein. A cysteine protease activity was associated only with the Gly+/Enz+ form. All these molecules exhibit a profile similar to natural Der p 1 with respect to IgE immunoreactivity, basophil activation and T cell recognition. Conclusion A tobacco plant expression system allows the production of various forms of mature Der p 1, which could be used for diagnostic or immunotherapeutic purposes. [source] Quantification of mitochondrial DNA mutation loadAGING CELL, Issue 5 2009Laura C. Greaves Summary Mitochondrial DNA (mtDNA) mutations are an important cause of genetic disease and have been proposed to play a role in the ageing process. Quantification of total mtDNA mutation load in ageing tissues is difficult as mutational events are rare in a background of wild-type molecules, and detection of individual mutated molecules is beyond the sensitivity of most sequencing based techniques. The methods currently most commonly used to document the incidence of mtDNA point mutations in ageing include post-PCR cloning, single-molecule PCR and the random mutation capture assay. The mtDNA mutation load obtained by these different techniques varies by orders of magnitude, but direct comparison of the three techniques on the same ageing human tissue has not been performed. We assess the procedures and practicalities involved in each of these three assays and discuss the results obtained by investigation of mutation loads in colonic mucosal biopsies from ten human subjects. [source] | |||||||||||||