RESUMO
SCRATCHY is a methodology for the construction of libraries of chimeras between genes that display low sequence homology. We have developed a strategy for library creation termed enhanced crossover SCRATCHY, that significantly increases the number of clones containing multiple crossovers. Complementary chimeric gene libraries generated by incremental truncation (ITCHY) of two distinct parental sequences are created, and are then divided into arbitrarily defined sections. The respective sections are amplified by skewed sets of primers (i.e. a combination of gene A specific forward primer and gene B specific reverse primer, etc.) allowing DNA fragments containing non-homologous crossover points to be amplified. The amplified chimeric sections are then subjected to a DNA shuffling process generating an enhanced crossover SCRATCHY library. We have constructed such a library using the rat theta 2 glutathione transferase (rGSTT2) and the human theta 1 glutathione transferase (hGSTT1) genes (63% DNA sequence identity). DNA sequencing analysis of unselected library members revealed a greater diversity than that obtained by canonical family shuffling or with conventional SCRATCHY. Expression and high-throughput flow cytometric screening of the chimeric GST library identified several chimeric progeny that retained rat-like parental substrate specificity.
Assuntos
Técnicas de Química Combinatória/métodos , Troca Genética/genética , Biblioteca Gênica , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Animais , Glutationa Transferase/química , Humanos , Ratos , Proteínas Recombinantes de Fusão/química , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Especificidade por SubstratoRESUMO
A computer model, based on homology, of the catalytic antibody 7F11 that catalyses the decomposition of the benzoate ester of a dioxetane resulting in chemiluminescence is reported. Antibody 7F11 has 89% identity in the V(L) domain, and 72% identity in the V(H) domain with hydrolytic antibodies 17E8 and 29G11 previously reported by Scanlan et al. These were also raised against a phosphonate containing hapten. The antigen-binding site of antibody 7F11 whilst similar to that of 17E8 has aspartic acids at positions 33H and 35H, reminiscent in position of the catalytic residues found in aspartate proteinases such as pepsin. AutoDock 3.0 has been used to identify the best binding mode for the hapten. Molecular dynamic simulations have also been undertaken to examine any major conformational changes induced by hapten binding. A mechanism for benzoate ester hydrolysis involving the aspartic acid side-chains is proposed. Construction of a single-chain variable fragment (scFv) of 7F11 is also reported.
