RESUMO
The likelihood for improvement in the catalytic properties of Escherichia coli alkaline phosphatase was examined using site-directed mutagenesis. Mutants were constructed by introducing sequence changes into nine preselected amino acid sites within 10 A of the catalytic residue serine 102. When highly conserved residues in the family of alkaline phosphatases were mutated, many of the resulting enzymes not only maintained activity, but also exhibited greatly improved kcat. Of approximately 170 mutant enzymes screened, 5% (eight mutants) exhibited significant increases in specific activity. In particular, a substitution by serine of a totally invariant Asp101 resulted in a 35-fold increase of specific activity over wild-type at pH 10.0. Up to 6-fold increases of the kcat/Km ratio were observed.
Assuntos
Fosfatase Alcalina/genética , Escherichia coli/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , Análise Mutacional de DNA , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Homologia de Sequência do Ácido Nucleico , Relação Estrutura-AtividadeRESUMO
A number of C5a modifications were tested to determine effects on receptor binding to polymorphonuclear leukocyte (PMNL) membrane receptors and triggering of PMNL chemokinesis and myeloperoxidase (MPO) release. Site-directed mutagenesis was used to probe relationships of key C-terminal residues, and suggested a role for additional sites, particularly Lys19-20. A synthetic peptide based on C5a 19-30, weakly inhibited C5a binding. Potency of the C-terminal octapeptide, a full agonist, was markedly improved by a single Phe substitution for His67, and a Phe point mutation at this site was shown to enhance activity of the full recombinant protein.
Assuntos
Complemento C5a/metabolismo , Neutrófilos/metabolismo , Receptores de Superfície Celular/metabolismo , Células Cultivadas , Humanos , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
A first totally synthetic Escherichia coli plasmid has been designed, constructed and shown to be a functional, stable, high-copy cloning vector. The FokI method of gene synthesis [Mandecki and Bolling, Gene 68 (1988) 101-107] was used to assemble the plasmid from 30 oligodeoxyribonucleotides. The plasmid contains synthetic modules for the beta-lactamase-encoding gene (bla), replication origin, lacZ gene fragment and multicloning site. The plasmid is patterned after the pUC-type plasmids and has a copy number similar to that of pUC plasmids. The major changes introduced include the removal of nearly 50% of the restriction sites present in pUC plasmids, reduction of plasmid size to 2050 bp, and introduction of transcription terminators downstream from both the bla gene and lacZ fragment. The changes facilitate a number of techniques, such as cloning, mutagenesis, expression and restriction analysis.
Assuntos
Escherichia coli/genética , Expressão Gênica , Mutagênese , Plasmídeos , Sequência de Bases , Clonagem Molecular/métodos , DNA Bacteriano/síntese química , Técnicas Genéticas , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Mapeamento por Restrição , Regiões Terminadoras Genéticas , Transcrição GênicaRESUMO
A method was developed for determination of the rate of undesired point mutations upon cloning of synthetic DNA. The method relies on cloning of an oligonucleotide(s) into the E. coli alkaline phosphatase gene inactivated due to a small deletion within the active site. The oligonucleotide adds back the deleted sequence, but simultaneously introduces a missense mutation at a critical position. The activity of the enzyme is restored only if there is a predefined sequence change within the codon specifying an essential residue of the active site. The clones carrying the reactivated gene are detected by colony color screening on plates. The method is fast and simple, does not require specialized equipment nor enzymatic reactions, although a separate oligonucleotide needs to be provided for each sequence change to be evaluated. The procedure allows for the use of crude extracts of oligonucleotides and distinguishes between different types of sequence changes.
Assuntos
Bioensaio , Clonagem Molecular , Genes Sintéticos , Mutação , Oligodesoxirribonucleotídeos/genética , Fosfatase Alcalina/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Vetores Genéticos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/síntese química , Proteínas Recombinantes/genéticaRESUMO
C5a is an inflammatory mediator potentially involved in a number of diseases. To help define which of its 74 residues are important for receptor binding and response triggering, changes in the amino acid sequence of C5a were introduced by site-directed mutagenesis. Synthetic C5a-encoding genes incorporating point mutations were expressed in Escherichia coli, and the mutant proteins were purified to homogeneity. Modifications of the C5a molecule causing parallel reductions in binding to polymorphonuclear leukocyte membranes and in stimulation of polymorphonuclear leukocyte locomotion (chemokinesis) suggest that carboxyl-terminal residues Lys-68, Leu-72, and Arg-74 interact with the receptor. Substitutions in the disulfide-linked core of C5a revealed involvement of Arg-40 or nearby residues, because potency losses were associated with only localized conformational changes as detected by NMR. Surprisingly, a substitution at core residue Ala-26, which did not alter C5a core structure, appeared from NMR results to reduce potency by causing a long-distance conformational change centered on residue His-15. Thus, at least three discontinuous regions of the C5a molecule appear to act in concert to achieve full potency.
