RESUMO
The ToxB gene was cloned and characterized from a race 5 isolate of Pyrenophora tritici-repentis from North Dakota. ToxB contains a 261-bp open reading frame that encodes a 23 amino acid putative signal peptide and a 64 amino acid host-selective toxin, Ptr ToxB. Analysis of Ptr ToxB from heterologous expression in Pichia pastoris confirms that ToxB encodes a host-selective toxin.
Assuntos
Ascomicetos/genética , Proteínas Fúngicas/genética , Triticum/microbiologia , Sequência de Aminoácidos , Ascomicetos/patogenicidade , Sequência de Bases , Proteínas Fúngicas/química , Dados de Sequência Molecular , Fases de Leitura Aberta , Pichia/genética , Doenças das Plantas/microbiologia , Sinais Direcionadores de Proteínas , Análise de Sequência de DNA , Análise de Sequência de ProteínaRESUMO
We used the surface protein expression (SPEX) system to express an anchored and a secreted form of staphylococcal nuclease A (NucA) from gram-positive bacteria. NucA is a small ( approximately 18 kDa), extracellular, monomeric enzyme from Staphylococcus aureus. A deletion of amino acids 114-119 causes monomeric NucA to form homodimers. The DNA sequence encoding either wild-type or deletion mutant NucA was cloned via homologous recombination into Streptococcus gordonii. S. gordonii strains expressing either anchored or secreted, monomeric or dimeric NucA were isolated and tested for enzymatic activity using a novel fluorescence enzyme assay. We show that active monomeric and dimeric NucA enzyme can be expressed either anchored on the cell surface or secreted into the culture medium. The activity of the dimer NucA was approximately 100-fold less than the monomer. Secreted and anchored, monomeric NucA migrated on SDS-polyacrylamide gels at approximately 18 or approximately 30 kDa, respectively. In addition, similar to S. aureus NucA, the S. gordonii recombinant NucA enzyme was dependent on CaCl(2) and was heat stable. In contrast, however, the recombinant NucA activity was maximal at pH 7.0-7.5 whereas S. aureus NucA was maximal at pH 9.0. These results show, for the first time, expression of active enzyme and polymeric protein in secreted and anchored forms using SPEX. This further demonstrates the utility of this gram-positive surface protein expression system as a potential commensal bacterial delivery system for active, therapeutic enzymes, biopharmaceuticals, or vaccines.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Nuclease do Micrococo/isolamento & purificação , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Western Blotting , Cloreto de Cálcio/química , Cromatografia em Gel , DNA/metabolismo , Dimerização , Estabilidade Enzimática , Fluorescência , Calefação , Concentração de Íons de Hidrogênio , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Nuclease do Micrococo/biossíntese , Nuclease do Micrococo/genética , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Streptococcus/genéticaRESUMO
Mutants of Streptococcus gordonii resistant to 5-fluorodeoxyuridine (FUdR(r)) were isolated. Each strain contained a point mutation resulting in the premature termination of the thymidine kinase (TK) open reading frame (tdk). In vitro translation of the mutant tdk coding regions resulted in synthesis of truncated TK polypeptides deficient in TK activity.