RESUMEN
To explore the physiological role of tetraheme cytochrome c(3) in the sulfate-reducing bacterium Desulfovibrio desulfuricans G20, the gene encoding the preapoprotein was cloned, sequenced, and mutated by plasmid insertion. The physical analysis of the DNA from the strain carrying the integrated plasmid showed that the insertion was successful. The growth rate of the mutant on lactate with sulfate was comparable to that of the wild type; however, mutant cultures did not achieve the same cell densities. Pyruvate, the oxidation product of lactate, served as a poor electron source for the mutant. Unexpectedly, the mutant was able to grow on hydrogen-sulfate medium. These data support a role for tetraheme cytochrome c(3) in the electron transport pathway from pyruvate to sulfate or sulfite in D. desulfuricans G20.
Asunto(s)
Grupo Citocromo c/genética , Grupo Citocromo c/metabolismo , Desulfovibrio/genética , Desulfovibrio/metabolismo , Mutación , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Conjugación Genética , Desulfovibrio/crecimiento & desarrollo , Datos de Secuencia Molecular , Periplasma/metabolismo , Plásmidos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Transcripción GenéticaRESUMEN
This study examined the global stability and activity properties of recombinant DNA-binding antibody fragments that were obtained from a bacteriophage combinatorial display library. The goal of this study was to determine whether the combinatorial approach of heavy and light chain assembly in E. coli and subsequent affinity selection preferentially selects for antibody fragments with unusual structural stabilities. Specifically, the binding properties and stability of recombinant antibody fragments with or without a C-terminal His tag to temperature, pH, and guanidine-HCI were examined. Both Fab exhibited almost identical Kd (120-130, 140-170, and 450-560 nM) and maximal fluorescence quenching (20-25%) values for binding to (dT)20, (dT)15, and (dT)10, respectively. Thermal denaturation data obtained by CD spectroscopy demonstrated that both Fab possessed structural properties comparable to well-folded proteins with defined tertiary structures which were stable below 70 degrees C (Tm 73 degrees C). These results were confirmed by differential scanning calorimetry. Both Fab exhibited the same rate of irreversible thermal inactivation (0.061-0.069 min-1) at 75 degrees C and could be reversibly renatured from guanidine-HCI and pH extremes. Crystallization trials with one recombinant DNA-binding Fab yielded diffraction quality crystals also suggesting a well-defined tertiary structure.