RESUMEN
Penicillin-binding protein 3 (PBP3) of Pseudomonas aeruginosa is the primary target of ß-lactams used to treat pseudomonas infections. Meanwhile, structure change and overproduction of PBP3 play important roles in the drug resistance of P. aeruginosa. Therefore, studies on the gene and structure of PBP3 are urgently needed. P. aeruginosa CMCC 10104 is a type culture strain common used in China. However, there is no report on its genomic and proteomic profiles. In this study, based on ftsI of P. aeruginosa PAO1, the gene encoding PBP3 was cloned from CMCC 10104. A truncated version of the ftsI gene, omitting the bases encoding the hydrophobic leader peptide (amino acids 1-34), was amplified by PCR. The cloned DNA shared 99.76% identity with ftsI from PAO1. Only four bases were different (66 C-A, 1020 T-C, 1233 T-C, and 1527 T-C). However, there were no differences between their deduced amino acid sequences. The recombinant PBP3 (rPBP3), containing a 6-histidine tag, was expressed in Escherichia coli BL21 (DE3). Immobilized metal affinity chromatography (IMAC) with Ni(2+)-NTA agarose was used for its purification. The purified rPBP3 was identified by SDS-PAGE and western blot analysis, and showed a single band at about 60kDa with purity higher than 95%. The penicillin-binding assay indicated that the obtained rPBP3 was functional and not hindered by the presence of the C-terminal His-tag. The protocol described in this study offers a method for obtaining purified recombinant PBP3 from P. aeruginosa CMCC 10104.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Proteínas de Unión a las Penicilinas/aislamiento & purificación , Pseudomonas aeruginosa/química , Eliminación de Secuencia , Secuencia de Aminoácidos , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Datos de Secuencia Molecular , Proteínas de Unión a las Penicilinas/biosíntesis , Proteínas de Unión a las Penicilinas/genética , Penicilinas/química , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Señales de Clasificación de Proteína/genética , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Resistencia betalactámica/genéticaRESUMEN
The urban cooling island (UCI) effect is put forward in comparison with the urban heat island effect, and emphasizes on landscape planning for optimization of function and way of urban thermal environment. In this paper, we summarized current research of the UCI effects of waters, green space, and urban park from the perspective of patch area, landscape index, threshold value, landscape pattern and correlation analyses. Great controversy was found on which of the two factors patch area and shape index has a more significant impact, the quantification of UCI threshold is particularly lacking, and attention was paid too much on the UCI effect of landscape composition but little on that of landscape configuration. More attention should be paid on shape, width and location for water landscape, and on the type of green space, green area, configuration and management for green space landscape. The altitude of urban park and human activities could also influence UCI effect. In the future, the threshold determination should dominate the research of UCI effect, the reasons of controversy should be further explored, the study of time sequence should be strengthened, the UCI effects from landscape pattern and landscape configuration should be identified, and more attention should be paid to spatial scale and resolution for the precision and accuracy of the UCI results. Also, synthesizing the multidisciplinary research should be taken into consideration.