Purification and in vitro characterization of the maltose-binding protein of the plant pathogen Xanthomonas citri.
The uptake of maltose and maltodextrins in gram-negative bacteria is mediated by an ATP-dependent transport complex composed of a periplasmic maltose-binding protein (MBP) and membrane-associated proteins responsible for the formation of a membrane pore and generation of energy to drive the translocation process. In this work, we report the purification and in vitro functional analysis of MBP, encoded by the malE gene, of the plant pathogen Xanthomonas citri, responsible for the canker disease affecting citrus plants throughout the world. The X. citri MBP is composed of 456 amino acids, displaying a low amino acid identity (16% throughout the sequence) compared to the Escherichia coli K12 ortholog. The X. citri malE gene was cloned into a pET28a vector, and the encoded protein was expressed and purified by affinity chromatography as a His-tag N-terminal fusion peptide produced by the E. coli BL21 strain. Enhanced levels of soluble protein were achieved with static cultures kept overnight at 23 degrees C. Ability to bind immobilized amylose, the emission of intrinsic fluorescence and circular dichroism spectra indicated that the purified recombinant protein preserved both conformation and biological activity of the native protein. The availability of the recombinant MBP will contribute to the functional and structural analysis of the maltose and maltodextrin uptake system of the plant pathogen X. citri.
Balan
Andrea
A
Departamento de Microbiologia, Instituto de Ciências Biomédicas II, Universidade de São Paulo, CEP 05508-000, Cidade Universitária, SP, Brazil.
de Souza
Cristiane Santos
CS
Moutran
Alexandre
A
Ferreira
Rita C Café
RC
Franco
Christian Suarez
CS
Ramos
Carlos Henrique I
CH
de Souza Ferreira
Luís Carlos
LC
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D048168:Escherichia coli K12 / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D010944:Plants / Q000382:microbiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014974:Xanthomonas / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity
D001426:Bacterial Proteins; D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins