Purification and in vitro characterization of the maltose-binding protein of the plant pathogen Xanthomonas citri

Protein Expr Purif. 2005 Oct;43(2):103-10. doi: 10.1016/j.pep.2005.03.018. Epub 2005 Apr 9.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification*
  • Bacterial Proteins / metabolism
  • Carrier Proteins / chemistry*
  • Carrier Proteins / genetics
  • Carrier Proteins / isolation & purification*
  • Carrier Proteins / metabolism
  • Escherichia coli K12 / genetics
  • Escherichia coli K12 / metabolism
  • Gene Expression
  • Maltose-Binding Proteins
  • Molecular Sequence Data
  • Plants / microbiology*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Xanthomonas* / genetics
  • Xanthomonas* / metabolism
  • Xanthomonas* / pathogenicity

Substances

  • Bacterial Proteins
  • Carrier Proteins
  • Maltose-Binding Proteins
  • Recombinant Fusion Proteins