title: [Protein interaction site of Toxoplasma gondii microneme protein 6 and aldolase determined by site-directed mutagenesis].
abstract: OBJECTIVE
  To identify the protein interaction site of Toxoplasma gondii microneme protein 6 (MIC6) and aldolase by using site-directed mutagenesis.
  
  
  METHODS
  Based on Toxoplasma gondii MIC6 gene sequence (GenBank Accession No. AF110270), the specific primers were designed. Tryptophan (W)-348 of MIC6 C terminus (MIC6C) was mutated to valine (V) via site-directed mutagenesis. MIC6C W/V gene was obtained from cDNA library by PCR amplification and subcloned into pGEX-4T-1. The mutant protein GST-MIC6C W/V was expressed in E. coli, induced by 0.8 mmol/L IPTG, and purified by affinity chromatography. Glutathione sepharose beads were incubated with GST-MIC6C W/V and GST-MIC6C, respectively, and then incubated with T. gondii tachyzoites lysate, and bound proteins were eluted using sample buffer. Bound products were resolved by SDS-PAGE and Western blotting. Glutathione sepharose beads were incubated with GST-MIC6C W/V and GST-MIC6C, respectively, and then incubated with aldolase-His6. After incubation, the resin was washed and subjected to SDS-PAGE.
  
  
  RESULTS
  The MIC6C W/N gene was obtained, and the recombinant plasmid MIC6C W/V/pGEX-4T-1 was successfully constructed. The mutant protein GST-MIC6C W/V was expressed and purified in vitro. SDS-PAGE analysis indicated that GST-MIC6C was co-precipitated with aldolase from T. gondii tachyzoites lysate or aldolase-His6, whereas GST-MIC6C W/V failed to precipitate aldolase from T. gondii tachyzoites lysate or aldolase-His6. Western blotting analysis using anti-aldolase antibody indicated that GST-MIC6C could pull-down aldolase from T. gondii tachyzoites lysate.
  
  
  CONCLUSIONS
  Tryptophan (W348) was the interaction site of MIC6 and aldolase in T. gondii.
authors.forename: Bin
authors.initials: B
authors.lastname: Zheng
authors.forename: Zhi-Kui
authors.initials: ZK
authors.lastname: Yin
authors.forename: Xi-Mei
authors.initials: XM
authors.lastname: Zhan
mesh_terms: D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005634:Fructose-Bisphosphate Aldolase / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D054730:Protein Interaction Domains and Motifs; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D014122:Toxoplasma / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins; D005634:Fructose-Bisphosphate Aldolase