Purification, crystallization and preliminary X-ray diffraction studies of recombinant class A non-specific acid phosphatase ofSalmonella typhimurium

Abstract

The phoN gene of Salmonella enterica sv. Typhimurium strain MD6001 was cloned in the multicopy plasmid pBluescript SK(-). The nucleotide sequence of the cloned gene differs from the corresponding S. typhimurium LT2 sequence at 23 residues, leading to 15 amino-acid differences, but was very close to the S. typhi phoN sequence (only three nucleotide and two amino-acid differences). The recombinant PhoN protein was purified to homogeneity. Two forms of crystals were harvested from a single crystallization condition. Diffraction intensity data were collected using a laboratory X-ray source to resolution limits of 2.5 and 2.8 A for crystals belonging to space group C2 and C222(1), respectively. Based on non-crystallographic symmetry, four monomers of PhoN are expected to be present in the asymmetric unit of the C2 unit cell. Two monomers of a biologically active dimer in the asymmetric unit of the C222(1) unit cell are expected from the Matthews coefficient.

Structure and Mutational Analysis of the PhoN Protein of Salmonella typhimurium Provide Insight into Mechanistic Details

“…Quaternary Structure. The PhoN crystals show phosphatase activity when soaked with a biochemical substrate like pNPP (19). In the crystallographic subunit, four copies of PhoN monomers form two independent dimers.…”

“…The mutant plasmids were subsequently transformed into the expression host individually for the overproduction of proteins. The proteins were expressed and purified to homogeneity as described earlier ( , ). The purified proteins were characterized by N-terminal amino acid sequencing and molecular mass determination by MALDI-TOF.…”

“…Crystallization and Data Collection. The recombinant purified PhoN protein crystallized in two forms with space groups C2 and C222 1 , respectively (19). Briefly, the native PhoN crystals were grown by the vapor diffusion method using crystallization condition (12% PEG 6000 and 2% glycerol) in the presence of 25 mM sodium phosphate.…”

Expression, purification, crystallization and preliminary X-ray diffraction studies of recombinant class B non-specific acid phosphatase ofSalmonella typhimurium

“…The pathogenic S. typhimurium strain MD6001 obtained from the Central Research Institute (CRI), Kasuali, India (CRI No. 5710) had previously been identi®ed in our laboratory based on the 16S and 23S rRNA gene sequences (Makde et al, 2003). The primers Apha3 (5 H -CCG-GCCTGACAATAGCTGTTGTGAT-3 H ) and Apha4 (5 H -CGTCTCCTGAATAGTGTGCA-GCAAG-3 H ), designed on the basis of the published sequence of the aphA gene and its anking regions in the S. typhimurium LT2 genome (McClelland et al, 2001; GenBank accession Nos.…”

“…The ligation mixture was used to transform the DH5 strain of E. coli and the transformants were selected on LB plates supplemented with ampicillin, X-Gal and IPTG. The white recombinant clones were monitored on phosphatase-indicator plates as described in Makde et al (2003). The phosphatasepositive clones were checked for the proper inserts by PCR and restriction-enzyme analysis.…”