Production, Purification and Characterization of the Recombinant Brucella Abortus Rp17 Protein

Abstract

Immunoreactive cytosolic P17 protein of Brucella abortus was produced in Escherichia coli as 6xHistidine taggedrecombinant protein (rP17) by cloning the p17 gene into pColdI cold-shock expression vector. DNA sequence analysis of thecloned p17 gene showed that the recombinant rP17 protein contains a total of 181 amino acids constituted of 83 hydrophobic,42 hydrophilic, 35 basic and 21 acidic residues. Its theoretical isoelectric point was calculated as 6.42 and GRAVY index of -0.097 indicates its solubility. The instability index classifies the rP17 as a stable protein expressed in the transformed E. coli cells byinducing with IPTG. Lysate of the induced and non-induced bacteria was analyzed by SDS-PAGE showing expression of therP17 with a relative molecular weight of 24 kDa. After two-step purification procedure, Ni-NTA affinity chromatography andelution from polyacrylamide gels following SDS-PAGE, the rP17 was highly purified and analyzed by Western blot. Preliminaryresults showed that the recombinant rP17 protein still preserves its immunoreactivity. In present, large scale production of the rP17 is carried out for evaluation of its diagnostic performance with a large panel of well-defined sera.