Production, Purification and Characterization of the Recombinant Brucella abortus rP17 Protein

Büyüktanır Yaş Ö., Genç O., Yurdusev N.

KAFKAS UNIVERSITESI VETERINER FAKULTESI DERGISI, cilt.17, sa.1, ss.135-140, 2011 (SCI-Expanded) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 17 Sayı: 1
  • Basım Tarihi: 2011
  • Dergi Adı: KAFKAS UNIVERSITESI VETERINER FAKULTESI DERGISI
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, TR DİZİN (ULAKBİM)
  • Sayfa Sayıları: ss.135-140
  • Anahtar Kelimeler: Brucella abortus, Bacterial expression, Purification, Recombinant P17 protein, LINKED-IMMUNOSORBENT-ASSAY, HUMORAL IMMUNE-RESPONSES, BOVINE BRUCELLOSIS, MONOCLONAL-ANTIBODIES, PERIPLASMIC PROTEIN, IMMUNOBLOT ANALYSIS, CELL-ENVELOPE, DIAGNOSIS, ANTIGEN, CLONING
  • Ondokuz Mayıs Üniversitesi Adresli: Evet

Özet

Immunoreactive cytosolic P17 protein of Brucella abortus was produced in Escherichia coli as 6xHistidine tagged recombinant protein (rP17) by cloning the p17 gene into pColdI cold-shock expression vector. DNA sequence analysis of the cloned 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 by inducing with IPTG. Lysate of the induced and non-induced bacteria was analyzed by SDS-PAGE showing expression of the rP17 with a relative molecular weight of 24 kDa. After two-step purification procedure, Ni-NTA affinity chromatography and elution from polyacrylamide gels following SDS-PAGE, the rP17 was highly purified and analyzed by Western blot. Preliminary results 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.