ÖZLEM BÜYÜKTANIR YAŞ, OKTAY GENÇ, Nevzat YURDUSEV

Production, purification and characterization of the recombinant Brucella abortus rP17 protein

Sitozolik immunoreaktif B. abortus P17 proteini, pCold I soğuk-şok ekspresyon vektörüne p17 geni klonlanarak Escherichia coli’de 6xHistidin kuyruklu rekombinant protein (rP17) şeklinde üretildi. Klonlanan p17 geninin DNA sekans analizi, rekombinant rP17 proteininin 83 hidrofobik, 42 hidrofilik, 35 bazik ve 21 asidik olmak üzere 181 amino asitten oluştuğunu gösterdi. Teorik olarak izoelektrik noktası 6.42 olarak belirlendi. -0.097 olan GRAVY indeksi proteinin eriyebilir olduğuna işaret etmektedir. İnstabilite indeksi, IPTG ile indüklenerek transforme E. coli hücrelerinde eksprese edilen rP17 proteininin stabil olduğunu göstermektedir. İndüklenmiş ve indüklenmemiş bakteri lizatlarının SDS-PAGE analizleri göreceli moleküler ağırlığı 24 kDa olan rP17 proteininin ekspresyonunu göstermektedir. rP17 proteini, Ni-NTA affinite kromatografisi ve SDS-PAGE sonrası poliakrilamid jelden elusyonu içeren iki aşamada saflaştırıldı ve Western blot yöntemi ile incelendi. Ön sonuçlar rekombinant rP17 proteininin immün reaktifliğini koruduğunu göstermiştir. Bu aşamada, tanımlanmış serumlardan oluşan kapsamlı bir koleksiyon ile diyagnostik performansının değerlendirilmesine yönelik olarak rP17 proteininin büyük ölçek üretimi gerçekleştirilmektedir.

Anahtar Kelimeler:

genler, Brucella, Brucella abortus, saflaştırma, bakteriyal protein

Rekombinant Brucella abortus rP17 proteininin üretilmesi, saflaştırılması ve karakterizasyonu

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.

Keywords:

genes, Brucella, Brucella abortus, purification, bacterial protein,

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