Year: 2021 | Month: August | Volume 11 | Issue 4
Expression and Purification of Exposed Outer Domain of Pseudomonas aeruginosa Opr86 protein
Niraj K. Singh
Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen that can form a biofilm that provides crucial drug resistance components. The bacteria have many immunogenic outer membrane proteins (OMP) including, Opr86. The Opr86 is essential for the viability of bacterium, as it has a significant role in OMP assembly and its depletion leads to alteration of cellular morphology. Therefore, Opr86 may serve as a vaccine candidate. Moreover, Opr86 may be used for raising hyperimmune serum, which may be a potential therapeutic candidate in P. aeruginosa infection. In the current study, gene fragment encoded exposed outer domain toward extracellular side of Opr86 (E-Opr86) was amplified by polymerase chain reaction (PCR), cloned into pJET1.2 vector, and sequenced. The gene was inserted into pET302/NT-His vector and was expressed in Rosetta-gami 2(DE3) pLysS cells. The recombinant E-Opr86 protein expression was confirmed by the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and subjected to purification using His-tag affinity chromatography. The purified protein (~18kDa) was confirmed by SDS–PAGE and by the Western blotting. This study successfully cloned and characterized Opr86 protein of P. aeruginosa.
- Gene encoding exposed outer domain of Pseudomonas aeruginosa Opr86 (E-Opr86) protein was cloned and sequenced.
- Recombinant E-Opr86 protein of approximetlt 18kDa size was expressed and purifesd using bacterial expression system.
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