Expression and characterization of functional single-chain variable fragment against norfloxacin in Pichia pastoris GS115

J. Mala, S. Puthong, H. Maekawa, Y. Kaneko, T. Palaga, K. Komolpis, S. Sooksai

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2 Citations (Scopus)

Abstract

A functional single-chain variable fragment (scFv) of mouse monoclonal antibody against norfloxacin was expressed in yeast Pichia pastoris GS115, purified and characterized. Gene encoding monoclonal antibody against norfloxacin was transformed to P. pastoris GS115 using the pJM01 plasmid. Integration of the plasmid into the genome was verified by PCR and DNA sequencing analysis. After screening for the transformant and production of the selected scFv, the norfloxacin-binding ability was determined by an enzyme linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) analysis. Eight transformats (O1-O8) were obtained from the transformation of P. pastoris GS115 with PmeI-linearized pJM01. PCR products detected by gel electrophoresis confirmed that pJM01 expression cassette was integrated at AOX1 promoter. After screening all transformants for high expression of the recombinant scFv, transformant O5 was selected for antibody production and purification. Detection of norfloxacin by ELISA with the recombinant scFv fragment was successful albeit with lower sensitivity compared to the original monoclonal antibody. In SPR analysis, our recombinant scFv fragments have the binding capability to norfloxacin equivalent to the original antibody with the average angle shift of 580 ± 133 and 505 ± 28 mDegree, respectively. The functional recombinant scFv of monoclonal antibody against norfloxacin successfully produced by methylotrophic yeast, P. pastoris GS115.

Original languageEnglish
Pages (from-to)1726-1732
Number of pages7
JournalInternational Food Research Journal
Volume25
Issue number4
Publication statusPublished - Jan 1 2018
Externally publishedYes

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All Science Journal Classification (ASJC) codes

  • Food Science

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