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

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

研究成果: ジャーナルへの寄稿記事

2 引用 (Scopus)

抄録

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.

元の言語英語
ページ(範囲)1726-1732
ページ数7
ジャーナルInternational Food Research Journal
25
発行部数4
出版物ステータス出版済み - 1 1 2018
外部発表Yes

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norfloxacin
Single-Chain Antibodies
Norfloxacin
Pichia pastoris
Pichia
monoclonal antibodies
Monoclonal Antibodies
surface plasmon resonance
Surface Plasmon Resonance
plasmids
Plasmids
Yeasts
Enzyme-Linked Immunosorbent Assay
enzyme-linked immunosorbent assay
yeasts
recombinant antibodies
screening
Polymerase Chain Reaction
binding capacity
antibody formation

All Science Journal Classification (ASJC) codes

  • Food Science

これを引用

Expression and characterization of functional single-chain variable fragment against norfloxacin in Pichia pastoris GS115. / Mala, J.; Puthong, S.; Maekawa, Hiromi; Kaneko, Y.; Palaga, T.; Komolpis, K.; Sooksai, S.

:: International Food Research Journal, 巻 25, 番号 4, 01.01.2018, p. 1726-1732.

研究成果: ジャーナルへの寄稿記事

Mala, J, Puthong, S, Maekawa, H, Kaneko, Y, Palaga, T, Komolpis, K & Sooksai, S 2018, 'Expression and characterization of functional single-chain variable fragment against norfloxacin in Pichia pastoris GS115', International Food Research Journal, 巻. 25, 番号 4, pp. 1726-1732.
Mala, J. ; Puthong, S. ; Maekawa, Hiromi ; Kaneko, Y. ; Palaga, T. ; Komolpis, K. ; Sooksai, S. / Expression and characterization of functional single-chain variable fragment against norfloxacin in Pichia pastoris GS115. :: International Food Research Journal. 2018 ; 巻 25, 番号 4. pp. 1726-1732.
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AU - Mala, J.

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AU - Palaga, T.

AU - Komolpis, K.

AU - Sooksai, S.

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