Expression, purification, and characterization of anti-plumbagin single-chain variable fragment antibody in sf9 insect cell

Seiichi Sakamoto, Futoshi Taura, Ryota Tsuchihashi, Waraporn Putalun, Junei Kinjo, Hiroyuki Tanaka, Satoshi Morimoto

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Plumbagin (PL; 5-hydroxy-2-methyl-1, 4-naphthoquinone) is an important secondary metabolite, mainly produced in the Plumbago zeylanica L. (Plumbaginaceae). A single-chain variable fragment (scFv) antibody, fusion of the variable regions of the heavy chain and light chain of immunogloblin against PL (PL-scFv) was expressed by Bac-to-Bac Baculovirus Expression System using Spodoptera frugiperda (Sf9) insect cells and characterized to investigate potential use of PL-scFv as a tool for plant immunomodulation. Functional PL-scFv expressed in the Sf9 insect cells were purified using cation exchange chromatography followed by immobilized metal ion affinity chromatography (IMAC). The yields of the purified PL-scFv in the culture supernatant and Sf9 insect cells were 2.0 mg and 5.2 mg per 1 liter of Sf9 culture medium, respectively. Recombinant purified PL-scFv was then characterized by the indirect competitive enzyme-linked immunosorbent assay (ELISA). The cross-reactivity and sensitivity of PL-scFv expressed in Sf9 insect cells were compared with PL-scFv expressed in Escherichia coli and its parental anti-plumbagin monoclonal antibody (MAb 3A3) secreted from hybridoma cells. Intriguingly, the specificity of the PL-scFv expressed in Sf9 insect cells was found to be different from that expressed in E. coli and parental MAb 3A3, although the detectable level (0.2-25 μg/mL) was the same in ELISA using each antibody. Even more interestingly, the characteristics of PL-scFv, which have wide cross-reactivity against 1,4-napththoquinone, suggest its potential use as a tool for plant immunomodulation not only for breeding Plumbaginacea family containing PL but also for breeding other medicinal plants containing bioactive naphthoquinones.

Original languageEnglish
Pages (from-to)481-488
Number of pages8
JournalHybridoma
Volume29
Issue number6
DOIs
Publication statusPublished - Dec 1 2010

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Sf9 Cells
Single-Chain Antibodies
Insects
Plumbaginaceae
Immunomodulation
Breeding
Enzyme-Linked Immunosorbent Assay
Escherichia coli
Naphthoquinones
Spodoptera
Baculoviridae
Hybridomas
Medicinal Plants
Affinity Chromatography
Culture Media
Cations
Chromatography
Metals
Monoclonal Antibodies
plumbagin

All Science Journal Classification (ASJC) codes

  • Immunology
  • Immunology and Allergy

Cite this

Expression, purification, and characterization of anti-plumbagin single-chain variable fragment antibody in sf9 insect cell. / Sakamoto, Seiichi; Taura, Futoshi; Tsuchihashi, Ryota; Putalun, Waraporn; Kinjo, Junei; Tanaka, Hiroyuki; Morimoto, Satoshi.

In: Hybridoma, Vol. 29, No. 6, 01.12.2010, p. 481-488.

Research output: Contribution to journalArticle

Sakamoto, Seiichi ; Taura, Futoshi ; Tsuchihashi, Ryota ; Putalun, Waraporn ; Kinjo, Junei ; Tanaka, Hiroyuki ; Morimoto, Satoshi. / Expression, purification, and characterization of anti-plumbagin single-chain variable fragment antibody in sf9 insect cell. In: Hybridoma. 2010 ; Vol. 29, No. 6. pp. 481-488.
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