Construction and expression of specificity-improved single-chain variable fragments against the bioactive naphthoquinone, plumbagin

Seiichi Sakamoto, Futoshi Taura, Waraporn Putalun, Benyakan Pongkitwitoon, Ryota Tsuchihashi, Satoshi Morimoto, Junei Kinjo, Yukihiro Shoyama, Hiroyuki Tanaka

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

We constructed a single-chain variable fragment (scFv) antibody against plumbagin (PL) with improved specific binding to PL. Variable heavy- and light-chain genes were cloned directly from the cDNA of hybridoma cell line 3A3 and assembled using the splice-overlap extension polymerase chain reaction (SOE-PCR) with specific primers including flexible peptide (Gly 4Ser)3 linker primers. The constructed scFv gene was ligated into the pET28a expression vector and transformed into Escherichia coli BL21 (DE3). The denatured protein expressed as inclusion bodies in E. coli was solubilized, purified, and refolded by a stepwise dialysis. Intriguingly, the refolded scFv against PL displayed higher PL-binding specificity than that of its parental monoclonal antibody, MAb 3A3, which suggests the possibility of improving the function by constructing the scFv antibody. These notable properties of the recombinant antibody against PL made it possible to develop an enzyme-linked immunosorbent assay (ELISA) for reliable determination of PL.

Original languageEnglish
Pages (from-to)434-439
Number of pages6
JournalBiological and Pharmaceutical Bulletin
Volume32
Issue number3
DOIs
Publication statusPublished - Mar 1 2009

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Naphthoquinones
Single-Chain Antibodies
Escherichia coli
Inclusion Bodies
Hybridomas
Genes
plumbagin
Dialysis
Complementary DNA
Enzyme-Linked Immunosorbent Assay
Monoclonal Antibodies
Light
Cell Line
Polymerase Chain Reaction
Peptides
Antibodies
Proteins

All Science Journal Classification (ASJC) codes

  • Pharmacology
  • Pharmaceutical Science

Cite this

Construction and expression of specificity-improved single-chain variable fragments against the bioactive naphthoquinone, plumbagin. / Sakamoto, Seiichi; Taura, Futoshi; Putalun, Waraporn; Pongkitwitoon, Benyakan; Tsuchihashi, Ryota; Morimoto, Satoshi; Kinjo, Junei; Shoyama, Yukihiro; Tanaka, Hiroyuki.

In: Biological and Pharmaceutical Bulletin, Vol. 32, No. 3, 01.03.2009, p. 434-439.

Research output: Contribution to journalArticle

Sakamoto, Seiichi ; Taura, Futoshi ; Putalun, Waraporn ; Pongkitwitoon, Benyakan ; Tsuchihashi, Ryota ; Morimoto, Satoshi ; Kinjo, Junei ; Shoyama, Yukihiro ; Tanaka, Hiroyuki. / Construction and expression of specificity-improved single-chain variable fragments against the bioactive naphthoquinone, plumbagin. In: Biological and Pharmaceutical Bulletin. 2009 ; Vol. 32, No. 3. pp. 434-439.
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