Efficient construction of a diabody using a refolding system: anti-carcinoembryonic antigen recombinant antibody fragment.

Efficient construction of a diabody using a refolding system: anti-carcinoembryonic antigen recombinant antibody fragment. Research Abstract Details 

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Efficient construction of a diabody using a refolding system: anti-carcinoembryonic antigen recombinant antibody fragment. Abstract Text:

Ryutaro Asano,Toshio Kudo,Yukihiro Nishimura,Koki Makabe,Hiroki Hayashi,Masanori Suzuki,Kouhei Tsumoto,Izumi Kumagai,

Recombinant fragments of the variable region of antibodies are useful in many experimental and clinical applications. However, it can be difficult to obtain these materials in soluble form after their expression in bacteria. Here, we report an efficient procedure for preparing several variable-domain fragments (Fv), single-chain Fv (scFv), and a diabody (the smallest functional bispecific antibody) of anti-carcinoembryonic antigen (CEA) antibody by overexpression in Escherichia coli in inclusion bodies, using a refolding system to obtain renatured proteins. Two types of refolded Fv were prepared: (i) Heavy and light chains of the immunoglobulin variable regions (VH and VL, respectively) were coexpressed with a dicistronic expression vector (designated Fv(co)); (ii) VH and VL were expressed separately, mixed stoichiometrically, and refolded (designated Fv(mix)). All samples refolded with high efficiency; Fv(co), Fv(mix), scFv, and the bispecific diabody bound to several CEA-positive cell lines, exactly as did soluble Fv fragments secreted by E. coli (Fv(sol)) and the parent IgG. The refolded fragments inhibited binding of the parent IgG to CEA-positive cell lines, indicating that their epitope is identical to that of IgG. The bispecific diabody, which combined variable-region fragments of anti-CEA antibody with variable-region fragments of anti-CD3 antibody, was also prepared using the refolding system. This refolded diabody could bind to lymphokine-activated killer cells. In addition, its cytotoxicity toward human bile duct carcinoma TFK-1 and other several other CEA-positive cell lines was concentration-dependent. Taken together, our results suggest that a refolding procedure can be used to prepare various functional antibody fragments (Fv, scFv, and diabody).

Efficient construction of a diabody using a refolding system: anti-carcinoembryonic antigen recombinant antibody fragment. Publishing Authors By Initials

R Asano,T Kudo,Y Nishimura,K Makabe,H Hayashi,M Suzuki,K Tsumoto,I Kumagai,

For similar proteins: recombinant proteins research abstracts see: proteins: recombinant proteins research

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Efficient construction of a diabody using a refolding system: anti-carcinoembryonic antigen recombinant antibody fragment. Journal Published:

PUBLICATION TYPE: Journal Article

Journal: Journal of biochemistry

VOLUME: 132

Page Numbers: 903-9

Journal Abbreviation: J. Biochem.

ISSN: 0021-924X

DAY: 19

MONTH: Dec

YEAR: 2002

Efficient construction of a diabody using a refolding system: anti-carcinoembryonic antigen recombinant antibody fragment. Information

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LANGUAGE: eng

NlmUniqueID: 376600

Efficient construction of a diabody using a refolding system: anti-carcinoembryonic antigen recombinant antibody fragment. Keywords Mesh Terms:

KEYWORDS: Recombinant Proteins

MESH TERMS: metabolism

Chemical & Substance for Abstract: Efficient construction of a diabody using a refolding system: anti-carcinoembryonic antigen recombinant antibody fragment. Information

Substance Name: Recombinant Proteins

Registry Number: 0

Grant and Affiliation Information for Efficient construction of a diabody using a refolding system: anti-carcinoembryonic antigen recombinant antibody fragment.

AFFILIATION: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-ku, Sendai 980-8579, Japan.

Country: Japan

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MEDLINETA: J Biochem

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