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Production and characterisation of a recombinant scFv reactive with human gastrointestinal carcinomas.

Kim DJ, Chung JH, Ryu YS, Rhim JH, Kim CW, Suh Y, Chung HK - Br. J. Cancer (2002)

Bottom Line: The use of the polymerase chain reaction, expression cloning technology and gene expression systems in E. coli has led to the production of SC142 single-chain fragments, which was similar in activity to the SC142 parent antibody confirmed by immunohistochemistry.Competitive ELISA showed that SC142 single-chain fragments originated from parent SC142 antibody.These results illustrate the potential application of these novel products as an immunodiagnostic and further immunotherapeutic reagent.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Cancer Research Institute, Seoul National University College of Medicine, Seoul, 110-799, Korea.

ABSTRACT
SC142-reactive antigen are highly glycosylated glycoproteins expressed on tissues of gastric and colon cancers but not on normal tissues. Murine SC142 antibody specific for the SC142-reactive antigen has been produced by immunisation with SNU16 stomach cancer cells. However, SC142 antibody has several potential problems such as high immunogenicity and poor tumour penetration owing to their large size. To improve tumour penetration potential in vivo, recombinant single-chain fragments have been produced using the original hybridoma cells as a source of variable heavy- and variable light-chain-encoding antibody genes. The use of the polymerase chain reaction, expression cloning technology and gene expression systems in E. coli has led to the production of SC142 single-chain fragments, which was similar in activity to the SC142 parent antibody confirmed by immunohistochemistry. Analysis by DNA sequencing, SDS-PAGE and Western blotting has demonstrated the integrity of the single-chain fragments. Competitive ELISA showed that SC142 single-chain fragments originated from parent SC142 antibody. BIAcore biosensor binding experiments showed that the SC142 single-chain fragments had an ideal dissociation rate constant as a tumour imaging reagent. These results illustrate the potential application of these novel products as an immunodiagnostic and further immunotherapeutic reagent.

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Related in: MedlinePlus

SDS–PAGE and Western blot analysis of purified SC142 scFv. Left panel of (A) is Coomassie blue stained SDS–PAGE and right panel of (A) is Western blot analysis of E. coli BL21 (DE3) cells overproducing SC142 scFv probed with anti-Xpress antibody. Lanes 1 and 3 are whole cell lysate and lane 2 and 4 are purified SC142 scFv. (B) chromatogram of molecular standards; (C) chromatogram of the purified and refolded SC142 scFv.
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fig3: SDS–PAGE and Western blot analysis of purified SC142 scFv. Left panel of (A) is Coomassie blue stained SDS–PAGE and right panel of (A) is Western blot analysis of E. coli BL21 (DE3) cells overproducing SC142 scFv probed with anti-Xpress antibody. Lanes 1 and 3 are whole cell lysate and lane 2 and 4 are purified SC142 scFv. (B) chromatogram of molecular standards; (C) chromatogram of the purified and refolded SC142 scFv.

Mentions: For this additional characterisation of SC142 scFv, we subcloned the scFv gene fragments into the pRSET SfiI/NotI vector, transformed it into E. coli BL21 cells, and produced SC142 scFv. Preliminary studies were performed in which whole-cell extracts, periplasmic extracts, and supernatants from cultures of clones were tested for the presence of scFv by SDS–PAGE and Western blotting. All of the clones expressed insoluble recombinant scFv in sufficient quantities to be revealed in the inclusion bodies by Western blotting (data not shown). Inclusion bodies were solubilised and renatured as described in Materials and Methods. Appropriate fractions were further analysed by SDS–PAGE and Western blotting. Figure 3AFigure 3


Production and characterisation of a recombinant scFv reactive with human gastrointestinal carcinomas.

Kim DJ, Chung JH, Ryu YS, Rhim JH, Kim CW, Suh Y, Chung HK - Br. J. Cancer (2002)

SDS–PAGE and Western blot analysis of purified SC142 scFv. Left panel of (A) is Coomassie blue stained SDS–PAGE and right panel of (A) is Western blot analysis of E. coli BL21 (DE3) cells overproducing SC142 scFv probed with anti-Xpress antibody. Lanes 1 and 3 are whole cell lysate and lane 2 and 4 are purified SC142 scFv. (B) chromatogram of molecular standards; (C) chromatogram of the purified and refolded SC142 scFv.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2376138&req=5

fig3: SDS–PAGE and Western blot analysis of purified SC142 scFv. Left panel of (A) is Coomassie blue stained SDS–PAGE and right panel of (A) is Western blot analysis of E. coli BL21 (DE3) cells overproducing SC142 scFv probed with anti-Xpress antibody. Lanes 1 and 3 are whole cell lysate and lane 2 and 4 are purified SC142 scFv. (B) chromatogram of molecular standards; (C) chromatogram of the purified and refolded SC142 scFv.
Mentions: For this additional characterisation of SC142 scFv, we subcloned the scFv gene fragments into the pRSET SfiI/NotI vector, transformed it into E. coli BL21 cells, and produced SC142 scFv. Preliminary studies were performed in which whole-cell extracts, periplasmic extracts, and supernatants from cultures of clones were tested for the presence of scFv by SDS–PAGE and Western blotting. All of the clones expressed insoluble recombinant scFv in sufficient quantities to be revealed in the inclusion bodies by Western blotting (data not shown). Inclusion bodies were solubilised and renatured as described in Materials and Methods. Appropriate fractions were further analysed by SDS–PAGE and Western blotting. Figure 3AFigure 3

Bottom Line: The use of the polymerase chain reaction, expression cloning technology and gene expression systems in E. coli has led to the production of SC142 single-chain fragments, which was similar in activity to the SC142 parent antibody confirmed by immunohistochemistry.Competitive ELISA showed that SC142 single-chain fragments originated from parent SC142 antibody.These results illustrate the potential application of these novel products as an immunodiagnostic and further immunotherapeutic reagent.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Cancer Research Institute, Seoul National University College of Medicine, Seoul, 110-799, Korea.

ABSTRACT
SC142-reactive antigen are highly glycosylated glycoproteins expressed on tissues of gastric and colon cancers but not on normal tissues. Murine SC142 antibody specific for the SC142-reactive antigen has been produced by immunisation with SNU16 stomach cancer cells. However, SC142 antibody has several potential problems such as high immunogenicity and poor tumour penetration owing to their large size. To improve tumour penetration potential in vivo, recombinant single-chain fragments have been produced using the original hybridoma cells as a source of variable heavy- and variable light-chain-encoding antibody genes. The use of the polymerase chain reaction, expression cloning technology and gene expression systems in E. coli has led to the production of SC142 single-chain fragments, which was similar in activity to the SC142 parent antibody confirmed by immunohistochemistry. Analysis by DNA sequencing, SDS-PAGE and Western blotting has demonstrated the integrity of the single-chain fragments. Competitive ELISA showed that SC142 single-chain fragments originated from parent SC142 antibody. BIAcore biosensor binding experiments showed that the SC142 single-chain fragments had an ideal dissociation rate constant as a tumour imaging reagent. These results illustrate the potential application of these novel products as an immunodiagnostic and further immunotherapeutic reagent.

Show MeSH
Related in: MedlinePlus