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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

Screening of scFv by scFv expression. E. coli BL21 transformed cells containing expressed scFv were lysed and subjected to SDS–PAGE (12%) and stained with Coomassie blue staining after separating proteins on reducing SDS–PAGE gels. (A) Protein expression pattern before and after IPTG induction. Left-oriented arrowheads indicate relatively highly expressed protein bands. (B) Western blot analysis of selected clones using anti-Xpress antibody.
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fig2: Screening of scFv by scFv expression. E. coli BL21 transformed cells containing expressed scFv were lysed and subjected to SDS–PAGE (12%) and stained with Coomassie blue staining after separating proteins on reducing SDS–PAGE gels. (A) Protein expression pattern before and after IPTG induction. Left-oriented arrowheads indicate relatively highly expressed protein bands. (B) Western blot analysis of selected clones using anti-Xpress antibody.

Mentions: Insertion of SC142 VH- and VL-specific DNA in the scFv assembly process was analysed by expression of the DNA from transformants of BL21 E. coli clones grown on ampicillin plates. These tests were performed using Coomassie blue staining and Western blot analysis with anti-Xpress antibody. The pRSET-Angiogenin SfiI/NotI expression vector uses a reporter sequence known as the Xpress-tag to show the presence of scFv (Figure 1). A murine antibody recognising the Xpress-tag peptide sequence allows the presence of expressed scFv to be visualised on Western blots. The use of this reporter, plus analysis of the product size through SDS–PAGE, reveals the presence of intact recombinant scFv. Of 10 randomly selected clones, three produced expression products of approximately 45 kDa in length (Figure 2Figure 2


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)

Screening of scFv by scFv expression. E. coli BL21 transformed cells containing expressed scFv were lysed and subjected to SDS–PAGE (12%) and stained with Coomassie blue staining after separating proteins on reducing SDS–PAGE gels. (A) Protein expression pattern before and after IPTG induction. Left-oriented arrowheads indicate relatively highly expressed protein bands. (B) Western blot analysis of selected clones using anti-Xpress antibody.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Screening of scFv by scFv expression. E. coli BL21 transformed cells containing expressed scFv were lysed and subjected to SDS–PAGE (12%) and stained with Coomassie blue staining after separating proteins on reducing SDS–PAGE gels. (A) Protein expression pattern before and after IPTG induction. Left-oriented arrowheads indicate relatively highly expressed protein bands. (B) Western blot analysis of selected clones using anti-Xpress antibody.
Mentions: Insertion of SC142 VH- and VL-specific DNA in the scFv assembly process was analysed by expression of the DNA from transformants of BL21 E. coli clones grown on ampicillin plates. These tests were performed using Coomassie blue staining and Western blot analysis with anti-Xpress antibody. The pRSET-Angiogenin SfiI/NotI expression vector uses a reporter sequence known as the Xpress-tag to show the presence of scFv (Figure 1). A murine antibody recognising the Xpress-tag peptide sequence allows the presence of expressed scFv to be visualised on Western blots. The use of this reporter, plus analysis of the product size through SDS–PAGE, reveals the presence of intact recombinant scFv. Of 10 randomly selected clones, three produced expression products of approximately 45 kDa in length (Figure 2Figure 2

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