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. 2005 Aug 21;11(31):4899-903.
doi: 10.3748/wjg.v11.i31.4899.

Disulfide-stabilized single-chain antibody-targeted superantigen: construction of a prokaryotic expression system and its functional analysis

Jian-Li Wang  1 , Yu-Ling Zheng, Ru Ma, Bao-Li Wang, Ai-Guang Guo, Yong-Qiang Jiang
Affiliations

Disulfide-stabilized single-chain antibody-targeted superantigen: construction of a prokaryotic expression system and its functional analysis

Jian-Li Wang et al. World J Gastroenterol. .

Abstract

Aim: To construct the expression vector of B3 (scdsFv)-SEA (D227A) and to identify its binding and cytotoxic ability to B3 antigen positive carcinoma cell lines.

Methods: This fusion protein was produced by a bacterial expression system in this study. It was expressed mainly in the inclusion body. The gene product was solubilized by guanidine hydrochloride, refolded by conventional dilution method, and purified using SP-sepharose cation chromatography.

Results: The expression vector B3 (scdsFv)-SEA-PET was constructed, the expression product existed mainly in the inclusion body, the refolding product retained the binding ability of the single-chain antibody and had cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37 degrees.

Conclusion: This genetically engineered B3 (scdsFv)-SEA fusion protein has bifunction of tumor targeting and tumor cell killing and shows its promises as an effective reagent for tumor-targeted immunotherapy.

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Figures

Figure 1
Figure 1
Schematic representation of B3scdsFv-SEA-pET22b expression plasmid. L: polypeptide linker; L1: (GlyGlyGlyGlySer)3; L2: (GlyGlyGlySer-GlyGlySer).
Figure 2
Figure 2
The SDS-PAGE analysis of expression products (A) and Western blot of inclusion body (B). A: Lane 1: B3scdsFv-SEA- pET22b before induction; lane 2: total protein after induction; lane 3: ultrasonic supernatant of B3scdsFv-SEA-pET22b induced by 1 mmol/L IPTG; lane 4: ultrasonic deposit after induction; lane M: low molecular weight protein marker; B: Lane 1: Western blot of B3scdsFv-SEA; lane 2: negative control (total protein before induction); lane M: low molecular weight protein marker.
Figure 3
Figure 3
Gel mobility of B3scdsFv-SEA under reducing (lane 1) and non-reducing condition (lane 2).
Figure 4
Figure 4
Binding assay of B3scdsFv-SEA to B3 antigen positive carcinoma cell lines.
Figure 5
Figure 5
Stability of B3scdsFv-SEA.
Figure 6
Figure 6
Cytotoxic effects of B3scdsFv-SEA on HT-29 cell line.
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