[Expression, purification, and characterization of single-chain disulfide-bond Fv (ScdsFv) antibody fused with targeted superantigen SEA (D227A)].
OBJECTIVE
To express, purify, and characterize scdsFv antibody fused with superantigen SEA(D227A).


METHODS
The expression plasmid of scdsFv-SEA(D227A) was constructed by standard molecular cloning procedures. The recombinant protein was induced to express in E. coli BL21plusS by IPTG and purified by Q Sepharose HP column and Hiprep 26/60 Sephacryl S-200 HR column. Formation of the intramolecular disulfide bond of the purified protein was analysed by AMS alkylation and PAGE electrophoresis. The binding activity, stability and killing activity of the purified protein were assayed by ELISA and MTS, respectively.


RESULTS
The recombinant protein was expressed as inclusion body, accounting for more than 30% of total bacterial protein. After purification by Q Sepharose HP and Hiprep 26/60 Sephacryl S-200 HR, the yield of the purified protein was 60 mg per liter of induced culture. AMS alkylation and PAGE electrophoresis analysis showed that intramolecular disulfide bond formed correctly in the recombinant protein. The purified protein had similar binding affinity as dsFv fused SEA and scFv fused SEA have and similar killing activity as native SEA has to human hepatoma cell line, but more stable, in vitro, as compared with dsFv fused SEA and scFv fused with SEA.


CONCLUSIONS
The scdsFv fused with SEA, as a novel form of immunotoxin, might be used in cancer treatment.
Hao
Huai-jie
HJ
Department of Molecular Biology, College of Life Science, Nanjing Normal University, Nanjing 210097, China.
Zheng
Yu-ling
YL
Yu
Duo-wei
DW
Ma
Ru
R
Jiang
Yong-qiang
YQ
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D010957:Plasmids / Q000235:genetics; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D018089:Superantigens / Q000235:genetics* / Q000276:immunology*
D000906:Antibodies; D004220:Disulfides; D011993:Recombinant Fusion Proteins; D018089:Superantigens