Construction and expression of disulphide stabilized anti-CD3/anti-Pgp diabody.
- Author:
Ye SU
1
;
Juanni LIU
;
Yingdai GAO
;
Li QIN
;
Ming YANG
;
Jinhong WANG
;
Yuanfu XU
;
Xiaofeng SHAO
;
Qing JI
;
Dongsheng XIONG
;
Chunzheng YANG
Author Information
1. State Key Laboratory of Experimental Hematology, Institute of Hematology & Hospital of Blood Diseases, Chinese Academic of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.
- Publication Type:Journal Article
- MeSH:
ATP Binding Cassette Transporter, Sub-Family B;
immunology;
Antibodies, Bispecific;
biosynthesis;
chemistry;
genetics;
immunology;
Binding, Competitive;
CD3 Complex;
immunology;
Cell Line;
Disulfides;
chemistry;
Drug Stability;
Escherichia coli;
genetics;
metabolism;
Humans
- From:
Chinese Journal of Biotechnology
2009;25(7):1042-1048
- CountryChina
- Language:Chinese
-
Abstract:
We constructed and expressed an anti-CD3/anti-Pgp (P-glycoprotein) diabody previously. However, the two chains of diabody are associated non-covalently, resulting in being capable of dissociating. The aim of this study is to enhance the stability of the diabody. We introduced cysteine residues into the CD3 or Pgp V-domain to covalently lock the two chains together. The disulphide crosslinked diabody were expressed by Escherichia coli (E. coli) 16C9 and purified by a cation exchange column and an anti-Etag affinity chromatography. The purified proteins were verified through SDS-PAGE. Flow cytometry (FCM) was used to analyse the binding properties, competitive binding capacity and stability in vitro. The dsPpg-diabody failed to form disulphide bond properly. The designed disulphide bridge between the different chains of dsCD3-diabody was formed correctly. FCM demonstrated the dsCD3-diabody has specific antigen binding activity, the same binding activity and competitive binding activity as its parent diabody. The dsCD3-diabody retained the full activity even after 72 h incubation at 37 degrees C in human serum, in contrast, the parent diabody began to lose activity after only 1 h and lose all its activity 24 hours later. The induced disulphide bond in the CD3 V-domain effectively enhanced the stability of anti-CD3/anti-Pgp diabody. The method of stabilizing a diabody by introducing a disulphide bond into is practical.
