Construction and expression of disulphide stabilized anti-CD3/anti-Pgp diabody / 生物工程学报
Chinese Journal of Biotechnology
; (12): 1042-1048, 2009.
Article
ي Zh
| WPRIM
| ID: wpr-296959
المكتبة المسؤولة:
WPRO
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.
النص الكامل:
1
الفهرس:
WPRIM
الموضوع الرئيسي:
Binding, Competitive
/
Cell Line
/
Chemistry
/
CD3 Complex
/
Antibodies, Bispecific
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ATP Binding Cassette Transporter, Subfamily B
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Disulfides
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Drug Stability
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Allergy and Immunology
/
Escherichia coli
المحددات:
Humans
اللغة:
Zh
مجلة:
Chinese Journal of Biotechnology
السنة:
2009
نوع:
Article