High expression and purification of the recombinant camelid anti-MUC1 single domain antibodies in Escherichia coli.
In contrast to the murine and human VHs, camels' single domain antibodies (sdAb) have sufficient solubility. These antigen-specific fragments are expressed well in Escherichia coli. Here, we report high expression and purification of sdAbs against MUC1 mucin. MUC1 is a high molecular weight glycoprotein with an aberrant expression profile in various malignancies. The sdAb genes were sub-cloned into a pET32a(+) vector to overexpress the protein coupled with fusion tags in E. coli BL21(DE3). The expressed single domain antibodies were purified by immobilized metal affinity chromatography and antigen affinity chromatography. Analysis by SDS-PAGE and Western blotting demonstrated the integrity of the sdAbs-tags, while ELISA results confirm that the activity of these molecules compare favorably with that of the parent recombinant antibodies. Enterokinase treated sdAb showed a band at the molecular weight around 12 kDa which demonstrated the naked protein in its natural structure with activities comparable to that of native protein. The high binding activity to MUC1 antigen purified from ascitic fluid (of patients with small-cell lung aggressive carcinoma and metastasis to peritoneum) and the very close similarity of these molecules to human VHs illustrated the potential application of these novel products as an immunodiagnostic and immunotherapeutic reagent.
Rahbarizadeh
Fatemeh
F
Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modarres University, Tehran, Iran.
Rasaee
Mohammad Javad
MJ
Forouzandeh-Moghadam
Mehdi
M
Allameh
Abdol-Amir
AA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D002162:Camelus / Q000276:immunology; D018288:Carcinoma, Small Cell / Q000175:diagnosis / Q000276:immunology / Q000556:secondary; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D007159:Immunologic Tests / Q000379:methods; D051379:Mice; D018396:Mucin-1 / Q000276:immunology; D010534:Peritoneal Neoplasms / Q000175:diagnosis / Q000276:immunology / Q000556:secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D018396:Mucin-1; D011994:Recombinant Proteins