title: Studies of thermostability in Camelus bactrianus (Bactrian camel) single-domain antibody specific for the mutant epidermal-growth-factor receptor expressed by Pichia.
abstract: Camelids have a unique immune system capable of producing heavy-chain antibodies lacking the light chains and CH1 (constant heavy-chain domain 1). It has been shown that, in contrast with conventional antibody fragments, the variable domains of these heavy-chain antibodies are functional at or after exposure to high temperatures. In the present study, the VHH (variable domain of heavy-chain antibody) camel antibody was subcloned into vector Ppiczc and expressed in Pichia pastoris. ORB1-83 VHH antibody recognizes the external domain of the mutant EGFR [EGF (epidermal growth factor) receptor], EGFR VIII. This tumour-specific antigen is ligand-independent, contains a constitutively active tyrosine kinase domain and has been shown to be present in a number of human malignancies. We report here that, although expression from P. pastoris resulted in a significantly increased level of expression of the anti-EGFR VIII VHH antibodies compared with Escherichia coli [Omidfar, Rasaee, Modjtahedi, Forouzandeh, Taghikhani, Bakhtiari, Paknejad and Kashanian (2004) Tumor Biol. 25, 179-187; Omidfar, Rasaee, Modjtahedi, Forouzandeh, Taghikhani and Golmakany (2004) Tumor Biol. 25, 296-305], this antibody selectively bound to the EGFR VIII peptide and reacted specifically with the immunoaffinity-purified antigen from non-small-cell lung cancer. Furthermore, thermal denaturation stability and CD spectra analysis of the Camelus bactrianus (Bactrian camel) VHH and heavy-chain antibodies at different temperature proved reversibility and binding activity after heat denaturation. Our results indicate that the P. pastoris expression system may be useful for the expression of camel single domain antibody and the ability of the expressed protein to reversibly melt without aggregation, allowing it to regain binding activity after heat denaturation.
authors.affiliation: Endocrinology and Metabolism Research Centre, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Kobra
authors.initials: K
authors.lastname: Omidfar
authors.forename: Mohhamad Javad
authors.initials: MJ
authors.lastname: Rasaee
authors.forename: Soheila
authors.initials: S
authors.lastname: Kashanian
authors.forename: Malieheh
authors.initials: M
authors.lastname: Paknejad
authors.forename: Zahra
authors.initials: Z
authors.lastname: Bathaie
mesh_terms: D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D001483:Base Sequence; D002162:Camelus; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D066246:ErbB Receptors / Q000235:genetics / Q000276:immunology*; D010843:Pichia / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000906:Antibodies; D017931:DNA Primers; D011994:Recombinant Proteins; D066246:ErbB Receptors