Biotechnol. Appl. Biochem. (2007) 46, 41–49 (Printed in Great Britain) doi:10.1042/BA20060104 41 Studies of thermostability in Camelus bactrianus (Bactrian camel) single-domain antibody specific for the mutant epidermal-growth-factor receptor expressed by Pichia Kobra Omidfar*, Mohhamad Javad Rasaee† 1 , Soheila Kashanian‡, Malieheh Paknejad§ and Zahra Bathaie† *Endocrinology and Metabolism Research Centre, Tehran University of Medical Sciences, Tehran, Iran, †Department of Biochemistry, School of Medical Sciences, Tarbiat Modarres University, Tehran, I.R. Iran, ‡Department of Chemistry, School of Sciences, Razie University, Kermanshah, I.R. Iran, and §Department of Biochemistry, School of Medical Sciences, Tehran University, Tehran, I.R. Iran Camelids have a unique immune system capable of pro- ducing heavy-chain antibodies lacking the light chains and CH1 (constant heavy-chain domain 1). It has been shown that, in contrast with conventional antibody frag- ments, the variable domains of these heavy-chain anti- bodies are functional at or after exposure to high tem- peratures. 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 ex- ternal domain of the mutant EGFR [EGF (epidermal growth factor) receptor], EGFR VIII. This tumour- specific antigen is ligand-independent, contains a con- stitutively active tyrosine kinase domain and has been shown to be present in a number of human malignan- cies. We report here that, although expression from P. pastoris resulted in a significantly increased level of ex- pression of the anti-EGFR VIII VHH antibodies compa- red 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 anti- body 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 denatu- ration. Our results indicate that the P. pastoris expres- sion system may be useful for the expression of camel single domain antibody and the ability of the expressed protein to reversibly melt without aggregation, allow- ing it to regain binding activity after heat denaturation. Introduction Camelids (camels, dromedaries, llamas, alpacas, guanacos and vicunas) are known to produce immunoglobulins devoid of light chains and CH1 (constant heavy-chain domain 1). Antigen-specific fragments of these heavy-chain IgGs [VHH (variable domain of heavy-chain antibody)] are of great interest in biotechnological applications. These heavy-chain antibodies provided a natural solution for size reduction of an antibody fragment. In contrast with VH (heavy-chain variable domain), the antigen-binding fragments (VHH) of these molecules are well expressed and were shown to overcome, to a large extent, the stability, aggregation and degradation problems often encountered with scFvs (single- chain variable domain fragments) [1–6]. In addition, VHHs have a close homology with human VH fragments, which may result in the reduction or absence of immunogenic VHH molecules in patients [1]. Single domain and heavy-chain antibodies have been produced in a wide variety of systems, including Escherichia coli [1,6,7], Saccharomyces cerevisiae [8], Aspergillus awamori [9] and myeloma cells [10]. However, all these expression systems have different advantages or disadvantages. Pichia pastoris has been widely used as a host organism in recent years, especially for the production of eukaryotic heterologous proteins [11,12], and, compared with other eukaryotic expression systems, offers many advantages, including protein processing, folding and post-translational modification. Pichia does not have the endotoxin problem associated with bacteria or viral contamination problems of proteins produced in animal cell cultures [11]. Key words: Camelus bactrianus (Bactrian camel), epidermal-growth-factor receptor VIII (EGFR VIII), immunoglobulin, Pichia, single-domain antibody, thermostability, variable domain of heavy-chain antibody (VHH). Abbreviations used: AOX1, alternative oxidase 1; CDR, complementary-determining region; EGF, epidermal growth factor; EGFR, EGF receptor; VH, heavy-chain variable domain; VL, light-chain variable domain; Fv, heterodimer of VH and VL; HRP , horseradish peroxidase; KLH, keyhole-limpet (Diodora aspera) haemocyanin; mAb, monoclonal antibody; MD medium, minimal dextrose medium; MM medium, minimal methanol medium; NSB, non-specific binding; NSCLC, non-small-cell lung cancer; scFv, single-chain variable domain fragment; VHH, variable domain of heavy-chain antibody; YNB, yeast nitrogen base. 1 To whom correspondence should be addressed (email rasaee m@modares.ac.ir). C  2007 Portland Press Ltd