TY - JOUR T1 - Optimal Production of a Fusion Protein Consisting of a Single-chain Variable Fragment Antibody Against a Tumor-associated Antigen and Interleukin-2 in Fed-batch Culture of <em>Pichia pastoris</em> JF - Anticancer Research JO - Anticancer Res SP - 3925 LP - 3935 VL - 34 IS - 8 AU - SAKORN ANULEEJUN AU - TANAPAT PALAGA AU - YOSHIO KATAKURA AU - MOTOMU KUROKI AU - MASAHIDE KUROKI AU - SUCHADA CHANPRATEEP NAPATHORN Y1 - 2014/08/01 UR - http://ar.iiarjournals.org/content/34/8/3925.abstract N2 - Background/Aim: The aim of the present study was to establish the strategy for producing a single-chain variable fragment (scFv) antibody fused with interleulin-2 (IL2) by Pichia pastoris and to optimize production during fed-batch cultivation in a 5-l fermenter. Materials and Methods: We constructed a fusion sequence consisting of an scFv gene derived from a mouse monoclonal antibody against a tumor-associated antigen (designated MK-1 antigen) and human interleulin-2 (IL-2) gene, ligated the sequences to expression vector pPICZα-A and separately transformed the constructs into Pichia pastoris strains GS115 and KM71H. Results: The highest concentration of secreted fusion protein, 738±44 mg/l, was obtained after a 60-h induction. To investigate the specific binding activity of the partially purified fusion protein, we used an enzyme-linked immunosorbent assay and antigen from a whole-cell lysate. Student's t-test showed that the specific binding activity of the partially-purified fusion protein to the lysate of Chinese hamster ovary cell lines expressing the MK-1 antigen was significantly higher than that of the lysate of CHO cell lines that do not express MK-1. Conclusions: The method described here permits the production of substantial amounts of the fusion protein for conducting functional studies on the biological role of these fusion proteins. ER -