PT  - JOURNAL ARTICLE
AU  - SAKORN ANULEEJUN
AU  - TANAPAT PALAGA
AU  - YOSHIO KATAKURA
AU  - MOTOMU KUROKI
AU  - MASAHIDE KUROKI
AU  - SUCHADA CHANPRATEEP NAPATHORN
TI  - 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>
DP  - 2014 Aug 01
TA  - Anticancer Research
PG  - 3925--3935
VI  - 34
IP  - 8
4099  - http://ar.iiarjournals.org/content/34/8/3925.short
4100  - http://ar.iiarjournals.org/content/34/8/3925.full
SO  - Anticancer Res2014 Aug 01; 34
AB  - 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.