(191ae) Extracellular Production of Soluble Single-Chain Variable Fragment (scFv) Using Recombinant E. coli By Precisely Controlled Fed-Batch Culture with Do Stat

Single-chain variable fragment (scFv) antibodies are currently utilized as affinity reagents for diagnostics instead of full-length antibodies. ScFvs can be easily expressed in recombinant E. coli, however, there are some drawbacks such as low productivity and the formation of inclusion bodies. In this study, the extracellular production of soluble scFv using recombinant E. coli b was aimed by employing a fed-batch culture with DO-stat to realize the cost-effective production of soluble scFvs.

Five E. coli horboring four different scFv genes with periplasmic secretion signal(pelB leader) were constructed employing pET Expression System. Using the strains, the influence of temperature, culture media and host strains on scFv expression was evaluated and the induction timing was optimized. Based on these results, fed-batch cultures for scFv production were performed. Glucose feeding of the fed-fed batch system was carried out based on the DO-stat with PID control.

Fed-batch cultures with DO-stat were successfully performed and the cell concentration was increased to approximately 130 (OD600) during 100 hrs cultivation. During the fed-batch cultivation, soluble scFv was gradually excreted from cells and accumulated in the borth. Finally, 3.1g/L of soluble Anti-human IgG scFv and 4.8g/L of soluble Anti-CRP scFv were extracellularly produced during 100 hrs fed-batch cultivation. Total scFv production including inclusion bodies of scFvs expressed in the periplasm was 6.0g/L and 8.7g/L, respectively. These results will greatly contribute to achieve the cost-effective production of scFvs because the soluble scFV does not require the refolding step during purification process of scFvs.