Production of single-chain variable fragment epidermal growth factor receptor (scFv-EGFR) antibody

• Main Author: Loh, Valerie Kai Li.
• Other Authors: Lim Sierin
• Format: Final Year Project
• Language: English
• Published: 2011
• Subjects: DRNTU::Engineering::Chemical engineering::Biotechnology
• Online Access: http://hdl.handle.net/10356/45746
• Institution: Nanyang Technological University

The project serves to identify a suitable prokaryotic expression system and protocol for routine production of single-chain variable fragment epidermal growth factor receptor (scFv-EGFR) clone B10 antibody. The antibody product is meant to bind to EGFRs and has diagnostic and potential therapeutic applications, for instance, conjugating to a carrier for drug delivery purposes. Initially, optimization of yield was attempted via use of different induction strategies, coupled with the use of various expression systems, including the use of commercially available pET vectors and BL21 host strain derivatives that have been well-published. Subsequently, the scFv-EGFR B10 gene sequence underwent codon optimization and a different expression system, pSYN1 vector and E. coli TG1 competent host strain, was used. Affinity chromatography and size-exclusion chromatography via Fast Protein Liquid Chromatography (FPLC) are the methods of purification and protein products are characterised using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and/or Western blot. Results show that the antibody product may not have been produced successfully using the initial expression system due to recurring inconclusive results from purification and characterisation procedures. However, with the use of pSYN1/TG1 expression system and protocol, Western blot results appear to show positive production of scFv-EGFR B10 (synthetic optimized sequence), and specific affinity of the scFv-EGFR B10 antibody product to EGFR is proven.