SUBCLONING OF DNA ENCODING ANTI-EGFRvIII SCFV ANTIBODY AND ITS PERIPLASMIC EXPRESSION IN ESCHERICHIA COLI

• Main Author: Sari Dewi, Kartika
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/45787
• Institution: Institut Teknologi Bandung

Epidermal growth factor receptor variant III (EGFRvIII) is a mutant of EGFR which expressed only in several types of cancers cells. This characteristic makes it ideal as molecular target for immunology-based cancer therapy. One of the potential delivery systems using EGFRvIII as a molecular target is single-chain variable fragment (scFv) antibody. Recombinant scFv antibody in its native form can be produced in periplasmic of Escherichia coli cell. Previous research has successfully constructed an open reading frame (ORF) of the DNA encoding anti- EGFRvIII scFv1 (VH-linker-VL) and scFv2 (VL-linker-VH) antibody for expression in Pichia pastoris. This research aims to subclone DNA encoding scFv1 and scFv2 into pJ414 expression vector and to analyze its expression product in periplasmic compartment of E. coli BL21(DE3). DNA encoding scFv1 and scFv2 were amplified from pJ201:57086-AFseq_optPp and pTz_scFv2 plasmid using spesific primers by PCR method. Each PCR product was cloned into pJ414 expression vector, followed by transformation of recombinant plasmids (pJ414_scFv1 and pJ414_scFv2) into E. coli TOP10. The recombinant plasmids were characterized by migration, restriction, PCR and DNA sequencing analysis. ScFv1 and scFv2 proteins were produced in recombinant E. coli BL21(DE3) with 0.1 and 1 mM final concentration of IPTG induction, respectively. Overproduction was performed at 20-23 °C for 20 hours. Total proteins were obtained from lysing of pellet cells using lyses buffer and heating. Periplasmic proteins were isolated from pellet cells using hypertonic solution and the spheroplast were lysed by freeze-thaw method to obtain cytoplasmic proteins. Total proteins, soluble periplasmic proteins and cytoplasmic proteins were analyzed by SDS-PAGE and Western Blot using anti-His primary antibody. The result from scFv1 and scFv2 protein analysis using both methods showed protein bands from periplasmic fraction with size of 28.76 and 29.81 kDa.