Hwan, JC Chung, HH Yu, J Chang, YJ Kim, HB Paek, SH Shin, DH Kim, KH 2024-01-21T15:32:00Z 2024-01-21T15:32:00Z 2021-09-05 1999-06 1017-7825 https://pubs.kist.re.kr/handle/201004/142161 Constructs, encoding a single-chain Variable fragment of a catalytic antibody 4f4f (scFv-4f4f) with glycosidase activity, were made by combining the coding sequences for the heavy and light chain variable domains with a sequence encoding a linker (GGGGS). Using three different plasmid systems, single-chain antibodies were expressed separately in Escherichia coli, demonstrating significant differences in the expression level and amounts in soluble form of the recombinant protein. The protein expression from pET3a-scFv-4f4f was up to 20% of the total soluble proteins and, more importantly, the proteins were mostly found in a soluble form. An SDS-PAGE analysis of the purified single-chain proteins, yielding higher than 5 mg from a 1-1 culture, showed a single band corresponding to its molecular weight of 29,100. A preliminary study shows that the expressed scFv-4f4f is catalytically active. The catalytic parameters for the hydrolysis of p-nitrophenyl-beta-D-glucopyranoside by scFv-4f4f are being investigated. English KOREAN SOC MICROBIOLOGY & BIOTECHNOLOGY TRANSITION-STATE ANALOG IN-VITRO IMMUNIZATION ESCHERICHIA-COLI FUSION PROTEINS BINDING IMMUNOTOXINS AFFINITY LINKER SITES Single-chain Fv fragment of catalytic antibody 4f4f with glycosidase activity: Design, expression, and purification Article 1 JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, v.9, no.3, pp.376 - 380 JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY 9 3 376 380 scie scopus 000081304400026 2-s2.0-0344780800 Biotechnology & Applied Microbiology Microbiology Biotechnology & Applied Microbiology Microbiology Article TRANSITION-STATE ANALOG IN-VITRO IMMUNIZATION ESCHERICHIA-COLI FUSION PROTEINS BINDING IMMUNOTOXINS AFFINITY LINKER SITES single-chain Fv fragment catalytic antibody engineering expresssion