caa a22 4500 397494122 CHVBK 20180308164516.0 cr unu---uuuuu 161202e199510 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a124986 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a124986 VI-Linker-Vh Orientation-Dependent Expression of Single Chain Fv Containing an Engineered Disulfide-Stabilized Bond in the Framework Regions [Elektronische Daten] [Dong Luo, Nancy Mah, Mark Krantz, Kristoffer Wilde, David Wishart, Yi Zhang, Fred Jacobs, Luis Martin] Single chain Fv fragments (scFv) derived from an antibody, MAb 174H.64 (Tru-ScintRSQTM kit, Biomira), were constructed in both orientations, i.e. Vh-linker-VI and VI-linker-Vh, but only the latter form could be expressed and secreted in the recombinant Pichia pastoris system. The secreted scFv protein showed specific anti-idiotype binding activity. Additionally, the molecular graphic modeling has been used to identify a possible site for the introduction of an interchain disulfide bond in the framework region of Fv. These Cys-modifications of the sites were done using a method of PCR-mediated mutagenesis. The engineered protein (disulfide-stabilized Fv: dsFv) was expressed and tested for its binding activity. It was found that dsFv was as active as the corresponding scFv and more stable as determined by competitive radioimmunoassay. © Oxford University Press Regular Papers nationallicence antibody engineering nationallicence disulfide bond nationallicence mutagenesis nationallicence Pichia pastoris nationallicence single-chain Fv nationallicence Luo Dong Research and Development Division, Biomira Inc. 2011-94 Street, Edmonton, Alberta, Canada T6N 1H1 aut Mah Nancy Research and Development Division, Biomira Inc. 2011-94 Street, Edmonton, Alberta, Canada T6N 1H1 aut Krantz Mark Research and Development Division, Biomira Inc. 2011-94 Street, Edmonton, Alberta, Canada T6N 1H1 aut Wilde Kristoffer Research and Development Division, Biomira Inc. 2011-94 Street, Edmonton, Alberta, Canada T6N 1H1 aut Wishart David Protein Engineering, Department of Biochemistry, University of Alberta Edmonton, Alberta, Canada aut Zhang Yi Research and Development Division, Biomira Inc. 2011-94 Street, Edmonton, Alberta, Canada T6N 1H1 aut Jacobs Fred Research and Development Division, Biomira Inc. 2011-94 Street, Edmonton, Alberta, Canada T6N 1H1 aut Martin Luis Research and Development Division, Biomira Inc. 2011-94 Street, Edmonton, Alberta, Canada T6N 1H1 aut The Journal of Biochemistry Oxford University Press 118/4(1995-10), 825-831 0021-924X 118:4<825 1995 118 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a124986 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a124986 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Luo Dong Research and Development Division, Biomira Inc. 2011-94 Street, Edmonton, Alberta, Canada T6N 1H1 aut NATIONALLICENCE 700 1- Mah Nancy Research and Development Division, Biomira Inc. 2011-94 Street, Edmonton, Alberta, Canada T6N 1H1 aut NATIONALLICENCE 700 1- Krantz Mark Research and Development Division, Biomira Inc. 2011-94 Street, Edmonton, Alberta, Canada T6N 1H1 aut NATIONALLICENCE 700 1- Wilde Kristoffer Research and Development Division, Biomira Inc. 2011-94 Street, Edmonton, Alberta, Canada T6N 1H1 aut NATIONALLICENCE 700 1- Wishart David Protein Engineering, Department of Biochemistry, University of Alberta Edmonton, Alberta, Canada aut NATIONALLICENCE 700 1- Zhang Yi Research and Development Division, Biomira Inc. 2011-94 Street, Edmonton, Alberta, Canada T6N 1H1 aut NATIONALLICENCE 700 1- Jacobs Fred Research and Development Division, Biomira Inc. 2011-94 Street, Edmonton, Alberta, Canada T6N 1H1 aut NATIONALLICENCE 700 1- Martin Luis Research and Development Division, Biomira Inc. 2011-94 Street, Edmonton, Alberta, Canada T6N 1H1 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 118/4(1995-10), 825-831 0021-924X 118:4<825 1995 118 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford