TY - JOUR
T1 - Linker modification introduces useful molecular instability in a single chain antibody
AU - Solar, Irit
AU - Gershoni, Jonathan M.
N1 - Funding Information:
We thank Professor Ephraim Katzir and Prof. Arieh Yaron for their discussions and critical comments, Dr Marc Whitlow and the Genex Corporation for providing the SCA 4-4-20/212 plasmid, Ecoli strain and preprints of his work, Ekatherina Baibikova for technical assistance and Jean-Michel Lebleautte for editorial assistance. This research was supported by the Rashi Foundation.
PY - 1995/7
Y1 - 1995/7
N2 - SCA 4-4-20/212, is a recombinant single chain antibody directed against fluorescein (Fl) composed of the variable light (VL) and variable heavy (VH) domains of the monoclonal antibody 4-4-20, tethered by a 14 amino acid linker. Binding of SCA 4-4-20/212 to Fl quenches its fluorescence, thus enabling the distinction between bound and free Fl. This was used to follow antibody denaturation which followed a two-step process: rapid selected and restricted denaturation followed by slow and progressive denaturation. This two-phase phenomenon might reflect selective susceptibility of the CDR loops to denaturation. Furthermore, a new SCA, SCA 4-4-20/9, was constructed by site-directed mutagenesis of SCA 4-4-20/212 using PCR methodology. SCA 4-4-20/9 was similar to SCA 4-4-20/212, but for a nine residue linker. The two SCAs were compared for Fl binding, heat stability, the effect of denaturing agents and susceptibility to proteolysis. The modification of the linker caused a general conformational rearrangement in the SCA molecule, rendering it more sensitive to denaturation and proteolysis. This molecular instability may find utility in the application of SCAs in analytical systems or as the recognition component in biosensors.
AB - SCA 4-4-20/212, is a recombinant single chain antibody directed against fluorescein (Fl) composed of the variable light (VL) and variable heavy (VH) domains of the monoclonal antibody 4-4-20, tethered by a 14 amino acid linker. Binding of SCA 4-4-20/212 to Fl quenches its fluorescence, thus enabling the distinction between bound and free Fl. This was used to follow antibody denaturation which followed a two-step process: rapid selected and restricted denaturation followed by slow and progressive denaturation. This two-phase phenomenon might reflect selective susceptibility of the CDR loops to denaturation. Furthermore, a new SCA, SCA 4-4-20/9, was constructed by site-directed mutagenesis of SCA 4-4-20/212 using PCR methodology. SCA 4-4-20/9 was similar to SCA 4-4-20/212, but for a nine residue linker. The two SCAs were compared for Fl binding, heat stability, the effect of denaturing agents and susceptibility to proteolysis. The modification of the linker caused a general conformational rearrangement in the SCA molecule, rendering it more sensitive to denaturation and proteolysis. This molecular instability may find utility in the application of SCAs in analytical systems or as the recognition component in biosensors.
KW - Biorecognition
KW - Protein engineering
KW - Recombinant antibody
KW - Single-chain antibody
UR - http://www.scopus.com/inward/record.url?scp=0028879775&partnerID=8YFLogxK
U2 - 10.1093/protein/8.7.717
DO - 10.1093/protein/8.7.717
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AN - SCOPUS:0028879775
SN - 1741-0126
VL - 8
SP - 717
EP - 723
JO - Protein Engineering, Design and Selection
JF - Protein Engineering, Design and Selection
IS - 7
ER -