3U6R

Three dimensional structure of broadly neutralizing anti - Hepatitis C virus (HCV) glycoprotein E2 single chain FV fragment 1:7

Summary for 3U6R

• Entry DOI: 10.2210/pdb3u6r/pdb
• Descriptor: Antibody 1:7 (Heavy chain), Antibody 1:7 (Light chain), SULFATE ION, ... (4 entities in total)
• Functional Keywords: ig-like domain, neutralizing single chain fv, immune system
• Biological source: Homo sapiens (human); More
• Total number of polymer chains: 4
• Total formula weight: 61993.89

Authors

Gilmartin, A.A.,Lamp, B.,Ruemenapf, T.,Persson, A.A.,Rey, F.A.,Krey, T. (deposition date: 2011-10-12, release date: 2012-01-11, Last modification date: 2024-11-27)

Primary citation

Gilmartin, A.A.,Lamp, B.,Rumenapf, T.,Persson, M.A.,Rey, F.A.,Krey, T.
High-level secretion of recombinant monomeric murine and human single-chain Fv antibodies from Drosophila S2 cells.
Protein Eng.Des.Sel., 25:59-66, 2012

PubMed Abstract: Single-chain variable fragment (scFvs) antibodies are small polypeptides (∼26 kD) containing the heavy (V(H)) and light (V(L)) immunoglobulin domains of a parent antibody connected by a flexible linker. In addition to being frequently used in diagnostics and therapy for an increasing number of human diseases, scFvs are important tools for structural biology as crystallization chaperones. Although scFvs can be expressed in many different organisms, the expression level of an scFv strongly depends on its particular amino acid sequence. We report here a system allowing for easy and efficient cloning of (i) scFvs selected by phage display and (ii) individual heavy and light chain sequences from hybridoma cDNA into expression plasmids engineered for secretion of the recombinant fragment produced in Drosophila S2 cells. We validated the method by producing five scFvs derived from human and murine parent antibodies directed against various antigens. The production yields varied between 5 and 12 mg monomeric scFv per liter of supernatant, indicating a relative independence on the individual sequences. The recombinant scFvs bound their cognate antigen with high affinity, comparable with the parent antibodies. The suitability of the produced recombinant fragments for structural studies was demonstrated by crystallization and structure determination of one of the produced scFvs, derived from a broadly neutralizing antibody against the major glycoprotein E2 of the hepatitis C virus. Structural comparison with the Protein Data Bank revealed the typical spatial organization of V(H) and V(L) domains, further validating the here-reported expression system.

PubMed: 22160929
DOI: 10.1093/protein/gzr058

Experimental method

X-RAY DIFFRACTION (2.67 Å)