Conformational stability, folding, and ligand-binding affinity of single-chain Fv immunoglobulin fragments expressed in Escherichia coli.
A fluorescein-binding single-chain Fv (scFv) was chosen as a model for the study of the physicochemical parameters associated with synthetic IgG fragments. Three such scFv proteins were designed from the primary sequences of one anti-fluorescyl monoclonal antibody (Mab 4.4.20). These were constructed with varying-length interdomain peptide linkers of between 12 and 25 residues, expressed in Escherichia coli, and the protein folding, stability, and antigen-binding characteristics were assessed. Efficient renaturation could be accomplished in vitro to yield approximately 26 mg of active scFv/L of fermentation. Scatchard analysis for fluorescein ligand binding revealed that the scFv designs come within 2-fold of the Ka = 1.99 (+/- 0.18) x 10(9) observed for the parental 4.4.20 Fab and have identical stoichiometries (n approximately 0.99). Reversible solvent denaturation studies demonstrated that the unfolding/refolding equilibria for the scFv proteins can be fit to a simple two-state model and that two of the scFv designs were found to be slightly more stable than single IgG domains (VL and CL) when assessed in terms of the free energy of unfolding, delta Gon-u, or nearly identical to other multiple domain immunoglobulin proteins such as light chains and Fab's when relative transition midpoints, Cm, are compared. Linkers which conferred conformational flexibility beyond the minimally required length of 12 residues were found to have a stabilizing effect. By these criteria of ligand-binding function and protein stability, the scFv proteins were found to be bona fide minimal replicas of their parental IgG molecules.
Genex Corporation, Gaithersburg, Maryland 20872.
M W
MW
Pantoliano
R E
RE
Bird
S
S
Johnson
E D
ED
Asel
S W
SW
Dodd
J F
JF
Wood
K D
KD
Hardman
D000595:Amino Acid Sequence; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics*; D005452:Fluoresceins; D005822:Genetic Vectors; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D007136:Immunoglobulins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D007163:Immunosorbent Techniques; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D012997:Solvents / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology
D005452:Fluoresceins; D006146:Guanidines; D007128:Immunoglobulin Fragments; D007136:Immunoglobulins; D011994:Recombinant Proteins; D012997:Solvents; C055966:immunoglobulin Fv; D014508:Urea; D019791:Guanidine