A soluble immunoglobulin variable domain without a disulfide bridge: construction, accumulation in the cytoplasm of E. coli, purification and physicochemical characterization.
Two amino acid exchanges (Y32H and C23V) were introduced sequentially into the immunoglobulin REIV, a human kappa variable domain. The first exchange stabilizes the folded state of the domain by 4.6 kJ/mol (1.1 kcal/mol), the second abolishes the central disulfide bridge and destabilizes the folded domain by 17.5 kJ/mol (4.2 kcal/mol). Introduction of the stabilizing exchange first is a necessary pre-requisite to the removal of the central disulfide bridge without collapse of the fold. The double mutant REIV-C23V/Y32H can be accumulated in the cytoplasmatic compartment of the E. coli cell, a finding that opens new possibilities in antibody engineering.
Frisch
C
C
Institut für Molekulare Genetik, Georg-August-Universität Göttingen, Germany.
Kolmar
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H
Fritz
H J
HJ
D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D013050:Spectrometry, Fluorescence
D004220:Disulfides; D007135:Immunoglobulin Variable Region