Recombinant antineuraminidase single chain antibody: expression, characterization, and crystallization in complex with antigen.
The variable heavy (VH) and variable light (VL) genes of NC10, a monoclonal antibody with specificity toward N9 neuraminidase (NA), were cloned and sequenced. A single chain Fv (scFv) fragment of NC10, consisting of VH and VL domains joined by a peptide linker, was designed, constructed and expressed in the E. coli expression vector pPOW. The N-terminal secretion signal PelB directed the synthesized protein into the periplasm where it was associated with the insoluble membrane fraction. An octapeptide (FLAG) tail was fused to the C-terminus of the single chain Fv to aid in its detection and remained intact throughout the protein purification process. NC10 scFv was purified by solubilization of the E. coli membrane fraction with guanidinium hydrochloride followed by column chromatography. The purified NC10 scFv showed binding affinity for its antigen, NA, 2-fold lower than that of the parent Fab. The complex between NA and the scFv has been crystallized by the vapor diffusion method. The crystals are tetragonal, space group P42(1)2, with unit cell dimensions a = b = 141 A, c = 218 A.
Malby
R L
RL
Biomolecular Research Institute, Parkville, Vic, Australia.
Caldwell
J B
JB
Gruen
L C
LC
Harley
V R
VR
Ivancic
N
N
Kortt
A A
AA
Lilley
G G
GG
Power
B E
BE
Webster
R G
RG
Colman
P M
PM
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics; D000936:Antigen-Antibody Complex / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D015870:Gene Expression; D005796:Genes; D005803:Genes, Immunoglobulin / Q000235:genetics; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000235:genetics / Q000276:immunology*; D009975:Orthomyxoviridae / Q000276:immunology; D011994:Recombinant Proteins; D014961:X-Ray Diffraction
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; D009439:Neuraminidase