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Title
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Recombinant antineuraminidase single chain antibody: expression, characterization, and crystallization in complex with antigen.
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Authors
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R.L.Malby,
J.B.Caldwell,
L.C.Gruen,
V.R.Harley,
N.Ivancic,
A.A.Kortt,
G.G.Lilley,
B.E.Power,
R.G.Webster,
P.M.Colman.
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Ref.
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Proteins, 1993,
16,
57-63.
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PubMed id
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Abstract
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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.
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