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PDBsum entry 3dgg
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Immune system
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PDB id
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3dgg
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References listed in PDB file
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Key reference
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Title
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A pipeline for the production of antibody fragments for structural studies using transient expression in hek 293t cells.
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Authors
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J.E.Nettleship,
J.Ren,
N.Rahman,
N.S.Berrow,
D.Hatherley,
A.N.Barclay,
R.J.Owens.
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Ref.
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Protein Expr Purif, 2008,
62,
83-89.
[DOI no: ]
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PubMed id
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Abstract
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We describe a pipeline for the rapid production of recombinant Fabs derived from
mouse monoclonal antibodies suitable for use in structural studies. The pipeline
is exemplified by the production of three Fabs derived from the monoclonal
antibodies OX108 (anti-CD200 receptor), OX117 and OX119 (anti-SIRPgamma). Heavy
and light chain variable domains were inserted into separate expression vectors
containing resident constant regions using In-Fusion PCR cloning. Following
transient co-expression in HEK 293T cells, secreted Fab fragments were purified
by metal chelate chromatography and gel filtration using an automated procedure
with yields of up to 4mg/L of cell culture. Following crystallization trials,
diffracting crystals were obtained for the recombinant Fabs of OX108 and OX117,
and their structures solved to 2.3A and 2.4A, respectively.
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