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* Residue conservation analysis
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DOI no:
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J Biol Chem
272:29784-29789
(1997)
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PubMed id:
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A single chain Fv fragment of P-glycoprotein-specific monoclonal antibody C219. Design, expression, and crystal structure at 2.4 A resolution.
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F.J.Hoedemaeker,
T.Signorelli,
K.Johns,
D.A.Kuntz,
D.R.Rose.
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ABSTRACT
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A construct encoding a single chain variable fragment of the anti-P-glycoprotein
monoclonal antibody C219 was made by combining the coding sequences for the
heavy and light chain variable domains with a sequence encoding the flexible
linker (GGGGS)3, an OmpA signal sequence, a c-myc identification tag, and a
five-histidine purification tag. The construct was expressed in Escherichia coli
and purified from the periplasmic fraction using a nickel chelate column and ion
exchange chromatography. Three-step Western blot analysis showed that the
construct retains binding affinity for P-glycoprotein. Crystals of 1.0 x 0.2 x
0.2 mm were grown in 100 mM citrate, pH 4.5, 21% polyethylene glycol 6000 in the
presence of low concentrations of subtilisin, resulting in proteolytic removal
of the linker and purification tags. The structure was solved to a resolution of
2.4 A with an R factor of 20.6, an Rfree of 28.5, and good stereochemistry. This
result could lead to a clinically useful product based on antibody C219 for the
diagnosis of P-glycoprotein-mediated multidrug resistance. The molecule will
also be useful in biophysical studies of functional domains of P-glycoprotein,
as well as studies of the intact molecule.
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Selected figure(s)
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Figure 4.
Fig. 4. Detail of the electron density at the V[L]-V[H]
interface, contoured at 1  . View is
"up" toward the CDRs along the pseudo-2-fold^ axis. Two
short-range hydrogen bonds between glutamines in the^ light and
heavy chains are indicated in green. Figs. 4 and 5A^ were
produced with SETOR (49).
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Figure 6.
Fig. 6. Helical wheel plot of the peptide epitope.
Hydrophobic residues are indicated with boldface lettering;
residues that are potentially important for binding are boxed
(13). The epitope^ sequence is extended by one helix turn
(VVQEALDKARE) to show the^ continuous amphiphilicity.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(1997,
272,
29784-29789)
copyright 1997.
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Figures were
selected
by the author.
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Literature references that cite this PDB file's key reference
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PubMed id
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Reference
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Q.Xie,
N.Tang,
R.Wan,
Y.Qi,
X.Lin,
and
J.Lin
(2011).
Recombinant snake venom cystatin inhibits the growth, invasion and metastasis of B16F10 cells and MHCC97H cells in vitro and in vivo.
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Toxicon,
57,
704-711.
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E.Crowley,
M.L.O'Mara,
I.D.Kerr,
and
R.Callaghan
(2010).
Transmembrane helix 12 plays a pivotal role in coupling energy provision and drug binding in ABCB1.
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FEBS J,
277,
3974-3985.
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X.Gu,
X.Jia,
J.Feng,
B.Shen,
Y.Huang,
S.Geng,
Y.Sun,
Y.Wang,
Y.Li,
and
M.Long
(2010).
Molecular modeling and affinity determination of scFv antibody: proper linker peptide enhances its activity.
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Ann Biomed Eng,
38,
537-549.
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E.Crowley,
M.L.O'Mara,
C.Reynolds,
D.P.Tieleman,
J.Storm,
I.D.Kerr,
and
R.Callaghan
(2009).
Transmembrane helix 12 modulates progression of the ATP catalytic cycle in ABCB1.
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Biochemistry,
48,
6249-6258.
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P.Lu,
and
M.G.Feng
(2008).
Bifunctional enhancement of a beta-glucanase-xylanase fusion enzyme by optimization of peptide linkers.
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Appl Microbiol Biotechnol,
79,
579-587.
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S.Matsuzawa,
M.Cuddy,
T.Fukushima,
and
J.C.Reed
(2005).
Method for targeting protein destruction by using a ubiquitin-independent, proteasome-mediated degradation pathway.
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Proc Natl Acad Sci U S A,
102,
14982-14987.
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R.K.Gaur,
M.B.Kupper,
R.Fischer,
and
K.M.Hoffmann
(2004).
Preliminary X-ray analysis of a human V(H) fragment at 1.8 A resolution.
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Acta Crystallogr D Biol Crystallogr,
60,
965-967.
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R.Niv,
Y.G.Assaraf,
D.Segal,
E.Pirak,
and
Y.Reiter
(2001).
Targeting multidrug resistant tumor cells with a recombinant single-chain FV fragment directed to P-glycoprotein.
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Int J Cancer,
94,
864-872.
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Y.Heike,
K.Kasono,
C.Kunisaki,
S.Hama,
N.Saijo,
T.Tsuruo,
D.A.Kuntz,
D.R.Rose,
and
D.T.Curiel
(2001).
Overcoming multi-drug resistance using an intracellular anti-MDR1 sFv.
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Int J Cancer,
92,
115-122.
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A.Goel,
D.Colcher,
J.S.Koo,
B.J.Booth,
G.Pavlinkova,
and
S.K.Batra
(2000).
Relative position of the hexahistidine tag effects binding properties of a tumor-associated single-chain Fv construct.
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Biochim Biophys Acta,
1523,
13-20.
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J.M.van Den Elsen,
D.A.Kuntz,
F.J.Hoedemaeker,
and
D.R.Rose
(1999).
Antibody C219 recognizes an alpha-helical epitope on P-glycoprotein.
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Proc Natl Acad Sci U S A,
96,
13679-13684.
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PDB code:
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The most recent references are shown first.
Citation data come partly from CiteXplore and partly
from an automated harvesting procedure. Note that this is likely to be
only a partial list as not all journals are covered by
either method. However, we are continually building up the citation data
so more and more references will be included with time.
Where a reference describes a PDB structure, the PDB
code is
shown on the right.
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