Beruflich Dokumente
Kultur Dokumente
Received: 10 December 2009, Revised: 23 February 2010, Accepted: 24 February 2010, Published online in Wiley Online Library: 4 May 2010
(wileyonlinelibrary.com) DOI:10.1002/jmr.1039
Keywords: antibody library; crystal structure; fab fragment; hapten; steroid hormone; twinning
J. Mol. Recognit. 2011; 24: 209–219 Copyright ß 2010 John Wiley & Sons, Ltd.
M. H. NIEMI ET AL.
MATERIALS AND METHODS 15 min 48C) and the culture supernatant concentrated to a final
volume of 1 L using a Pellicon concentrator (Millipore) and a
Isolation of the 5F2 anti-testosterone clone cellulose filter with a 10 kDa cut-off (Millipore). During the
concentration, the buffer was exchanged to PBS (pH 7.4). The Fab
The VTT naı̈ve human scFv phage display libraries VHVk and VHVl fragment was purified using a Cu2þ-Chelating Sepharose
(both containing approximately 108 clones) have been cloned Streamline column (Amersham-Pharmacia) and a linear gradient
from a pool of human lymphocytes of 50 healthy donors elution (20–500 mM imidazole in PBS, pH 7.4). Fractions
(Reinman et al., 2003; Pulli et al., 2005; Weber et al., 2005; Turunen containing the Fab fragment were pooled and the buffer was
et al., 2009). scFv libraries were enriched against a testoster- exchanged to PBS.
one-3-CMO-BSA conjugate (TES–BSA, Sigma) in two panning The affinity of 5F2 Fab was determined by ELISA by coating
rounds. Briefly, libraries were electroporated into XL-1 blue microtiter plate wells with 0.3 mg of TES–BSA in a 0.1 M bicarbonate
Escherichia coli cells, and phage particles were rescued by buffer (pH 9.6) for 16 h at 48C. The wells were then washed three
infection of the bacteria with the helper phage VCSM13 times with PBS and blocked with 200 ml of PBS–0.5% BSA for 1 h at
(Stratagene). Libraries were incubated for 18 h at þ48C in BSA RT and washed. The samples containing 5F2 Fab with soluble
coated wells (to deplete the BSA binders) and transferred into testosterone were prepared by mixing 900 ng Fab 5F2 in 450 ml of
(TES–BSA, 1 mg/100 ml/well) coated wells. After 1 h incubation at PBS-0.5% BSA and 50 ml of increasing concentrations of testoster-
RT, the wells were washed 20 times with PBS and the bound one (0, 106, 105, 104, 103, 102, 101, 1, 10, 100 and 10 000 mM)
phages were eluted with 100 mM glycine–HCl buffer pH 2.2 and in PBS in test tubes. An aliquot of 100 ml of each sample was
after neutralization used to reinfect E. coli. pipetted into wells, four parallel measurements were made. The
samples were incubated at room temperature for 1 h. After the
ELISA screening of E. coli colonies producing scFv-fragments washing step (three times with PBS), the detection was carried out
After the second selection round, single colonies were picked by with a goat anti-human F(ab)2 alkaline phosphatase conjugate
the QPix colony picker (Genetix) into 96-well plates containing (Rockland) diluted by 1:2000 in PBS–0.5% BSA. After a 1 h incubation
200 ml/well of SB-media with 100 mg/ml of ampicillin, 10 mg/ml of at RT, the wells were washed and 100 ml of 2 mg/mL of
tetracycline and 1% glucose. The colonies were grown o/n at p-nitrophenylphosphate (Sigma) in diethanolamine–MgCl2 buffer
þ308C with 700 rpm and 85% humidity in a shaker (InforsMulti- (Reagena) was added to the wells. The absorbance was measured at
tron). 150 ml of the culture suspension was transferred into new 405 nm after 30 min (Varioscan, Thermo Electron Corporation).
plates. The o/n cultivation plates containing the rest of the
inoculm cultivation (50 ml) were stored as the master plates at Crystallization and X-ray data collection
48C. Culture suspensions transferred to the new plates were
centrifuged for 10 min at 2000 g (Eppendorf Centrifuge 5810R) The anti-testosterone 5F2 Fab fragment was co-crystallized
and the culture supernatant was removed. The cells were with testosterone by using the hanging-drop vapour-diffusion
resuspended into fresh SB-media (200 ml) supplemented with method. Favourable crystallization conditions were found using
ampicillin (100 mg/ml), tetracycline (10 mg/ml) and 1 mM iso- the modified PEG3350 series for antibodies (Valjakka et al., 2000).
propyl b-D-thiogalactopyranoside and the plates were grown for 5F2 Fab crystals suitable for X-ray analysis were obtained by
20 h, as before. The binding activities of the soluble scFv–myc–pIII combining the streak-seeding method with nanospheres (Kallio
fusion proteins released into the culture supernatant were et al., 2009). Briefly, the crystallization droplets contained 2 ml of
analysed by ELISA. As a control for the screening assay, the protein solution (8.3 mg/ml in 20 mM HEPES buffer, pH 7.0), 0.5 ml
anti-testosterone 77 Fab fragment was inoculated into four wells of testosterone solution (5 mM in 50% ethanol), 1 ml of nano-
per plate and used as a control for the testosterone binding. ELISA sphere solution (50 nm in diameter, 1:1 dilution to water) and 2 ml
was performed using TES–BSA coated wells and mouse anti-myc of precipitant solution (12% v/w PEG3350, 0.1 M sodium citrate
antibody (clone 9E10, ATCC # CRL-1729) and goat anti-mouse at pH 4.7). The protein droplets were allowed to pre-equilibrate
IgG-AP conjugated antibody (Bio-Rad) for detection (detecting for 24 h and were then seeded by transferring crystal nuclei from
also the expressed 77 Fab fragment). previously grown 5F2 Fab crystal clusters. Single crystals reached
The scFv coding regions of the positive clones were sequenced the approximate dimensions of 0.2 0.1 0.1 mm3.
by an ABI 3100 Genetic Analyser using a BigDye 3.0 Sequence For data collection, the crystals were transferred to a
Termination Kit (Applied Biosystems). cryoprotective solution (12% PEG3350, 20% glycerol, 0.1 M
sodium citrate). After cryoprotection, crystals were frozen by
plunging them into liquid nitrogen. The X-ray data were collected
Production, purification and characterization of the
from a single crystal that diffracted to a resolution of 1.5 Å on the
anti-testosterone 5F2 Fab fragment
beamline ID-29 at the ESRF (Grenoble, France). The crystal was
For further characterization, the 5F2 scFv was converted to a Fab exposed for 2 s with a 1.08 oscillation per frame. Reflections were
fragment by cloning the genes encoding human IgG1 CH1- and recorded with an ADSC Q315R detector. The diffraction data were
Ck-constant regions in frame with VH- and VL-regions, respect- processed with XDS and scaled with XSCALE (Kabsch, 1993).
ively. The genes encoding the 5F2 Fab fragment were cloned into
the E. coli expression vector (pKKtac) containing a hexahistidine
Structure solution from twinned 5F2 Fab crystal
tag at the C-terminus of the heavy chain constant region for
immobilized metal ion affinity chromatography (IMAC)- The data were initially processed in the monoclinic space group
purification. High cell density cultivation of the 5F2 Fab fragment C2 with the unit cell parameters a ¼ 101.7, b ¼ 87.4, c ¼ 66.7 Å
was carried out in a 4.5-L fermenter using E. coli RV308 strain and b ¼ 117.88. The diffraction intensity statistics analysis
(ATCC 31608), as described (Nevanen et al., 2001). After the performed with the program TRUNCATE from the CCP4 package
cultivation, the cells were harvested by centrifugation (3963 g, (Collaborative Computational Project, Number 4, 1994) did not
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wileyonlinelibrary.com/journal/jmr Copyright ß 2010 John Wiley & Sons, Ltd. J. Mol. Recognit. 2011; 24: 209–219
THE TESTOSTERONE BINDING MECHANISM
J. Mol. Recognit. 2011; 24: 209–219 Copyright ß 2010 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jmr
M. H. NIEMI ET AL.
Figure 1. Screening individual scFv clones and analysis of the binding affinity of the 5F2 Fab by ELISA. (A) The culture supernatant samples of 92
individual clones producing soluble scFv–myc–pIII fusion proteins and four anti-testosterone 77 Fab producing colonies (marked with a black circle) were
analysed on TES–BSA coated wells. Five scFv clones (marked with an asterisk) showing higher binding response than 77 Fab were selected for further
characterization. (B) The percentage of the total binding represents the value obtained by dividing the mean binding signal, an average of four parallel
measurements, at each testosterone concentration by the binding response without the competing testosterone. The IC50value of 5F2 Fab, equal to the
testosterone concentration at which the binding response is 50% of the maximum, is 900 nM.
domains (Lefranc et al., 2003). The light chain of 5F2 Fab belongs to side-chain of Trp118H and the main-chain oxygen of Met115H
the l class. All three CDR loops of the light chain and the CDR-1 and stabilize the bulged conformation in the torso region (Morea et al.,
CDR-2 loops of the heavy chain could be classified into the 1998). The tip of the CDR-H3 loop contains two hydrophobic
common canonical structures of immunoglobulins (Al-Lazikani residues, Leu110H and Trp111H, which form a hydrophobic patch
et al., 1997). CDR-L1 loop (9 residues) represents canonical class 2 on the antibody surface.
with a slightly distorted a-helix. CDR-L2 loop consists of three
residues and belongs to canonical class 1. CDR-L3 contains 11
Testosterone binding site
residues and resembles the structures of canonical class 2, though
Pro115L breaks the typical hydrogen bond pattern. The heavy Testosterone is bound in a deep pocket, which is located between
chain CDR-H1 (8 residues) has canonical structure 1. Arg80H from the variable domains of the light chain and the heavy chain of an
b-strand 4 is important for the structure of this loop, by forming antibody (Figure 3). The contacts between 5F2 Fab and
hydrogen bonds with main chain oxygens of Phe30H and Tyr37H. testosterone are given in Table 2. The interface area of 5F2
In CDR-H2 (8 residues), three glycine residues (Gly–Ser–Gly–Gly) Fab in complex structure is 233 Å2, covering 37 Å2 (16%) of the
are found between the Ser57H and Ser64H and the loop was light chain and 196 Å2 (84%) of the heavy chain molecular
categorized into canonical class 3. The CDR-H3 loop of the surfaces. Thus, the heavy chain interacts predominantly with the
antibody consists of 15 amino acid residues. A salt bridge between hapten, making 76% of all atom-atom contacts. The D-ring of
Arg106H and Asp116H and a hydrogen bond between the the testosterone molecule is directed toward the bottom of the
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THE TESTOSTERONE BINDING MECHANISM
binding pocket and the amino acids interact with the A-ring and
the B-ring of the steroid skeleton. The 3-keto group oxygen of
the testosterone A-ring is close (3.3 Å) to the hydroxyl group of
the Tyr66H side chain. However, because of an unfavourable
geometry, this hydrogen bond is weak.
DISCUSSION
A 5F2 anti-testosterone antibody with a moderate testosterone
binding affinity was isolated from a naı̈ve human scFv library
(108 clones). The affinity of the 5F2 clone correlates well with
the other published examples of steroid binding antibodies
isolated from naı̈ve or designed sources of similar diversities
(Dörsam et al., 1997; Persson et al., 2006). In order to obtain an
insight into the testosterone binding mechanism of this
non-affinity maturated antibody, the crystal structure of the
5F2 Fab in complex with testosterone was determined.
J. Mol. Recognit. 2011; 24: 209–219 Copyright ß 2010 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jmr
M. H. NIEMI ET AL.
Figure 3. The binding of testosterone to 5F2 Fab. (A) The binding of testosterone in stereo. The 5F2 Fab light chain is in blue, the heavy chain in green.
The residues interacting with testosterone are shown in light grey and testosterone in dark grey. (B) The chemical structure of testosterone and an
overview of the binding site interactions.
the CDR-H1 is in contact with testosterone through Ser35. Upon The light chain of the 5F2 Fab has only few contacts with the
hapten binding, 88 Å2 of the light chain (38%) and 146 Å2 of the testosterone molecule. Two residues are forming part of the
heavy chain (62%) solvent accessible surfaces of 77 Fab are testosterone binding site both in 5F2 and 77 Fab. Pro115L of 5F2
buried. Fab corresponds to Val114L in 77 Fab, and Tyr116L is replaced by
Three of the testosterone contacting residues of the 5F2 Fab proline in 77 Fab. However, when the side-chain of Arg32L has a
heavy chain are identical with the Fab 77 residues. Trp52H, Tyr prominent role in the formation of the binding site in 77 Fab, this
66H and Gly114H, contribute to the hapten binding in both position is occupied by the side-chain of Tyr113H in 5F2 Fab
antibodies. The positions of three contacting residues are (Figure 4).
common in 5F2 and 77 Fabs. Ala55H of 5F2 Fab corresponds 77 Fab binds testosterone in sandwich between the aromatic
to Ser55H in 77 Fab, as well as Met115H corresponds to Leu115H, amino acid residues Trp52H and Tyr112H. In the 5F2 Fab
respectively. In 5F2 Fab, Asp107H forms a hydrogen bond with structure the amino acid residues Trp52H and Tyr112aH are in
the testosterone and it is significant for the recognition of ligand the corresponding positions at the binding site. However, the
but 77 Fab has Ala107H at this position. A comparison of the orientation of the bound testosterone is different between
heavy chain contacts of 5F2 and 77 Fabs revealed also some these two testosterone binding Fab fragments. In the 5F2 Fab
differences. Both 5F2 and 77 Fab have Ser40H, but this residue is the testosterone is turned almost 908C in comparison with the
in contact with testosterone only in 77 Fab (Table 2, Figure 6). The location of testosterone in the binding site of 77 Fab (Figures 4
CDR-H3 loop is three residues longer in 5F2 compared to 77 Fab, and 5A). In 77 Fab, the a-side of testosterone packs against
in consequence, the conformation of the CDR-H3 loops are Tyr112H and the methyl groups on the b-side of testosterone
different. However, Tyr112aH of 5F2 Fab superimposes well with point toward the framework residue Trp52H, whereas in 5F2
Tyr112H of 77 Fab. Tyrosine at position 113H has a substantial Tyr112aH and Trp52H are in contact with the edges of the
effect on the shape of the binding pocket of 5F2. In Fab 77, this testosterone molecule. The different orientation of testoster-
position is occupied by a non-contacting residue valine (Figures 4 one in the binding pocket of 5F2 Fab most probably is due
and 6). to the amino acid residues of Tyr116L and Tyr113H, whose
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THE TESTOSTERONE BINDING MECHANISM
location enables favourable interactions with the a-side of buried water molecule. Thus, the hydrogen bond interactions and
testosterone. especially better shape complementarity seems to explain the
The relative testosterone binding affinity of 77 Fab is higher testosterone binding capacity of 77 Fab.
approximately 3 1010 M, which is around 300-fold higher
than that of 5F2 Fab (Kd 107 M). In the 77 Fab-testosterone
Comparison to other steroid-binding antibodies
complex, one direct hydrogen bond between the main chain
oxygen of Gly114H and the 17-hydroxyl group of testosterone To date, several crystals structures of steroid binding antibodies
D-ring is formed. The 17-hydroxyl group of testosterone also have been solved. The three-dimensional structures have been
makes indirect hydrogen bonds to Ala38H and Ser40H through a determined for three anti-estradiol antibodies [57-2, 17E12 and
Figure 4. Comparison of the testosterone binding site of two antibodies. (A) The binding site of the 5F2 Fab. (B) The binding site of the 77 Fab. The
molecular surface of the light chain is in blue and the heavy chain in green. Testosterone is shown as a grey stick model.
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M. H. NIEMI ET AL.
Figure 5. Antibody–steroid complexes, superimposed with 5F2 Fab–testosterone complex (shown in grey). (A) 77/testosterone (PDB code 1vpo),
(B) 1E9dm/5b-androstane-3,17-dione (2o5z), (C) 26-10/digoxin (1igj) and (D) 10G6/estradiol-6-CMO (1jnh).
10G6 (Lamminmäki and Kankare, 2001; Monnet et al., 2002)], an mutated residue Trp52H is not in direct contact with haptens, but
anti-progesterone antibody DB3 (Arevalo et al., 1994), two it has a substantial effect on the shape of the binding pocket of
anti-digoxin antibodies (26-10 and 40-50 (Jeffrey et al., 1993; 1E9dm Fab. The depth of the binding pocket and the relative
Jeffrey et al., 1995), a glucuronide binding antibody Fv4155 position of 5b-androstane-3,17-dione with the D-ring being
fragment (Trinh et al., 1997) and a mutated catalytic Diel- deepest buried, resembles the 5F2 Fab-testosterone complex
s-Alderase antibody Fab fragment 1E9dm (Verdino et al., 2008). structure (Figure 5B). Similarly, with 5F2 Fab, the role of the
The relative orientation of the bound steroid molecule, number of antibody heavy chain in the complex formation is significant in
hydrogen bonds between the hapten and antibody and the 1E9dm-steroid structures (72 and 28% for the heavy and the light
contribution of the antibody heavy chain to steroid binding vary chain for both structures).
among these structures. By comparing the overall binding The 26-10 Fab-digoxin complex (Jeffrey et al., 1993) is another
orientation of steroid molecules three antibodies, 1E9dm, 20-10, example of a steroid binding antibody, where the heavy chain
and 10G6, show overall similarity with 5F2 Fab and were selected is responsible for the majority of the contacts between the
for more detailed comparison. In these antibody structures, the antibody and the hapten. Upon hapten binding, up to 82% of
steroid skeleton of the hapten molecule is approximately parallel the heavy chain surface buries. Two heavy chain CDR loops make
to the interface area of the variable domains (Jeffrey et al., 1995). contacts with a digoxin molecule, while the light chain of the
The anti-progesterone antibody DB3 and the catalytic antibody is in contact with the hapten through two residues from
Diels-Alderase antibody 1E9 derived from the same germ line the CDR-L3 loop. Also, in this case, the recognition of digoxin
family share a high sequence identity (91%) with distinct by Fab 26-10 is based on the surface complementarity, since
functional characters. Two mutations (Leu52HTrp and hydrogen bonds or salt bridges between the antibody and
Arg113HTrp) directed to the binding site residues of 1E9 Fab hapten are not formed. The location of a steroid skeleton of the
were sufficient to improve the steroid binding affinity of 1E9 Fab. hapten molecule is close to the testosterone in the 5F2 Fab
The crystal structures of the 1E9 double mutant Fab (1E9dm) in structure (Figure 5C). In the 26-10 Fab-digoxin complex, the
complex with progesterone and 5b-androstane-3,17-dione D-ring of the steroid molecule with the C-17 attached lactone ring
showed the major role of hydrophobic interactions in the is buried deepest in the binding pocket.
complex formation (Verdino et al., 2008). In both 1E9dm Fab The location of a bound steroid molecule is also similar in
complex structures, the hapten molecule is bound in a sandwich monoclonal anti-estradiol Fab 10G6, which has been crystallized
between the amino acid residues Trp55H and Trp113H. The in complex with estradiol-6-CMO (Monnet et al., 2002). In this
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THE TESTOSTERONE BINDING MECHANISM
Figure 6. Sequence comparison of the variable regions of heavy and light chains of 5F2 antibody with four other steroid-binding antibodies. The
residues participating in the steroid binding are shown in red and locations of the CDRs are indicated.
structure, 87% of the estradiol part of the hapten molecule is shortest CDR-H3 loops (7 and 9 residues) are found in two
buried upon binding. The D-ring with the O17-hydroxyl group is monoclonal anti-estradiol antibodies, Fab 17E12 and Fab 57-2. In
at the bottom of the binding pocket and two hydrogen bonds are these antibodies, the light chain contributes as much as the
formed between the 10G6 and estradiol-6-CMO. Three trypto- heavy chain to the hapten binding. The length of the CDR-H3
phan residues, Trp38H, Trp52H and Trp107L, surround the bound loop of the 5F2 Fab is longer, 15 residues. This is in line with the
hapten but they do not form a sandwich structure (Figure 5D). finding that human antibodies frequently have longer CDR-H3
Also, in this case, the heavy chain dominates the interactions loops than corresponding mouse antibodies (Collis et al., 2003).
between the antibody and hapten. The amino acid residues that are involved in steroid binding
The sequence comparison of the Fab region of the 5F2 can often be found in particular positions. Most of the
antibody with four other steroid binding antibodies is shown in steroid-specific antibodies are in contact with the hapten
Figure 6. The comparison reveals the importance of the CDR-H3 molecule through residues L107, L116, H40, H55, H107 and
loop, which is a commonly observed feature in antigen/hapten H115 (IMGT numbering). Another common feature in steroid
binding. binding is the utilization of aromatic residues. In particular, two
The average length of the CDR-H3 region in steroid binding positions from the heavy chain frequently carry aromatic
antibodies is 12 amino acid residues. The lengths of CDR-H3 loops side chains, 52H and 113H (Trp and Tyr in 5F2, respectively).
vary from 7 residues (Fab 17E12) to 15 residues (Fab 40-50). The The indole side-chain of tryptophan at position 113H is a part of
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M. H. NIEMI ET AL.
REFERENCES
Adams PD, Grosse-Kunstleve RW, Hung L-W, Ioerger TR, McCoy AJ, immunoglobulin and T cell receptor variable domains and IG super-
Moriarty NW, Read RJ, Sacchettini JC, Sauter NK, Terwilliger TC. family V-like domains. Dev. Comp. Immunol. 27: 55–77.
2002. PHENIX: building new software for automated crystallographic Lefranc M-P, Pommié C, Kaas Q, Duprat E, Bosc N, Guiraudou D, Jean C, Ruiz
structure determination. Acta Crystallogr. D58: 1948–1954. M, Da Piédade I, Rouard M, Foulquier E, Thouvenin V, Lefranc G. 2005.
Al-Lazikani B, Lesk AM, Chothia C. 1997. Standard conformations for the IMGT unique numbering for immunoglobulin and T cell receptor
canonical structures of immunoglobulins. J. Mol. Biol. 273: 927–948. constant domains and Ig superfamily C-like domains. Dev. Comp.
Arevalo JH, Hassig CA, Stura EA, Sims MJ, Taussig MJ, Wilson IA. 1994. Immunol. 29: 185–203.
Structural analysis of antibody specificity-detailed comparison of five Matthews BW. 1968. Solvent content of protein crystals. J. Mol. Biol. 33:
Fab0 -steroid complexes. J. Mol. Biol. 241: 663–690. 491–497.
Collaborative Computational Project, Number 4. 1994. The CCP4 suite: Monnet C, Bettsworth F, Stura EA, Le Du M-H, Ménez R, Derrien L,
programs for protein crystallography. Acta Crystallogr. D50: 760–763. Zinn-Justin S, Gilquin B, Sibai G, Battail-Poirot N, Jolivet M, Ménez
Collis AVJ, Brouwer AP, Martin ACR. 2003. Analysis of the antigen com- A, Arnaud M, Ducancel F, Charbonnier JB. 2002. Highly specific
bining site: correlations between length and sequence composition anti-estradiol antibodies: structural characterisation and binding
of the hypervariable loops and the nature of the antigen. J. Mol. Biol. diversity. J. Mol. Biol. 315: 699–712.
325: 337–354. Morea V, Tramontano A, Rustici M, Chothia C, Lesk AM. 1998. Confor-
Dörsam H, Rohrbach P, Kürschner T, Kipriyanov S, Renner S, Braunagel M, mations of the third hypervariable region in the VH domain of
Welschof M, Little M. 1997. Antibodies to steroids from a small human immunoglobulins. J. Mol. Biol. 275: 269–294.
naive IgM library. FEBS Lett. 414: 7–13. Mueller U, Muller YA, Herbst-Irmer R, Sprinzl M, Heinemann U. 1995.
Hemminki A, Niemi S, Hautoniemi L, Söderlund H, Takkinen K. 1998a. Fine Disorder and twin refinement of RNA heptamer double helices. Acta
tuning of an anti-testosterone antibody binding site by stepwise Crystallogr. D55: 1405–1413.
optimisation of the CDRs. Immunotechnology 4: 59–69. Nevanen TK, Söderholm L, Kukkonen K, Suortti T, Teerinen T, Linder M,
Hemminki A, Niemi S, Hoffrén A-M, Hakalahti L, Söderlund H, Takkinen K. Söderlund H, Teeri TT. 2001. Efficient enantioselective separation of
1998b. Specificity improvement of a recombinant anti-testosterone drug enantiomers by immobilised antibody fragments. J. Chroma-
Fab fragment by CDRIII mutagenesis and phage display selection. togr. A925: 89–97.
Protein Eng. 11: 311–319. Persson H, Lantto J, Ohlin M. 2006. A focused antibody library for
Hoogenboom HR. 2005. Selecting and screening recombinant antibody improved hapten recognition. J. Mol. Biol. 357: 607–620.
libraries. Nat. Biotechnol. 23: 1105–1116. Persson H, Wallmark H, Ljungars A, Hallborn J, Ohlin M. 2008. In vitro
Jeffrey PD, Strong RK, Sieker LC, Chang CYY, Campbell RL, Petsko GA, evolution of an antibody fragment population to find high-affinity
Haber E, Margolies MN, Sheriff S. 1993. 26-10 Fab-Digoxin Complex: hapten binders. Protein Eng. Des. Sel. 21: 485–493.
affinity and specificity due to surface complementarity. Proc. Natl. Pope A, Pritchard K, Williams A, Roberts A, Hackett JR, Mandecki W,
Acad. Sci. 90: 10310–10314. Johnson KS. 1996. In vitro selection of a high affinity antibody to
Jeffrey PD, Schildbach JF, Chang CYY, Kussie PH, Margolies MN, Sheriff S. oestradiol using a phage display human antibody library. Immuno-
1995. Structure and specificity of the anti-digoxin antibody 40-50. J. technology 2: 209–217.
Mol. Biol. 248: 344–360. Pulli T, Höyhtyä M, Söderlund H, Takkinen K. 2005. One-step homo-
Kabsch WJ. 1993. Automatic processing of rotation diffraction data from geneous immunoassay for small analytes. Anal. Chem. 77: 2637–2642.
crystals of initially unknown symmetry and cell constant. J. Appl. Reinman M, Jäntti J, Alfthan K, Keränen S, Söderlund H, Takkinen K. 2003.
Cryst. 26: 795–800. Functional inactivation of the conserved Sem1p in yeast by intra-
Kallio JM, Hakulinen N, Kallio JP, Niemi MH, Kärkkäinen S, Rouvinen J. 2009. bodies. Yeast 20: 1071–1084.
The contribution of polystyrene nanospheres towards the crystal- Trinh CH, Hemmington SD, Verhoeyen ME, Phillips SEV. 1997. Antibody
lization of proteins. PLoS ONE 4: e4198. fragment Fv4155 bound to two closely related steroid hormones: the
Karpusas M, Ferrant J, Weinreb P, Carmillo A, Taylor F, Garber E. 2003. structural basis of fine specificity. Structure 5: 937–948.
Crystal structure of the alpha 1 beta 1 integrin I domain in complex Turunen L, Takkinen K, Söderlund H, Pulli T. 2009. Automated panning and
with an antibody Fab fragment. J. Mol. Biol. 327: 1031–1041. screening procedure on microtiterplates for antibody generation
Krebs B, Rauchenberger R, Reiffert S, Rothe C, Tesar M, Thomassen E, Cao from phage display libraries. J. Biomol. Screen. 14: 282–293.
M, Dreier T, Fischer D, Höss A, Inge L, Knappik A, Marget M, Pack P, Vagin A, Teplyakov A. 1997. MOLREP: an automated program for mol-
Meng XQ, Schier R, Söhlemann P, Winter J, Wölle J, Kretzschmar T. ecular replacement. J. Appl. Crystallogr. 30: 1022–1025.
2001. High-throughput generation and engineering of recombinant Valjakka J, Hemminki A, Teerinen T, Takkinen K, Rouvinen J. 2000. X-ray
human antibodies. J. Immunol. Methods 254: 67–84. studies of recombinant anti-testosterone Fab fragments: the use of
Lamminmäki U, Kankare JA. 2001. Crystal structure of a recombinant PEG 3350 in crystallization. Acta Crystallogr. D56: 218–221.
anti-estradiol Fab fragment in complex with 17b-estradiol. J. Biol. Valjakka J, Takkinen K, Hemminki A, Niemi S, Söderlund H, Rouvinen J.
Chem. 276: 36687–36694. 2002a. Crystal structure of an in vitro affinity- and specificity-matured
Lefranc M-P, Pommié C, Ruiz M, Giudicelli V, Foulquier E, Truong L, anti-testosterone Fab in complex with testosterone. J. Biol. Chem.
Thouvenin-Contet V, Lefranc G. 2003. IMGT unique numbering for 277: 44021–44027.
218
wileyonlinelibrary.com/journal/jmr Copyright ß 2010 John Wiley & Sons, Ltd. J. Mol. Recognit. 2011; 24: 209–219
THE TESTOSTERONE BINDING MECHANISM
Valjakka J, Takkinen K, Teerinen T, Söderlund H, Rouvinen J. 2002b. Verdino P, Aldag C, Hilvert D, Wilson IA. 2008. Closely related antibody
Structural insights into steroid hormone binding. Crystal structure receptors exploit fundamentally different strategies for steroid recog-
of a recombinant anti-testosterone Fab fragment in free and testos- nition. Proc. Natl. Acad. Sci. 105: 11725–11730.
terone-bound forms. J. Biol. Chem. 277: 4183–4190. Weber V, Linsberger I, Ettenauer M, Loth F, Höyhtyä M, Falkenhagen D.
Vaughan TJ, Williams AJ, Pritchard K, Osbourn JK, Pope AR, Earnshaw 2005. Development of specific adsorbents for human tumor necrosis
JC, McCafferty J, Hodits RA, Wilton J, Johnson KS. 1996. Human factor-alpha: influence of antibody immobilization on performance
antibodies with sub-nanomolar affinities isolated from a large and biocompatibility. Biomacromolecules 6: 1864–1870.
non-immunized phage display library. Nat. Biotechnol. 14: Westbrook J, Feng Z, Burkhardt K, Berman HM. 2003. Validation of protein
309–314. structures for protein data bank. Methods Enzymol. 374: 370–385.
219
J. Mol. Recognit. 2011; 24: 209–219 Copyright ß 2010 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jmr