X-ray studies of recombinant anti-testosterone Fab fragments: the use of PEG 3350 in crystallization

Valjakka, Jarkko; Hemminki, Ari; Teerinen, Tuija; Takkinen, Kristiina; Rouvinen, Juha

doi:10.1107/s0907444999016224

Cited by 10 publications

(9 citation statements)

References 8 publications

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“…Therefore, it was necessary to collect data by using cryotechniques. After soaking in the cryoprotectant solution (18), the crystals were quickly placed in a nitrogen gas coldstream maintained at 120 K using an Oxford Cryosystems Cryostream cooler. The crystal to image plate distance was 120 mm, and the oscillation range was 0.5°in the bound crystal and 1°in the free crystal.…”

Section: Table II Affinities and Cross-reactivities Of The Mutant Antmentioning

confidence: 99%

Crystal Structure of an in Vitro Affinity- and Specificity-matured Anti-testosterone Fab in Complex with Testosterone

Valjakka

Hemminki

Niemi

et al. 2002

Journal of Biological Chemistry

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A highly selective, high affinity recombinant anti-testosterone Fab fragment has been generated by stepwise optimization of the complementarity-determining regions (CDRs) by random mutagenesis and phage display selection of a monoclonal antibody (3-C 4 F 5 ). The best mutant (77 Fab) was obtained by evaluating the additivity effects of different independently selected CDR mutations. The 77 Fab contains 20 mutations and has about 40-fold increased affinity (K d ‫؍‬ 3 ؋ 10 ؊10 M) when compared with the wild-type (3-C 4 F 5 ) Fab. To obtain structural insight into factors, which are needed to improve binding properties, we have determined the crystal structures of the mutant 77 Fab fragment with (2.15 Å) and without testosterone (2.10 Å) and compared these with previously determined wild-type structures. The overall testosterone binding of the 77 Fab is similar to that of the wild-type. The improved affinity and specificity of the 77 Fab fragment are due to more comprehensive packing of the testosterone with the protein, which is the result of small structural changes within the variable domains. Only one important binding site residue Glu-95 of the heavy chain CDR3 is mutated to alanine in the 77 Fab fragment. This mutation, originally selected from the phage library based on improved specificity, provides more free space for the testosterone D-ring. The light chain CDR1 of 77 Fab containing eight mutations has the most significant effect on the improved affinity, although it has no direct contact with the testosterone. The mutations of CDR-L1 cause a rearrangement in its conformation, leading to an overall fine reshaping of the binding site.Steroid hormones have remained a great challenge for immunodiagnostics. They are small, rigid, hydrophobic molecules with only a few functional groups capable of specific interactions with antibodies. The number of different closely related steroids in human serum is high, their in vivo concentrations are low (down to a picomolar level), and their relative concentrations can vary greatly, even between normal healthy individuals. Furthermore, steroids are poorly immunogenic in mice and rats; two species from which monoclonal antibodies are usually generated, making it extremely difficult to produce high affinity and specificity anti-steroid monoclonal antibodies. In most diagnostic immunoassays of steroid hormones, rabbit polyclonal antibodies are used, despite the many drawbacks associated with the use of antisera. The supply of good polyclonal antibody reagents of uniform quality is a severe problem for the immunodiagnostic industry and requires continuous immunization of many laboratory animals.Antibody engineering provides excellent tools to tailor the properties of antibodies with respect to affinity, specificity, and performance for different applications. An in vitro process of antigen-driven selection, based on the display of antibody fragments on the surface of a filamentous bacteriophage, has been shown to be a powerful method for the selection of specific or improved...

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Section: Table II Affinities and Cross-reactivities Of The Mutant Antmentioning

confidence: 99%

Crystal Structure of an in Vitro Affinity- and Specificity-matured Anti-testosterone Fab in Complex with Testosterone

Valjakka

Hemminki

Niemi

et al. 2002

Journal of Biological Chemistry

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“…Crystallization-Crystals of the free and testosterone-complexed 3-C 4 F 5 Fab fragment were grown as described previously (16). Crystals without testosterone were grown by the hanging-drop method, from 9 mg/ml protein solutions buffered with 0.1 M BTP (Bis-Tris propane) (pH 5.6) and using 15% PEG3350 as the precipitant.…”

Section: Methodsmentioning

confidence: 99%

Structural Insights into Steroid Hormone Binding

Valjakka¹,

Takkinenz²,

Teerinen³

et al. 2002

Journal of Biological Chemistry

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The monoclonal anti-testosterone antibody (3-C 4 F 5 ) has a relatively high affinity (3 ؋ 10 8 M ؊1 ) with an overall good specificity profile. However, the earlier characterized binding properties have shown that both the affinity and specificity of this antibody must be improved if it is intended for use in clinical immunoassays. In this paper, the crystal structures of the recombinant antitestosterone (3-C 4 F 5 ) Fab fragment have been determined in the testosterone-bound and free form at resolutions of 2.60 and 2.72 Å, respectively. The high affinity binding of the (3-C 4 F 5 ) Fab is mainly determined by shape complementarity between the protein and testosterone. Only one direct hydrogen bond is formed between the hydroxyl group of the testosterone D-ring and the main-chain oxygen of Gly100 J H. The testosterone is deeply bound in a hydrophobic pocket, and the close shape complementarity is mainly formed by the third complementarity-determining regions ( The number of different, closely related steroid hormones in human serum is high, and their relative concentrations, which usually are very low, can vary greatly even between normal healthy individuals. Testosterone, the main male sex hormone, is one of the structurally similar steroid hormones. Measurement of serum testosterone levels is important in the evaluation of conditions associated with hyperandrogenism in women (1, 2) and to diagnose hypogonadism, impotence, and problems in spermatogenesis and pubertical development in men (3, 4). Development of monoclonal antibodies that would fulfill the high affinity and selectivity requirements needed for the accurate measurement of testosterone levels in serum samples has not been successful with the use of hybridoma technology. Rabbit polyclonal antibodies are used in most current immunoassays of steroid hormones. However, the supply of polyclonal reagents with consistent quality is a severe problem for the immunodiagnostic industry and requires continuous immunization of many animals. The inherent batch-to-batch variation of the polyclonal sera makes it necessary to optimize the assay parameters for each new serum batch.The three-dimensional structures of steroid binding Fab fragments are important for a general understanding of the molecular basis of specific interactions between antibodies and hydrophobic molecules such as steroids. The molecular basis of steroid antibody interactions has been studied in detail by determining the structures of a progesteronebinding antibody (DB3) in its free and bound form and with different high affinity cross-reactive progesterone derivatives (5-7). The binding site of the DB3 antibody is a hydrophobic pocket, and the binding affinity and specificity are determined by van der Waals interactions and a few hydrogen bonds formed with the progesterone hapten (5). The high affinity binding mode of a number of progesterone derivatives is due to two alternate docking orientations for the steroid skeleton (6). In the cases of the anti-digoxin 26-10 and 40-50 antibodies, the h...

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“…The droplets were allowed to equilibrate against 500 ml reservoir solution. Initial crystallization conditions were found using the modified crystal screen PEG 3350 series for antibodies (Valjakka et al, 2000). Needlelike crystals (Fig.…”

Section: Crystallizationmentioning

confidence: 99%

Characterization and crystallization of a recombinant IgE Fab fragment in complex with the bovine β-lactoglobulin allergen

et al. 2007

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A D1 Fab fragment containing the allergen-binding variable domains of the IgE antibody was characterized by ESI FT-ICR mass spectrometry and crystallized with bovine -lactoglobulin (BLG) using the hanging-drop vapour-diffusion method at 293 K. X-ray data suitable for structure determination were collected to 2.8 Å resolution using synchrotron radiation. The crystal belonged to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 67.0, b = 100.6, c = 168.1 Å . The three-dimensional structure of the D1 Fab fragment-BLG complex will provide the first insight into IgE antibody-allergen interactions at the molecular level.

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X-ray studies of recombinant anti-testosterone Fab fragments: the use of PEG 3350 in crystallization

Cited by 10 publications

References 8 publications

Crystal Structure of an in Vitro Affinity- and Specificity-matured Anti-testosterone Fab in Complex with Testosterone

Crystal Structure of an in Vitro Affinity- and Specificity-matured Anti-testosterone Fab in Complex with Testosterone

Structural Insights into Steroid Hormone Binding

Characterization and crystallization of a recombinant IgE Fab fragment in complex with the bovine β-lactoglobulin allergen

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