Mutations that cover the sequence of Bacillus stearothermophilus alpha-amylase were produced by an efficient in vitro enzymatic random mutagenesis method and the mutant alpha-amylases were expressed in Escherichia coli, which also secreted the product. Ninety-eight mutants were identified by sequencing and their enzyme activities were classified into three classes: wild-type, reduced or null. A molecular model of the enzyme was constructed using the coordinates of Takaamylase A and a consensus alignment of mammalian, plant, and bacterial alpha-amylases. The location of mutant amino acids on the model indicate that mutations which destroy or decrease the catalytic activity are particularly clustered: (i) around the active site and along the substrate-binding groove and (ii) in the interface between the central alpha/beta barrel and the C-terminal domain. Exposed loops are typically tolerant towards mutations.

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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The monoclonal antibodies so far developed by hybridoma technology have not had high enough specificity or affinity to distinguish the closely related steroid hormones in routine clinical assays. We have employed random mutagenesis and phage display approaches to improve the specificity of one anti-testosterone monoclonal antibody (3-C4F5). The affinity of the antibody is 0.3 x 10(9) M(-1) and the cross-reactivities with most of the related steroids are low. However, the antibody cross-reacts about 1% with dehydroepiandrosterone sulfate (DHEAS) and owing to the high DHEAS serum concentration this is about 1000-fold too high for clinical immunoassays. The complementarity-determining regions (CDRs) of the heavy and light chains, which were predicted by molecular modelling to be in close contact with the testosterone (TES) ligand, were randomized and mutant Fab libraries were cloned into a phagemid vector. Binders were selected by a competitive panning procedure. By combining the identified light and heavy chain CDRIII mutations the TES affinity was preserved at the wild-type level but DHEAS cross-reactivity was decreased to 0.03%. An important finding was that by the competitive panning procedure the overall binding specificity of the 3-C4F5 antibody was refined, since the cross-reactivities to related steroids were also significantly decreased in the combined mutant.

Recombinant anti-testosterone wild-type Fab fragment and mutant Fab fragments with high binding selectivity developed by protein engineering have been crystallized with and without ligands. Crystals of these Fab fragments were obtained by the vapour-diffusion technique at room temperature using solutions of PEG 3350 with various biological buffers and with a wide pH range. So far, five data sets have been collected from crystals of three Fab-antigen complexes and from two uncomplexed Fab fragments, with resolutions ranging from 2.10 to 3.1 A. Crystallization conditions for Fab fragments were found by using modifications of the low ionic strength PEG 3350 series. Suitable concentrations of PEG 400, MPD and glycerol solutions for use as cryoprotectants in PEG 3350 solutions have been determined. One useful observation was that PEG 3350 is able to work alone as a cryoprotectant. The screening protocol used requires a smaller amount of protein material to achieve auspicious pre-crystals than previously. Results support the claim that PEG 3350 is more suitable for the crystallization of Fab fragments than higher molecular weight PEGs.