Thesis (Ph. D.)--California Institute of Technology, 1993. UM #93-25,374. / Advisor names found in the Acknowledgements pages of the thesis. Title from home page. Viewed 01/15/2010. Includes bibliographical references.

Directed evolution studies were done with PTE for the enhancement of hydrolysis of both sarin and soman analogs. Particular attention was focused on the toxic SpRc and SpSc isomers of the soman analog, and the Sp isomer of the sarin analog. Double substitution libraries yielded several mutants that had enhanced activity for the substrates. Among them was the double mutant, H257Y-L303T, which displayed a 462-fold increase in activity for hydrolysis of the most toxic SpSc isomer of the GD analog in comparison to the wild type. Several other mutants such as the triple mutants H254R-H257A-L303T and H254R-H257S-L303T had enhancements of between 150- and 200-fold, and had also displayed a different order of stereoselectivity relative to the wild type. For these mutants, the order of preferential hydrolysis was such that the SpRc isomer was preferentially hydrolyzed first. In contrast, the order of preferential hydrolysis for the wild type was that the RpRc was hydrolyzed first, followed by the RpSc, SpRc, and then the SpSc isomer. The reversal of stereoselective preference was also seen with the double substitution library members for hydrolysis of the sarin analog isomers. However, there were no significant improvements for sarin analog hydrolysis in these libraries. Among the best mutants obtained were H254G-H257W, H254G-H257R, and H257Y, all of which had catalytic efficiencies on the order of 106 M-1 s-1 for hydrolysis of the Sp isomer. The toxicity for analogs of sarin, soman, and VX was evaluated using Hydra attenuata as a model organism. The toxicity of each compound was assessed quantitatively by measuring the minimal effective concentration within 92 h in H. attenuata. There was a positive correlation between the molecular hydrophobicity of the compound and its ability to cause toxicity. Results from this study indicate the potential for application of this assay in the field of organophosphate nerve agent detection, as well as for the prediction of toxicity of structurally similar organophosphate compounds. The minimal effective concentration for two of the VX analogs was 2 orders of magnitude more toxic than the analog for soman and four orders of magnitude more toxic than the analog for sarin.

Protein engineering is a powerful tool to modify proteins to generate novel and desired properties that could be applied in biotechnological, diagnostics and therapeutic areas. In this thesis, both rational design and library based engineering principles have been exploited to develop affinity proteins with desired traits. One study was focused on the use of site-directed mutagenesis to obtain variants of the staphylococcal protein A-derived 58-residue immunoglobulin binding Z domain with improved affinity for mouse IgG1 Fc. Screening of ca. 170 constructed variants revealed one variant with a single F5I amino acid substitution, denoted ZF5I, with a ten-fold higher affinity. The Fc binding ZF5I variant was further investigated for use in affinity-driven site-specific covalent photoconjugation to mIgG1 monoclonal antibodies. Here, nine candidate positions in the domain were investigated for introduction of a UV-activatable maleimide benzophenone (MBP) group via conjugation to an introduced cysteine residue. The best photo-conjugation results were obtained for a variant in which the MBP was introduced at position 32, denoted ZF5I-Q32C-MBP, which could be conjugated at high yields to all nineteen mouse IgG1s tested. The use of a biotinylated Z-based probe for biotinylation via photoconjugation of a monoclonal anti-interferon gamma antibody resulted in a higher antigen binding activity than if a conventional amine directed biotinylation strategy was used. In a second study, the goal was to develop a new homogeneous immunoassay for quick antigen detection, based on split-protein complementation and pairs of antigen recognizing proteins. In one of the formats investigated, separate fragments of a split-beta-lactamase enzyme reporter were genetically linked to ZF5I-Q32C-MBP units which were individually photo-conjugated to two different mAbs recognizing different epitopes on a human interferon gamma model target analyte. Simultaneous binding of the two mAb-enzyme half probes to the analyte resulted in an analyte concentration-dependent enzyme fragment complementation which could be spectrophotometrically detected using a nitrocefin substrate. Using ribosome display technology, Z-domain based binders to mouse IgG1 were selected from an affibody library. One binder denoted Zmab25 was shown capable of selective binding to mouse IgG in a background of bovine IgG, and could be used for species-selective recovery of monoclonal antibodies from complex samples, resembling hybridoma culture supernatants. Epitope mapping experiments showed that that the binding site on mouse IgG was located in the Fab fragment and was overlapping with that of streptococcal protein G. In a final study, phage display technology was used to select affibodies binding to human interleukin 6 (IL-6), for potential use in rheumatoid arthritis (RA) therapy via blocking of the signaling involving the ternary complex between IL-6, the IL-6 receptor α (IL-6R α) and the gp130 co-receptor. Several affibodies were shown to be capable of blocking the interaction between gp130 and preformed complexes of IL-6 and soluble IL-6R α (IL-6/sIL-6R α) in vitro, corresponding to the so-called trans-signaling interaction. One of these affibody variants denoted ZIL-6_13 showed a KD of approx. 500 pM for IL-6 and was genetically fused to different chain ends of the monoclonal anti-TNF antibody adalimumab to build bi-specific “AffiMab” constructs. One construct, ZIL-6_13-HCAda,in which the affibody was fused to the N-terminus of the adalimumab heavy chain had the most optimal properties in different cell assays and was also evaluated in vivo in an acute serum amyloid A (SAA) mouse RA model, involving a dual challenge of animals with both IL-6 and TNF. Compared to adalimumab that could only reduce SAA levels to 50% at the highest dose, the bi-functional AffiMab reduced SAA levels to below the detection level. / <p>QC 20150416</p>

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by Wong Kam Bo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 149-153). / Acknowledgments --- p.i / Abstract --- p.ii / Contents --- p.iii / Abbreviations --- p.vii / Short names for mutants --- p.viii / One letter symbol for amino acids --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1. --- Chemical and Physical Properties of Trichosanthin --- p.1 / Chapter 1.2. --- Activities of Trichosanthin at the cellular level --- p.2 / Chapter 1.3. --- Activities of Trichosanthin at the molecular level --- p.3 / Chapter 1.4. --- Objective and Strategy of Protein engineering of Trichosanthin --- p.8 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1. --- General Techniques --- p.15 / Chapter 2.1.1. --- Ethanol Precipitation of DNA and RNA --- p.15 / Chapter 2.1.2. --- Spectrophotometric quantification of DNA and RNA --- p.15 / Chapter 2.1.3. --- Minipreparation of Plasmid DNA --- p.15 / Chapter 2.1.4. --- Preparation of Plasmid DNA using Qiagen-pack 100 Cartridge --- p.16 / Chapter 2.1.5. --- Preparation of Plasmid DNA using Magic´ёØ Minipreps DNA Purification kit from Promega --- p.17 / Chapter 2.1.6. --- Preparation and Transformation of Escherichia coli Competent Cell --- p.18 / Chapter 2.1.7. --- Agarose Gel Electrophoresis of DNA --- p.19 / Chapter 2.1.8. --- Purification of DNA from Agarose Gel using GeneClean® (BIO 101 Inc.) kit --- p.20 / Chapter 2.1.9. --- Polymerase Chain Reaction (PGR) --- p.21 / Chapter 2.1.10. --- Restriction Digestion of DNA --- p.23 / Chapter 2.1.11. --- Ligation of DNA fragments --- p.23 / Chapter 2.1.12. --- Autoradiography --- p.24 / Chapter 2.1.13. --- SDS-Polyacrylamide Gel Electrophoresis (SDS- PAGE) --- p.24 / Chapter 2.1.14. --- Staining of Protein in polyacrylamide gel --- p.25 / Chapter 2.1.15. --- Western Blot detection of TCS --- p.25 / Chapter 2.1.16. --- Liquid Scintillation Counting --- p.27 / Chapter 2.1.17. --- Minimization of Ribonuclease (RNAase) activity in experiments involving RNA --- p.27 / Chapter 2.2. --- Site-Directed Mutagenesis of Trichosanthin --- p.28 / Chapter 2.2.1. --- "Construction of E160D,El60A and SEAAR deletion mutants" --- p.28 / Chapter 2.2.2. --- Construction of E189A mutant and El60A E189A double mutant --- p.31 / Chapter 2.2.3. --- Construction of E189D mutant and El60A E189D double mutant --- p.36 / Chapter 2.2.4. --- Construction of Q156A mutant --- p.38 / Chapter 2.2.5. --- Construction of Q156A El60A mutant (Fig. 2.7) --- p.41 / Chapter 2.2.6. --- Construction of Q156A El89A mutant (Fig. 2.8) --- p.43 / Chapter 2.3. --- DNA sequencing --- p.45 / Chapter 2.3.1. --- DNA Sequencing Reaction --- p.45 / Chapter 2.3.2. --- DNA Sequencing Electrophoresis --- p.46 / Chapter 2.3.3. --- Resolving GC band compression --- p.48 / Chapter 2.4. --- Overexpression of mutated TCS in Escherichia coli --- p.48 / Chapter 2.5. --- Purification of mutated TCS --- p.49 / Chapter 2.6. --- Ribosome inactivating activity Assay using Rabbit Reticulocyte Lysate In Vitro Translation system --- p.50 / Chapter 2.7. --- N-glycosidase activity Assay --- p.51 / Chapter 2.7.1. --- Inactivation of ribosome in rabbit reticulocyte lysate --- p.51 / Chapter 2.7.2. --- RNA extraction --- p.51 / Chapter 2.7.3. --- Aniline Degradation --- p.52 / Chapter 2.7.4. --- Electrophoresis of RNA in Agarose Gel containing Formamide --- p.52 / Chapter 2.8. --- Reagents and buffers --- p.53 / Chapter 2.8.1. --- Nucleic Acid Electrophoresis Buffers --- p.53 / Chapter 2.8.2. --- Reagents for preparation of plasmid DNA --- p.54 / Chapter 2.8.3. --- Media for bacterial culture --- p.54 / Chapter 2.8.4. --- Reagents for SDS-PAGE --- p.55 / Chapter 2.8.5. --- Reagents for western blot --- p.56 / Chapter 2.8.6. --- Reagents for DNA sequencing --- p.57 / Chapter Chapter 3 --- Construction of TCS mutants / Chapter 3.1. --- Introduction --- p.59 / Chapter 3.2. --- Results --- p.59 / Chapter 3.2.1. --- "Construction of E160D,El60A and ASEAAR" --- p.59 / Chapter 3.2.2. --- Construction of E189A and E160AE189A mutants --- p.66 / Chapter 3.2.3. --- Construction of E189D and E160AE189D mutants --- p.80 / Chapter 3.2 --- A Construction of Q156A mutant --- p.82 / Chapter 3.2.5. --- Construction of Q156AE160A and Q156AE189A --- p.86 / Chapter 3.3. --- Discussion --- p.90 / Chapter Chapter 4 --- Expression and Purification of mutated TCS proteins / Chapter 4.1. --- Introduction --- p.94 / Chapter 4.2. --- Results --- p.95 / Chapter 4.2.1. --- Expression and purification of E160D and El60A mutants --- p.95 / Chapter 4.2.2. --- Expression and purifcation of E189D and E160AE189D mutants --- p.99 / Chapter 4.2.3. --- Expression and purifcation of E189A and E160AE189A mutants --- p.104 / Chapter 4.2.4. --- Expression and purifcation of Q156A and Q156AE160A mutants --- p.109 / Chapter 4.2.5. --- Expression and purifcation of Q156AE189A mutant --- p.114 / Chapter 4.2.6. --- Analysis of protein purity by SDS-PAGE and Western immunoblotting --- p.114 / Chapter 4.3. --- Discussion --- p.119 / Chapter Chapter 5 --- Biological Assay of mutated proteins / Chapter 5.1. --- Introduction --- p.125 / Chapter 5.2. --- Results --- p.125 / Chapter 5.2.1. --- Ribosome inactivating activity assay --- p.125 / Chapter 5.2.2. --- N-glycosidase activities of El60AE189A mutant --- p.131 / Chapter 5.3. --- Discussion --- p.133 / Chapter 5.3.1. --- Role of glutamate-160 --- p.133 / Chapter 5.3.2. --- A putative mechanism for N-glycosidase activity of TCS --- p.137 / Chapter 5.3.3. --- Role of glutamate-189 and glutamine-156 --- p.143 / Chapter 5.3.4. --- Prospective and future studies --- p.145 / Chapter 5.4. --- Concluding remarks --- p.147 / Appendix / Chapter A.l --- Size of molecule weight markers --- p.148 / Chapter A.2 --- Reference --- p.149