An alpha-amino ester hydrolase (AEH) applicable to synthesis of semi-synthetic antibiotics was cloned from the genomic DNA of Xanthomonas campestris pv. campestris sp. strain ATCC 33913. AEHs catalyze the synthesis and hydrolysis of alpha-amino beta-lactam antibiotics. The enzyme was characterized for thermodynamic and kinetic parameters. The enzyme shows optimal ampicillin hydrolytic activity at 25C and pH 6.8. The AEH enzymes have been shown to have excellent synthetic capability. Additionally, we demonstrated the first fully aqueous enzymatic one-pot synthesis of ampicillin direct from the natural product penicillin G eliminating the isolation of the intermediate 6-APA. Lastly, to improve the thermostability of the AEH a modified structure-guided consensus model of seven homologous enzymes was generated along with analysis of the B-factors from the available crystal structures of the known AEH from Xanthomonas citri. Our best variant, which is a quadruple mutant, E143H/A275P/N186D/V622I, which has a T_50_30, the temperature at which the half-life is 30 minutes, of 34C and 1.3-fold activity compared to wild-type. Overall, we have successfully improved the understanding of the AEH class of enzymes and applied a novel cascade application, demonstrating AEHs unique applicability in the synthesis of beta-lactam antibiotics. The improved thermostability will further improve the industrial relevance of AEHs.

Submitted in fulfilment for the requirement of a Degree of Doctor of Technology: Biotechnology, Durban University of Technology, 2010. / The aim of this study was to evaluate T. lanuginosus for the production of hemicellulases, its yield enhancement using mutagenesis and application of a selected xylanase on bagasse pupl to assess the improvement of pulp properties. The objectives were: To determine the localization of hemicellulases in T. lanuginosus strains, To develop high yielding strains of T. lanuginosus through mutagenensis, To investigate the synthesis of xylanase by T. lanuginosus MC134, To optimize the medium components and cultural conitions of T. lanuginosus MC134 strain, To study the influence of agitation and aeration on the production of xylanase by T. lanuginosus MC134 in a fermenter, To evaluate the bleach boosting abilities of T. lanuginosus xylanase on bagasse pulp, To evaluate simultaneous xylanase production and biobleaching potential of T. lanuginosus.

The availability of cost-effective high throughput screening assays combined with an enhanced understanding of oncogenesis has driven the development of more potent, specific, and less toxic anti-cancer agents. At the forefront of these advances are immunoglobulin molecules and their fragments. However, difficulties in producing antibodies in sufficient quantity and quality for commercial application have driven the development of alternative systems that can produce antibodies efficiently and cost-effectively. This thesis focuses on the engineering of an antibody fragment referred to as a single chain variable fragment (scFv), which consists of antibody light and heavy chain variable domains fused together by a peptide linker. Although the use of scFvs circumvents many of the issue of full-length antibody production, they still possess their own unique set of difficulties, including stability. In this thesis, we explored the following strategies to increase scFv stability. First, we increased the number of linkers used to join the variable light and heavy domains. We constructed two linear and two cyclic permutated scFvs that contained additional peptide linkers. Two linear permutated scFvs, named Model 1 and Model 3, showed increased stability with calculated melting temperatures (Tms) exceeding that of the unpermutated scFv. The two cyclic scFvs were less stable with Tms less than that of the unpermutated scFv. Second, we mutated light and heavy variable domains by introducing prolines or mutating glycine to alanine in the variable domain framework regions. Sites for proline mutations and glycine to alanine mutations were identified and scFvs containing the mutations were purified and their thermal stability tested. Unfortunately, there were no discernible differences between purified scFv mutants and the control scFv. Third, we designed a new selection/screening strategy using phage display and yeast two-hybrid assays to identify complementarity determining regions on scFvs that increased intracellular stability. We used this strategy to isolate anti-Abl-SH3 scFvs. Transient expression of scFvs in K562 cells indicated that two anti-Abl-SH3 scFv decreased viability.

Human drug metabolites are frequently biologically active, with many implications for human health. Pharmaceutical companies have become increasingly aware of the need to identify and test these metabolites. The P450 BM3 enzyme from Bacillus megaterium offers substantial advantages to the current methods of metabolite synthesis, as its soluble, catalytically self-sufficient nature, coupled with its high catalytic activity, make P450 BM3 ideal for engineering towards specificity for human drugs. The highly-active I401P BM3 mutant was characterized for its reactivity towards human drugs and for the development of a human P450-like metabolite profile. The I401P mutant exhibits binding to molecules including alkaloids, steroids, and azole drugs, along with many other compounds. I401P binds/oxidizes human CYP substrates, including alosetron, phenacetin, caffeine, nicotine and diclofenac. LC-MS product identification shows that I401P BM3 forms 4OH-diclofenac, the major human metabolite for diclofenac. I401P BM3 also produces nornicotine, the second major human metabolite of nicotine. I401P BM3 also forms theophylline, theobromine and paraxanthine, the three major human metabolites of caffeine. Thermostability (DSC) data show that the I401P mutation destabilizes the BM3 heme domain in both its substrate-free and substrate-bound forms. The I401P heme domain X-ray crystal structure reinforces previous structural observations that the Pro401 mutation causes the BM3 protein to adopt a high-spin, "substrate-bound" state, with a displaced heme iron axial water, producing a "catalytically primed" mutant with greater diversity in substrate selectivity. The destabilisation of the BM3 heme domain structure due to the Pro401 mutation increases conformational plasticity in this mutant, allowing it to function as a platform for future mutagenesis aimed at improved binding and metabolite yield from specific drug substrates. Further proline mutations (A330P, A330P/I401P and A82F/F87V/I401P) were examined for increased affinity for drug substrates. The A330P mutant shows no novel drug substrate specificity, despite its reported affinity for small molecules. The A330P/I401P double mutant demonstrates weak binding to WT BM3 and I401P substrates, but no synergistic effects were obtained by combining the two mutations. The double mutant exhibits very low solvent tolerance and significant structural destabilisation. DSC data confirms this, with the double mutant destabilising the BM3 heme domain by up to 20 °C. Initial work with the A82F/F87V/I401P mutant showed increased affinity for A82F/F87V- and I401P-type substrates, including diclofenac. LC-MS product analysis confirms that the A82F/F87V/I401P mutant oxidises diclofenac into its major human metabolite 4OH-diclofenac. These data indicate that human-like oxidation reactions are feasible with BM3 mutants. In this work, proline insertion mutants were generated that introduced novel affinity for biotechnologically relevant substrates. In particular the I401P mutant offers an excellent platform for future biotechnological engineering.

abstract: Synthetic manipulation of chromatin dynamics has applications for medicine, agriculture, and biotechnology. However, progress in this area requires the identification of design rules for engineering chromatin systems. In this thesis, I discuss research that has elucidated the intrinsic properties of histone binding proteins (HBP), and apply this knowledge to engineer novel chromatin binding effectors. Results from the experiments described herein demonstrate that the histone binding domain from chromobox protein homolog 8 (CBX8) is portable and can be customized to alter its endogenous function. First, I developed an assay to identify engineered fusion proteins that bind histone post translational modifications (PTMs) in vitro and regulate genes near the same histone PTMs in living cells. This assay will be useful for assaying the function of synthetic histone PTM-binding actuators and probes. Next, I investigated the activity of a novel, dual histone PTM binding domain regulator called Pc2TF. I characterized Pc2TF in vitro and in cells and show it has enhanced binding and transcriptional activation compared to a single binding domain fusion called Polycomb Transcription Factor (PcTF). These results indicate that valency can be used to tune the activity of synthetic histone-binding transcriptional regulators. Then, I report the delivery of PcTF fused to a cell penetrating peptide (CPP) TAT, called CP-PcTF. I treated 2D U-2 OS bone cancer cells with CP-PcTF, followed by RNA sequencing to identify genes regulated by CP-PcTF. I also showed that 3D spheroids treated with CP-PcTF show delayed growth. This preliminary work demonstrated that an epigenetic effector fused to a CPP can enable entry and regulation of genes in U-2 OS cells through DNA independent interactions. Finally, I described and validated a new screening method that combines the versatility of in vitro transcription and translation (IVTT) expressed protein coupled with the histone tail microarrays. Using Pc2TF as an example, I demonstrated that this assay is capable of determining binding and specificity of a synthetic HBP. I conclude by outlining future work toward engineering HBPs using techniques such as directed evolution and rational design. In conclusion, this work outlines a foundation to engineer and deliver synthetic chromatin effectors. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2019

Going down the folding funnel, proteins may sample a wide variety of conformations, some being outright detrimental to the organism. Yet, the vast majority of polypeptide molecules avoid such pitfalls. Not only do they reach the native minimum of the energy landscape; they do so via blazingly fast, biased, routes. This specificity and speed is remarkable, as the surrounding solution is filled to the brim with other molecules that could potentially interact with the protein and in doing so stabilise non-native, potentially toxic, conformations. How such incidents are avoided while maintaining native structure and function is not understood.  This doctoral thesis argues that protein structure and function can be separated in the folding code of natural protein sequences by use of multiple partly uncoupled factors that act in a concerted fashion. More specifically, we demonstrate that: i) Evolutionarily conserved functional and regulatory elements can be excised from a present day protein, leaving behind an independently folded protein scaffold. This suggests that the dichotomy between functional and structural elements can be preserved during the course of protein evolution. ii) The ubiquitous charges on soluble protein surfaces are not required for protein folding in biologically relevant timescales, but are critical to intermolecular interaction. Monomer folding can be driven by hydrophobicity and hydrogen bonding alone, while functional and structural intermolecular interaction depends on the relative positions of charges that are not required for the native bias inherent to the folding mechanism. It is possible that such uncoupling reduces the probability of evolutionary clashes between fold and function. Without such a balancing mechanism, functional evolution might pull the carpet from under the feet of structural integrity, and vice versa. These findings have implications for both de novo protein design and the molecular mechanisms behind diseases caused by protein misfolding. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

The application of magnetic resonance imaging (MRI) to non-invasively assess disease biomarkers has been hampered by lack of desired contrast agents with high relaxivity, targeting capability, and optimized pharmacokinetics. We developed a novel MRI probe which targets HER2, a biomarker for various cancers and a target for anti-cancer therapies. This multimodal HER2-targeted MRI probe integrates a rationally designed protein contrast agent with a high affinity HER2 affibody and near IR dye. Our probe can differentially monitor tumors with different HER2 levels in both cells and xenograft mice. In addition to its 10-fold higher dose efficiency compared to clinically-approved agent DTPA, our developed agent also exhibits advantages in crossing the endothelial boundary, tissue distribution, and tumor tissue retention as demonstrated by even distribution of the imaging probe across the entire tumor mass. Additionally, a second series of protein contrast agents that included affibody against EFGR developed with the capability to specifically target EGFR. These contrast agents have been utilized to monitor drug treatments and quantitatively analyze biomarker expression level. Furthermore, we anticipate these agents will provide powerful tools for quantitative assessment of molecular markers, and improved resolution for diagnosis, prognosis and drug discovery.

<p>Biosensors incorporating proteins as molecular recognition elements for analytes are used in clinical diagnostics, as biological research tools, and to detect chemical threats and pollutants. This work describes the application of protein engineering techniques to address three aspects in the design of protein-based biosensors; the transduction of binding into an observable, the manipulation of affinities, and the diversification of specificities. The periplasmic glucose-binding protein from the hyperthermophile Thermotoga maritima (tmGBP) was fused with green fluorescent protein variants to construct a fluorescent ratiometric sensor that is sufficiently robust to detect glucose up to 67°C. Ligand-binding affinities of tmGBP were changed by altering a C-terminal helical domain that tunes ligand binding affinity through conformational coupling effects. This method was extended to the Escherichia coli arabinose-binding protein. Computational design techniques were used to diversify the specificity of the E. coli maltose-binding protein (ecMBP) to bind ibuprofen, a non-steroidal antiinflammatory drug. These designs ranged in affinity from 0.24 to 0.8 mM and function as reagentless fluorescent sensors. The ligand affinities of ecMBP are tuned by complex interactions that control conformational coupling. These experiments demonstrate that long-range conformational effects as well as molecular recognition interactions need to be considered in the design of high-affinity receptors.</p> / Dissertation

Thesis (Ph.D)--Chemistry and Biochemistry, Georgia Institute of Technology, 2009. / Committee Chair: Doyle, Donald; Committee Member: Barry, Bridgette; Committee Member: Bommarius, Andreas; Committee Member: Ledoux, Joe; Committee Member: Matsumura, Ichiro; Committee Member: Oyelere, Adegboyega. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Several polymer systems including P(MAA-g-EG) and P(MAA-co-NVP) with crosslinking agents TEGDMA and PEGDMA1000, monomer-to-solvent ratios of 67:33, 60:40, and 50:50, and particle sizes of <75 microns, 90-150 microns, and 150-212 microns were synthesized for use with protein-transporter conjugates. All synthesized systems were characterized by SEM which demonstrated the visual size, surface features, and surface textures of the polymer microparticles. Insulin-transferrin and calcitonin-transferrin conjugates were successfully synthesized using the protein crosslinker SPDP, binding the two proteins with a disulfide bond. The multi-step conjugation reactions used to create the conjugates were analyzed by the use of UV spectroscopy and HPLC to ensure the quality of the final products. In both conjugation reactions, the final product yield was found to be over 70%. The in vitro loading and release characteristics for insulin-transferrin and calcitonin-transferrin were separately investigated. By testing loading and release using a number of different polymer systems with different synthesis parameters, it was possible to optimize the hydrogel carriers for use with each of the conjugates independently. Upon optimization, the ideal system for use with insulin-transferrin and calcitonin-transferrin was found to be P(MAA-g-EG) microparticles of <75 microns formed using a PEGDMA1000 crosslinker and a 50:50 monomer-to-solvent ratio for both conjugates through separate optimization processes. This optimized polymer carrier was found to release upwards of 50% of loaded insulin-transferrin conjugate and near 90% of loaded calcitonin-transferrin conjugate. The insulin-transferrin conjugate was further evaluated through the use of cellular and animal models. Using cellular models, the insulin-transferrin conjugate was shown to increase transport relative to insulin by a factor of 7, achieving an apparent permeability of 37 x 10⁹ cm/s. Also, in the presence of polymer microparticles, the insulin-transferrin conjugate increased transport by a factor of 14 times relative to insulin, achieve an apparent permeability of 72.8 x 10⁹ cm/s. The presence of the microparticles near the cells was found to improve conjugate transport by nearly 100%. The preliminary animal studies verified the successful synthesis of the insulin-transferrin conjugate as well as demonstrated the bioactivity of the insulin portion of the molecule by achieving a drop in blood glucose level upon subcutaneous injection. / text