Creating receptors for control of transcription with arbitrary small molecules has widespread applications including gene therapy, biosensors, and enzyme engineering. Using the combination of high throughput docking, codon randomization, and chemical complementation, we have created new receptors to control transcription with small molecules. Chemical complementation, a new method of protein engineering, was used to discover retinoid X receptors (RXR) variants that are activated by compounds that do not activate wild-type RXR. A first library of 32,768 RXR variants was designed for the synthetic retinoid-like compound LG335. The library produced ligand-receptor pairs with LG335 that have a variety of EC50s and efficacies. One engineered variant has essentially the reverse ligand specificity of wild-type RXR and is transcriptionally active at 10 and #64979;fold lower LG335 concentration than wild-type RXR with 9cRA in yeast. The activity of this variant in mammalian cells correlates with its activity in yeast. A second library of 262,144 RXR variants was designed for two purposes: (i) to develop a high-throughput chemical complementation method to select variants that have high efficacies and low EC50s; and (ii) to find variants which are activated by small molecules not known to bind RXR variants. Selection conditions were manipulated to find only variants with high efficacies and low EC50s. This library was also selected for variants that activate transcription specifically in response to gamma-oxo-1-pyrenebutyric acid (OPBA), which is different from any known RXR ligand. OPBA was chosen as a potential ligand using high-throughput docking with the software program FlexX. Two variants are activated by OPBA with an EC50 of 5 mM. This is only ten-fold greater than the EC50 of wild type RXR with its ligand 9cRA (500 nM) in yeast. An improved method synthesizing LG335 and a method for quantifying intracellular ligand concentrations were developed. Although the LG335 synthetic method has an additional step, the overall yield was improved to 8% from 4% in the original publication. Liquid chromatography and mass spectrometry was used to quantify the intracellular concentration of LG335, which was found to be within four fold of the LG335 concentration in the media.

Background: Trypsinogen is the inactive precursor of trypsin, a serine protease that cleaves proteins and peptides after arginine and lysine residues. In this study, human trypsinogen was used as a model protein to study the influence of electrostatic forces on protein–protein interactions. Trypsinogen is active only after its eight-amino-acid-long activation peptide has been cleaved off by another protease, enteropeptidase. Trypsinogen can also be autoactivated without the involvement of enteropeptidase. This autoactivation process can occur if a trypsinogen molecule is activated by another trypsin molecule and therefore is based on a protein–protein interaction. Results: Based on a rational protein design based on autoactivation-defective guinea pig trypsinogen, several amino acid residues, all located far away from the active site, were changed to modify the surface charge of human trypsinogen. The influence of the surface charge on the activation pattern of trypsinogen was investigated. The autoactivation properties of mutant trypsinogen were characterized in comparison to the recombinant wild-type enzyme. Surface-charged trypsinogen showed practically no autoactivation compared to the wild-type but could still be activated by enteropeptidase to the fully active trypsin. The kinetic parameters of surface-charged trypsinogen were comparable to the recombinant wild-type enzyme. Conclusion: The variant with a modified surface charge compared to the wild-type enzyme showed a complete different activation pattern. Our study provides an example how directed modification of the protein surface charge can be utilized for the regulation of functional protein–protein interactions, as shown here for human trypsinogen.

Die rekombinante Expression des Vier-Helix-Bündelproteins Sulerythrin, aus Sulfolobus tokodaii-Stamm 7, wurde etabliert und für die Produktion von metallfreiem Apo-Protein optimiert. Durch Metall-Rekonstitution konnten Eisen, Nickel, Kobalt, Mangan und Eisen-57 als Cofaktoren des homo-bimetallischen Zentrums eingebracht werden. Es wurden Zusammenhänge zwischen Eigenschaften, Reaktivitäten und Strukturen verschiedener diMe-SulE-Proteine untersucht. Die Affinität der Bindungen der vier Metalle an das SulE-wt wurde mittels ITC untersucht und deutet auf eine hochaffine und eine niedrigaffine Bindungsstelle im Zentrum hin, was durch Kristallisation von mono-Fe-SulE bestätigt wurde. Die Me-SulE-wt-Proteine zeigten unterschiedliche UV-vis-Spektren im reduzierten und oxidierten Zustand. Es wurden diFe-SulE-Varianten der Koordinationssphäre des aktiven Zentrums erzeugt und deren Oxidationsraten mit Sauerstoff bei 325 nm bestimmt, wobei diFe-SulE-wt langsamer war als alle Varianten (beobachtete Geschwindigkeitskonstanten teilweise 40 bis 50-mal höher). Die reduzierten Zustände der diFe-SulE-Varianten wurden durch Mößbauer-Spektroskopie untersucht (80-100 %ige Fe(II) im high-spin Zustand). Für die diMe-SulE-wt-Varianten wurde die Katalaseaktivität gemessen. Durch die Inkubation von diCo-SulE-wt-Kristallen mit H2O2 konnte ein verbrückender μ2-η2-Peroxid-Ligand in der Proteinstruktur modelliert werden und ein Reaktionsintermediat nachgewiesen werden, dessen O-O-Bindungslänge mit 1,50 Å nahezu identisch mit H2O2 ist. Der Metall-Metall-Abstand im zweikernigen Metallzentrum der Varianten reichte von 3,97 Å für bis 3,15 Å. Anhand der Strukturen der aktiven Stellen der Varianten lassen sich strukturelle Unterschiede erkennen, die als Ursache für die unterschiedlichen Reaktivitäten und Oxidationsraten vermutet werden. Die Daten zeigen eine klare Korrelation zwischen einem abnehmenden Metall-Metall-Abstand und einem Anstieg der Geschwindigkeitskonstante für verschiedene Varianten. / The recombinant expression of the four-helix bundle protein Sulerythrin, from Sulfolobus tokodaii strain 7, was established and optimized for the production of metal-free apo-protein. By metal reconstitution, iron, nickel, cobalt, manganese and iron-57 were incorporated as cofactors of the homo-bimetallic center. Relationships between characteristics, reactivities and structures of different diMe-SulE proteins were investigated. The affinity of the bonds of the four metals to the SulE-wt was investigated by ITC and indicated a high-affinity and a low-affinity binding site in the active site, which was confirmed by crystallization of mono-Fe-SulE. The Me-SulE-wt proteins showed different UV-vis spectra in the reduced and oxidized states. diFe-SulE variants of the active site coordination sphere were generated and their oxidation rates with oxygen at 325 nm were determined, with diFe-SulE-wt being slower than all variants (observed rate constants 40 to 50 times higher in some cases). The reduced states of the diFe-SulE variants were investigated by Mössbauer spectroscopy (80-100 % Fe(II) in high-spin state). The catalase activity was measured for diMe-SulE-wt variants. By incubating diCo-SulE-wt crystals with H2O2, a bridging μ2-η2 peroxide ligand could be modeled in the protein structure and a reaction intermediate was detected whose O-O bond length of 1.50 Å is almost identical to H2O2. The metal-to-metal distance in the binuclear metal center of the variants ranged from 3.97 Å for to 3.15 Å. Based on the structures of the active sites of the variants, structural differences can be identified, which are assumed to be the cause of the different reactivities and oxidation rates. The data further provide a clear correlation between a decreasing distance between the metals and an increase in the rate constant for different variants.

Thesis (M.Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 vi, 92 leaves / Interest in xylanases from different microbial sources has increased markedly in the past decade, in part because of the application of these enzymes in a number of industries, the main area being the pulp and paper industry. While conventional methods will continue to be applied to enzyme production from micro-organisms, the application of recombinant DNA techniques is beginning to reveal important information on the molecular basis and this knowledge is now being applied both in the laboratory and commercially. In this study, a directed evolution strategy was used to select an enzyme variant with high thermostability. This study describes the use of error-prone PCR to modify the xylanase gene from Thermomyces lanuginosus DSM 5826, rendering it tolerant to temperatures in excess of 80°C. Mutagenesis comprised of different concentrations of nucleotides and manganese ions. The variants were generated in iterative steps and subsequent screening for the best mutant was evaluated using RBB-xylan agar plates. The optimum temperature for the activity of xylanases amongst all the enzyme variants was 72°C whilst the temperature optimum for the wild type enzyme was 70°C. Long term thermostability screening was therefore carried out at 80°C and 90°C. The screen yielded a variant which had a 38% improvement in thermostability compared to the wild type xylanase from pX3 (the unmutated gene). Successive rounds of error-prone PCR were carried out and in each round the progeny mutant displayed better thermostability than the parent. The most stable variant exhibited 71% residual activity after 90 minutes at 80˚C. Sequence analysis revealed four single amino acid residue changes that possibly enhanced their thermostabilities. This in vitro enzyme evolution technique therefore served as an effective tool in improving the thermostable property of this xylanase which is an important requirement in industry and has considerable potential for many industrial applications.

Submitted in fulfillment for the requirement of a Degree of Master of Technology: Biotechnology, in the Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa, 2009. / Protein engineering has provided a key for adapting naturally-occurring enzymes for industrial processes. However, several obstacles have to be overcome after these proteins have been adapted, the main one being finding a suitable host to over-express these recombinant protein. This study investigated Saccharomyces cerevisiae, Pichia pastoris and Escherichia coli as suitable expression hosts for a previously modified fungal xylanase, which is naturally produced by the filamentous fungus, Thermomyces lanuginosus. A xylanase variant, NC38, that was made alkaline-stable using directed evolution was cloned into four different vectors: pDLG1 with an ADH2 promoter and pJC1 with a PGK promoter for expression in S. Cerevisiae, pBGP1 with a GAP promoter for expression in P. pastoris and pET22b(+) for expression in E. Coli BL21 (DE3). S. Cerevisiae clones with the p DLG1-NC38 combination showed very low activity on the plate assay and were not used for expression in liquid media as the promoter was easily repressed by reducing sugars used during production experiments. S. cerevisiae clones carrying pJC1-NC38 were grown in media without uracil while P. Pastoris clones were grown in YPD containing the antibiotic, zeocin and E. Coli clones were grown in LB with ampicillin. The levels of xylanase expression were then compared between P. Pastoris, S. cerevisiae and E. coli. The highest recombinant xylanase expression was observed in P. Pastoris with 261.7U/ml, followed by E.coli with 47.9 U/ml and lastly S. cerevisiae with 13.2 U/ml. The localization of the enzyme was also determined. In the methylotrophic yeast, P. Pastoris, the enzyme was secreted into the culture media with little or no contamination from the host proteins, while the in other hosts, the xylanase was located intracellularly. Therefore in this study, a mutated alkaline stable xylanase was successfully expressed in P. Pastoris and was also secreted into the culture medium with little or no contamination by host proteins, which favours the application of this enzyme in the pulp and paper industry. / National Research Foundation

Submitted in complete fulfillment for the Degree of Master of Technology (Biotechnology)in the Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa, 2009. / Protein engineering has been a valuable tool in creating enzyme variants that are capable of withstanding the extreme environments of industrial processes. Xylanases are a family of hemicellulolytic enzymes that are used in the biobleaching of pulp. Using directed evolution, a thermostable and alkaline stabl xylanase variant (S340) was created from the thermophilic fungus, Thermomyces lanuginosus. However, a host that was capable of rapid growth and high-level expression of the enzyme in large amounts was required. The insert containing the xylanase gene was cloned into a series a pET vectors in Escherichia coli BL21 (DE3) pLysS and trimmed from 786 bp to 692 bp to remove excess fungal DNA upstream and downstream of the open reading frame (ORF). The gene was then re-inserted back into the pET vectors. Using optimized growth conditions and lactose induction, a 14.9% increase in xylanase activity from 784.3 nkat/ml to 921.8 nkat/ml was recorded in one of the clones. The increase in expression was most probably due to the removal of fungal DNA between the vector promoter and the start codon. The distribution of the xylanase in the extracellular, periplasmic and cytoplasmic fractions was 17.3%, 51.3% and 31.4%, respectively. The modified enzyme was then purified to electrophoretic homogeneity using affinity chromatography. The xylanase had optimal activity at pH 5.5 and 70°C. After 120 min at 90°C and pH 10, S340 still displayed 39% residual activity. This enzyme is therefore well suited for its application in the pulp and paper industry. / National Research Foundation

Cocaine abuse is a world-wide public health and social problem without a U.S. Food and Drug Administration (FDA)-approved medication. An ideal anti-cocaine medication would accelerate cocaine metabolism producing biologically inactive metabolites by administration of an efficient cocaine-specific exogenous enzyme. Recent studies in our lab have led to discovery of the desirable, highly efficient human cocaine hydrolases (hCocHs) that can efficiently detoxify and inactivate cocaine without affecting normal functions of central nervous system (CNS). Preclinical and clinical data have demonstrated that these hCocHs are safe for use in humans and effective for accelerating cocaine metabolism. However, the actual therapeutic use of a hCocH in cocaine addiction treatment is limited by the short biological half-life (e.g. 8 hours or shorter in rats) of the hCocH. In the investigation described in this thesis, we have demonstrated that mCocH and hCocH have improved the catalytic efficiency of mBChE and hBChE against cocaine by ~8- and ~2000-fold, respectively, although the catalytic efficiencies of mCocH and hCocH against other substrates, including acetylcholine (ACh) and butyrylthiocholine (BTC), are close to those of the corresponding wild-type enzymes mBChE and hBChE. In addition, we have identified the first benzoylecgonine-metabolizing enzymes that can hydrolyze benzoylecgonine and accelerate its clearance in rats. The developed LC-MS/MS method has enabled us to simultaneously determine cocaine and nine cocaine-related metabolites in whole blood samples. In development of the long-acting hCocHs, we have designed and discovered a novel hCocH form, catalytic antibody analog, which is an Fc-fused hCocH dimer (hCocH-Fc). The hCocH-Fc has not only a high catalytic efficiency against cocaine, but also a considerably longer biological half-life. A single dose of hCocH-Fc was able to accelerate cocaine metabolism in rats even after 20 days and, thus, block cocaine-induced hyperactivity for a long period of time. In consideration of the general observation that the biological half-life of a protein drug in humans is significantly longer than that in rodents, the hCocH-Fc could allow dosing once every 2-4 weeks, or longer for cocaine addiction treatment in humans.

ESF1 is a 2.052 kDa antimicrobial peptide, mimicking the charge distribution and amphipathic alpha-helical structure of magainin, pGLa, a naturally occurring antimicrobial peptide. ESF1 has been reported to display high activity against Fusarium oxysporum f. sp lycopersici and F. oxysporum f. sp cubense race 4, the tomato and banana crop plant, wilt-causing pathogens, respectively. To assess whether this synthetic peptide can be heterologously expressed in yeast in significant quantities, and still retain full bioactivity, within a eukaryotic system, the ESF1 gene was designed and synthesized from five oligonucleotides, and cloned into pUC18. From the pUC18/ESF1 recombinant plasmid, the ESF1 gene sequence was amplified and cloned into the pBluescript-based vector, pVD4, downstream of the yeast pheromone mating factor alpha (MFa1) promoter, and in frame with the MFa1 signal peptide sequence for expression and secretion in yeast. The expression cassette comprising the MFa1 promoter and signal peptide sequence, and ESF1 gene was subsequently cloned into the yeast/ E. coli shuttle vector, pTG3828 and transformed into Saccharomyces cerevisiae. Chicken IgY antibodies against ESF1 peptide were raised and immunoaffinity purified. Following this, western dot blot analysis and mass spectrometry confirmed the presence of ESF1 in partial HPLC purified fractions of the recombinant yeast culture supernatant. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

The development of enzymatic biosensors and enzymatic biofuel cells (EBFCs) has been a significant area of research for decades. Enzymatic catalysis can provide for specific, reliable sensing of target analytes as well as the continuous generation of power from physiologically present fuels. However, the broad implementation of enzyme-based devices is still limited by low operational/storage stabilities and insufficient power densities. Approaches to improving upon these limitations have focused on the optimization of enzyme activity and electron transfer kinetics at enzyme-functionalized electrodes. Currently, such optimization can be performed through enzyme structural engineering, improvement of enzyme immobilization methodologies, and fabrication of advantageous electrode materials to enhance retained enzyme activity density at the electrode surface and electron transfer rates between enzymes and an electrode. In this work, varying electrode materials were studied to produce an increased understanding on the impacts of material properties on resulting biochemical, and electrochemical performances upon enzyme immobilization and an additional method of electroactive enzyme-based optimization was developed through the use of polymer-based protein engineering (PBPE). First, graphene/single-wall carbon nanotube cogels were studied as supports for membrane- and mediator-free EBFCs. The high available specific surface area and porosity of these materials allowed the rechargeable generation of a power density within one order of magnitude of the highest performing glucose-based EBFCs to date. Second, two additional carbon nanomaterial-based electrode materials were fabricated and examined as EBFC electrodes. Graphene-coated single-wall carbon nanotube gels and gold nanoparticle/multi-wall carbon nanotube-coated polyacrylonitrile fiber paddles were utilized as electroactive enzyme supports. The performance comparison of these three materials provided an increased understanding of the impact of material properties such as pore size, specific surface area and material surface curvature on enzyme biochemical and electrochemical characteristics upon immobilization. Third, PBPE techniques were applied to develop enzyme-redox polymer conjugates as a new platform for enzymatic biosensor and EBFC optimization. Poly(N-(3-dimethyl(ferrocenyl) methylammonium bromide)propyl acrylamide) (pFcAc) was grown directly from the surface of glucose oxidase (GOX) through atom-transfer radical polymerization. Utilization of the synthesized GOX-pFcAc conjugates led to a 24-fold increase in current generation efficiency and a 4-fold increase in EBFC power density compared to native GOX. GOX-pFcAc conjugates were further examined as working catalysts in carbon paper-based enzymatic biosensors, which provided reliable and selective glucose sensitivities and allowed a systematic analysis of sources of instability in enzyme-polymer conjugate-based biosensors and EBFCs. The knowledge gained through these studies and the in-depth characterization of an additional layer of optimization capacity using PBPE could potentially enhance the progress of enzymatic biosensor and EBFC development.

<p>We have harnessed two reactions catalyzed by the enzyme sortase A and applied them to generate new methods for the purification and site-selective modification of recombinant protein therapeutics. </p><p>We utilized native peptide ligation —a well-known function of sortase A— to attach a small molecule drug specifically to the carboxy-terminus of a recombinant protein. By combining this reaction with the unique phase behavior of elastin-like polypeptides, we developed a protocol that produces homogenously-labeled protein-small molecule conjugates using only centrifugation. The same reaction can be used to produce unmodified therapeutic proteins simply by substituting a single reactant. The isolated proteins or protein-small molecule conjugates do not have any exogenous purification tags, eliminating the potential influence of these tags on bioactivity. Because both unmodified and modified proteins are produced by a general process that is the same for any protein of interest and does not require any chromatography, the time, effort, and cost associated with protein purification and modification is greatly reduced.</p><p>We also developed an innovative and unique method that attaches a tunable number of drug molecules to any recombinant protein of interest in a site-specific manner. Although the ability of sortase A to carry out native peptide ligation is widely used, we demonstrated that Sortase A is also capable of attaching small molecules to proteins through an isopeptide bond at lysine side chains within a unique amino acid sequence. This reaction —isopeptide ligation— is a new site-specific conjugation method that is orthogonal to all available protein-small conjugation technologies and is the first site-specific conjugation method that attaches the payload to lysine residues. We show that isopeptide ligation can be applied broadly to peptides, proteins, and antibodies using a variety of small molecule cargoes to efficiently generate stable conjugates. We thoroughly assessed the site-selectivity of this reaction using a variety of analytical methods and showed that in many cases the reaction is site-specific for lysines in flexible, disordered regions of the substrate proteins. Finally, we showed that isopeptide ligation can be used to create clinically-relevant antibody-drug conjugates that have potent cytotoxicity towards cancerous cells</p> / Dissertation