Magister Scientiae - MSc / This thesis describes the cloning and recombinant expression of domains from the human RBBP6 protein for future in vitro binding studies with pRb and p53. RBBP6 is a splicing-associated protein that is known to interact with both p53 and the Retinoblastoma gene product (pRb), and has recently been shown to be highly upregulated in oesophageal cancer. The pRb binding domain (RbBD) and the p53 binding domain (p53BD) were each expressed using the glutathione-S-transferase (GST) tag affinity system, and affinity purified using a glutathione-linked agarose column. Purified fusion proteins were cleaved to separate the target protein from GST using PreScission™ Protease, for which there is a recognition sequence located immediately upstream of the multiple cloning site on the pGEX-6P series of plasmids. The pRb binding and p53 binding domains were further purified using cation exchange chromatography. Mass spectrometry confirmed that the RbBD was expressed as a single species of the expected molecular weight. However preliminary NMR analysis suggested that the domain was not fully folded. A total yield of 8 mg of protein was achieved from 1l of culture, which make it feasible to express 15N and 12C labelled samples for NMR. The p53BD was found to be expressed at lower levels and subject to C-terminal degradation, which suggest that the C-terminus is unstructured most likely due to the presence of poly-lysine tail. Human pRb protein was also successfully expressed and purified using the GST affinity system. Human p53 protein was expressed but was found to be insoluble and attempts to purify it were not pursued. Attempts to confirm the interactions between human RBBP6 and p53 and pRb proteins are on-going but fall outside the scope of this thesis. Expression constructs for the RING and zinc finger domains from human RBBP6 were also cloned into the pGEX system for future structural studies using NMR. Both domains were found to be expressed as soluble fusion proteins in preliminary expression studies. / South Africa

Cytochrome P450 monooxygenases, one of the most important classes of heme-thiolate proteins, have attracted considerable interest in the biochemical community because of its catalytic versatility, substrate diversity and great number in the superfamily. Although P450s are capable of catalyzing numerous difficult oxidation reactions, the relatively low stability, low turnover rates and the need of electron-donating cofactors have limited their practical biotechnological and pharmaceutical applications as isolated enzymes. The goal of this study is to tailor such heme-thiolate proteins into efficient biocatalysts with high specificity and selectivity by protein engineering and to better understand the structure-function relationship in cytochromes P450. In the effort to engineer P450cam, the prototype member of the P450 superfamily, into an efficient peroxygenase that utilizes hydrogen peroxide via the “peroxide-shunt” pathway, site-directed mutagenesis has been used to elucidate the critical roles of hydrophobic residues in the active site. Various biophysical, biochemical and spectroscopic techniques have been utilized to investigate the wild-type and mutant proteins. Three important P450cam variants were obtained showing distinct structural and functional features. In P450camV247H mutant, which exhibited almost identical spectral properties with the wild-type, it is demonstrated that a single amino acid switch turned the monooxygenase into an efficient preoxidase by increasing the peroxidase activity nearly one thousand folds. In order to tune the distal pocket of P450cam with polar residues, Leu 246 was replaced with a basic residue, lysine, resulting in a mutant with spectral features identical to P420, the inactive species of P450. But this inactive-species-like mutant showed catalytic activities without the facilitation of any cofactors. By substituting Gly 248 with a histidine, a novel Cys-Fe-His ligation set was obtained in P450cam which represented the very rare case of His ligation in heme-thiolate proteins. In addition to serving as a convenient model for hemoprotein structural studies, the G248H mutant also provided evidence about the nature of the axial ligand in cytochrome P420 and other engineered hemoproteins with thiolate ligations. Furthermore, attempts have been made to replace the proximal ligand in sperm whale myoglobin to construct a heme-thiolate protein model by mimicking the protein environment of cytochrome P450cam and chloroperoxidase.

Protein engineering has been used to alter the stability of proteins for several decades withmuch success, one approach being to introduce two cysteine residues that together form adisulfide bridge. The disulfide bridge can increase the Gibbs free energy of the transitionstate, thus increasing energy difference between the folded state and the unfolding transitionstate, leading to increased kinetic stability of the protein. Subtilisin Carlsberg is a serineprotease that has widespread applications within the industry but has also been tried in biogasprocesses to increase the biomethane yield from proteinaceous substrates. Subtilisin’s activitylifetime was found to be short in the biogas process, which prompted the need to increase theenzyme’s kinetic stability, meaning that the introduction of a disulfide bridge could be asolution. The aim of this project was to increase the kinetic stability of Subtilisin Carlsbergwith the use of introduced disulfide bridges.The production of Subtilisin Carlsberg has traditionally been done using the source organismBacillus Licheniformis, but here a successful method for expressing Subtilisin, and fourdisulfide variants of it, as an inclusion-body protein is presented. Also, a method forpurifying and refolding the protein under denaturing conditions is presented with a significantprotein yield.Thermal stability analysis of the WT enzyme and its four variants (A24C/S86C,N122C/A227C, K12C/E270C, V26C/A231C) was performed using NanoDSF, and showedthat the thermal stability was practically unchanged for A24C/S86C at 67.9 ℃, decreased by5.6 ℃ for N122C/A227C, increased by 8.2 ℃ for K12C/E270C, and increased by 11.5 ℃ forV26C/A231C.The kinetic stability of Subtilisin and its variants was analysed using stopped-flowmeasurements of the proteins’ denaturation rate at various GuHCl concentrations. The resultsshowed that N122C/A227C and V26C/A231C were more kinetically stable than the WTenzyme, while A24C/S86C and K12C/E270C were less stable. N122C/A227C had anactivation energy for unfolding of 5.217 kJ/mol higher than WT Subtilisin. V26C/A231C hadan activation energy for unfolding of 1.220 kJ/mol higher than WT Subtilisin. The resultsthereby show that two disulfides bond mutations achieved the desired outcome of increasedkinetic stability. Thereby, the aim of the project was fulfilled.

No description available.

Enzymes can catalyze reactions with high selectivity under mild conditions, and are therefore especially suited for the upgrading of C1 feedstocks into value-added products. Linear carbon ligation routes are of particular interest due to their simplicity and potential for high carbon efficiencies. A linear carbon fixation pathway can be constructed using a combination of NADH-dependent oxidoreductase enzymes and a formaldehyde carboligation enzyme, through which CO₂ is upgraded into C₂ and C₃ products. The stoichiometric NADH requirement imposed by the oxidoreductase enzymes and the poor performance of two core pathway enzymes (formaldehyde dehydrogenase and formolase) are the main obstacles to the efficient application of this linear carbon fixation pathway. In this dissertation, a host of fundamental enzyme engineering and characterization techniques are applied to study and address the thermodynamic, transport, and kinetic challenges arising from the use of enzyme cascades for multistep catalysis. In Chapter 2, a modular approach for the design of cofactor-independent transhydrogenases was explored and developed to enable catalysis and cofactor recycling in a single protein. Individual, unmodified active sites were modularly assembled and their activity catalytically coupled using biomimetic PEG-NAD(H) swing arms. Protein engineering and molecular design were used to increase the swing arm content and increase the activity of the transhydrogenases without detriment to their selectivity, circumventing the typical tradeoffs associated with modifications to the active site. The modularity of this approach was illustrated through the creation and characterization of four novel transhydrogenase enzymes with behavior that was predictable from that of the parent enzyme active sites. In Chapter 3, the kinetic behavior of the formolase (FLS) enzyme was comprehensively characterized to facilitate its use in carbon fixation cascades. A mechanistic rate equation and theory-based figures of merit were derived from first principles and used to capture and rank the full catalytic performance of 8 FLS variants under different conditions. The transition state specificity constant derived in this chapter was used to quantify product preference. In Chapter 4, the limiting performance of the formaldehyde dehydrogenase enzyme was explored within the context of a NADH-dependent pathway for the reduction of CO₂ to methanol. Protein engineering experiments targeting the elongated cofactor binding loop of the enzyme were investigated as a means of enhancing NADH-dependent formate reductase activity, but all mutations in the loop region dramatically reduced protein expression levels. Pathway flux was increased through the substitution of the formaldehyde dehydrogenase with a better performing homolog. In Chapter 5, a model carboligation pathway for the conversion of formaldehyde to glycerol and ethylene glycol was built using the FLS, glycerol dehydrogenase, and phosphite dehydrogenase enzymes. The impact of the low activity and substrate affinity of the FLS enzyme on pathway carbon and energy efficiencies was examined in purified proteins and crude lysates. High energy efficiencies, as quantified through the efficiency of NADH utilization, were achieved only with purified proteins. Cofactor regeneration was also shown to lower the cofactor requirement from stoichiometric to catalytic concentrations. In this work, we utilized a range of characterization techniques to study the limitations and challenges involved in the use of both NADH-dependent oxidoreductases and designed enzymes for multistep catalysis, and used protein engineering to address them. The insights gained from this work will facilitate the efficient use of these enzymes for linear carbon fixation as well as other biocatalysis applications.

Abstract Laccases are members of the multicopper oxidases, having the ability to catalyze the oxidation of a wide spectrum of phenolic compounds. These enzymes are capable of oxidizing lignin-related compounds and highly resistant environmental pollutants, and hence can be used in wastewater treatment and detoxification. Laccases are mostly derived from fungi, bacteria, and plants. In general, fungal laccases are known for their high redox potential, while bacterial laccases have a better tolerance to temperature. A combination of these properties is ideal for industrial processes. Directed evolution and the consensus method are often used to engineer protein stability. However, they are time-consuming and expensive. Ancestral sequence reconstruction (ASR), an approach whereby probable ancestral sequences are obtained, is known to provide thermostable variants of modern proteins as the output. In this study, ASR was used to design a laccase with high thermostability and high redox potential. ASR was performed through the web tool Phylobot, where fungal laccases were used as the input to obtain a phylogenetic tree with ancestral variants of the fungal laccases. For rooting the phylogenetic tree, multiple outgroups were tested, and the ‘Three-outgroups’ scenario proved to be the most efficient. In all cases, the phylogenetic tree was branched into major clusters of thermostable clades and non-thermostable clades. Nodes representing ancestral sequences were selected based on their sequence length and proximity to the thermostable clades. Sequence alignment of the selected nodes to the fungal laccases showed that the selected nodes have a high percentage of identity to well-known, highly thermostable laccases like Trametes pubescens and Basidiomycete PM1. All selected nodes have some common conserved motifs in the vicinity of the copper ligands. It also shows that the number of prolines in nodes 229 and 345 is more than in other selected nodes. The gene for the most promising candidate, Node 345, was synthesized, cloned into the pPICZ A vector, and transformed into Pichia pastoris cells. In the future, the protein will be expressed and characterized. Furthermore, the engineered laccases from the directed evolution method and the ancestral sequence reconstruction method will be compared for their activity and thermostability. This work would pave the way for using ASR as a less resource-intensive and time-consuming method for protein engineering. 2.12.0.0 / <p></p><p></p><p></p><p></p><p></p><p></p><p></p><p>2.12.0.0</p>

Natural cellular networks are very good at processing diverse inputs, generating complicated responses, and confounding researchers with their complexities. As an alternative to traditional cellular engineering approaches, the field of synthetic biology attempts to avoid the complexities of natural systems by focusing on the bottom-up construction of artificial cellular circuits. By rationally building up circuit complexity, synthetic biologists hope to both create novel systems capable of programming unique cellular responses, and gain insights into the design principles of natural systems. Circuits that allow for the programming of intercellular responses are of particular interest, and researchers have focused on the use of bacterial communication mechanisms (quorum sensing) to construct such circuits. At their most basic, quorum-sensing systems are composed of three main components, making them amenable to genetic manipulation. These components, however, have properties that have been finely tuned through evolution to function in very specific ways, and repurposing them for our own uses requires methods to overcome their naturally evolved properties. This thesis details our work in the construction and engineering of synthetic circuits based on components of the LuxI-LuxR quorum-sensing system. Using these components, we demonstrate methods for altering both the sensitivity and the form of the quorum-sensing response through the creation of three unique systems: an ultrasensitive positive feedback loop, a logical AND gate, and a coupled feedback loop oscillator. Construction and tuning of each circuit's properties were achieved through a mixture of rational and evolutionary approaches, with particular emphasis on the directed evolution of the LuxR transcriptional activator. Mathematical modeling was also used during the construction of the more complex circuits to predict the properties that were essential to their functionalities. With the construction and characterization of these circuits, we have provided both well-defined modules that can be used in the construction of more complex systems, and developed methods that will allow for the creation and engineering of additional synthetic circuits.

No description available.

No description available.

No description available.