Omics approaches to assess the effect of agro-food nutritive extracts for pcDNA-FLAG-p53 biosynthesis

In the last years, plasmid DNA has been used as a vector for gene therapy and DNA vaccines, and for this reason, the ability to produce large quantities of plasmid DNA is important concerning the DNA vaccines production process, on an industrial scale. Thus, the objective of this research was to study the metabolism and proteome of Escherichia coli (strain VH35) during the pcDNA- FLAG-p53 biosynthesis using as alternative media agro–food industry compounds, as cheese whey, corn steep liquor and yeast extract. Combining these alternative compounds as a growth medium, and using strain VH35 as a host, the optimization of plasmid DNA production was performed in terms of yield and quality. After this optimization, it was analyzed the consumption of sugars in the fermentation medium overtime, and the proteome changes that occur due to metabolic adaptations. The developed work suggests that strain VH35 uses agro-food media as an energy source, thus consuming the lactose present in the agro-food medium, since it does not contain PTS–sugars. Due to the fact that this alteration can lead to proteome modifications in the cell, proteomic analysis was performed using two-dimensional electrophoresis, which showed that the protein composition of strain VH35 was different among the compared growth mediums. Lastly, these proteome changes were analyzed by MALDI-TOF/TOF, and it was possible to identify differentially expressed proteins, such as anthranilate synthase component 1, chaperone protein ClpB, deoxyribose-phosphate aldolase, that are related principally to metabolic pathways and nucleotides synthesis.Nos últimos anos, o DNA plasmídico tem sido usado como um vetor em terapia génica e em vacinas de DNA, e por essa razão, a capacidade de produzir grandes quantidades de plasmídeo é importante no que diz respeito ao processo de produção de vacinas de DNA, em larga escala. Assim, o objetivo deste trabalho foi o estudo metabólico e proteómico da Escherichia coli (estirpe VH35) durante a síntese de pcDNA-FLAG-p53, usando como meio de fermentação compostos alternativos provenientes da indústria agro–alimentar, como o soro de queijo, o “corn steep licor” e o extrato de levedura. Combinando estes compostos alternativos como meio de crescimento, e usando a estirpe VH35 como hospedeiro, procedeu-se então à otimização da produção de DNA plasmídeo, em termos de rendimento e qualidade. Após essa otimização, analisou-se o consumo de açúcares no meio de fermentação ao longo do tempo, e as alterações proteómicas que ocorrem devido a adaptações metabólicas. O trabalho desencolvido sugere que a estirpe VH35 utiliza meios agro-alimentares como fonte de energia, consumindo assim a lactose presente no meio, uma vez que estes meios não possuem açúcares PTS (fosfoenolpiruvato). Devido ao facto desta alteração poder levar a modificações no proteoma da célula, foi feita uma análise proteómica por eletroforese bidimensional, que revelou variações no proteoma da célula quando comparado com diferentes meios de crescimento. Por fim, essas variações proteómicas foram analisadas por MALDI-TOF/TOF, e foi possível identificar proteínas diferencialmente expressas, como por exemplo a proteína MreB, a desoxirribose fosfato – aldolase, chaperonina ClpB,as quais se encontram principalmente relacionadas com o metabolismo e a síntese de nucleótidos

Exploring the Biophysical Properties of Domains Within Multi-Domain Proteins

The past few decades, considerable progress has been made in understanding the biophysical properties of proteins using small modular domains such as SH3 domains. However, there is a surprising lack of knowledge regarding how these properties are affected when the domain is placed back within its full-length multi-domain protein. Using a combination of expressed protein ligation (EPL) and in vivo amino acid replacement of tryptophans with tryptophan (Trp) analogues, we have developed an integrated approach that allows the domain-specific incorporation of optical probes into large recombinant proteins. The Src homology 3 (SH3) domain from the c-Crk-l adaptor protein has been labeled with a Trp analogue, 7-azatryptophan (7AW), using E.coli Trp auxotrophs. Biophysical analysis shows that incorporation of 7AW does not significantly perturb the structure or function of the isolated domain. Ligation of 7AW labeled SH3 domain to the c-Crk-l Src homology 2 (SH2) domain, via EPL, generated the multi-domain protein, c-Crk-l, with a domain specific label. Studies on this labeled protein show that the biochemical and thermodynamic properties of the SH3 domain do not change within the context of a larger multi-domain protein. We have also utilized this technique and segmental isotopic labeling to study the Cterminal SH3 (cSH3) domain of the signaling adaptor protein, Crk-ll. Several studies suggest that the cSH3 domain plays an important regulatory role in the protein. However, no structural information is available on this domain and relatively little is known about its binding partners. We have solved the solution NMR structure of the C-terminal SH3 domain and it adopts the standard SH3 fold comprising a fivestranded p-barrel. Thermodynamic and kinetic studies show that the domain folds in a reversible two-state manner and that the stability of the fold is similar to that observed for other SH3 domains. Studies on the cSH3 domain specifically labeled within Crk-ll have provided the strongest evidence yet that the cSH3 domain interacts in an intramolecular fashion to regulate Crk-ll. The techniques developed here should be applicable to many other multi-domain proteins. This sets the stage for a better understanding of the biophysical properties of domains within these complex systems

Protein engineering of E.coli phosphofruktokinasE

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Structure, function and mechanism of the alternative oxidases

The alternative oxidase (AOX) is the terminal protein in the alternative oxidation pathway found in plants, fungi and some protozoa. One of the more prominent protozoa that contain AOX within the bloodstream form is Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT), in which the parasite has demonstrated to be totally dependent upon the protein for continued respiration. Given the lack of AOX in mammalian cells, the protein represents an attractive chemotherapeutic target for trypanosidal activity. Ascofuranone is a known inhibitor of AOX, but its complex synthesis precludes it from industrial production. To this end colletochlorin B, an analogue of ascofuranone, was synthesised and its inhibitory efficacy against AOX examined. IC50 values obtained demonstrate that removal of the problematic furanone ring does not reduce inhibitor efficacy to a large degree. Derivatives of colletochlorin B were synthesised to assess the importance of structural moieties present. The compounds were also tested against the cytochrome bc1 complex, an important respiratory chain complex, and compared with known fungicides. Using these compounds assays against fungal species has yielded promising results for the use of colletochlorin B as a lead fungicide. Recombinant wild type trypanosomal alternative oxidase (TAO) and Sauromatum guttatum alternative oxidase (SgAOX) have been expressed in E.coli. in addition to a number of mutants. Respiratory activities of these mutants were measured in order to assess the importance of highly conserved amino acids, with all mutants showing a decline in specific activity. Three of the mutants generated were also shown to affect the apparent affinity for oxygen, the implications of which are discussed. Recent crystallisation of TAO has enabled a more detailed examination of the structure of all AOXs. Work is presented relating structure to the overall function of the protein, taking into account conservation throughout the entire AOX family. Comparisons to other di-iron proteins revealed a conserved His-Asp-Tryp motif that could facilitate proton coupled electron transport. A full catalytic cycle based on these findings has been postulated

Investigation of recombinant protein production by Escherichia coli : expression of Green fluorescent protein and a co-factor dependent flavinated enzyme

This thesis summarises work done on the Escherichia coli strain MG1655 expressing a Green Fluorescent Protein (GFP) and the flavo-protein N-methyl-L-tryptophan oxidase (MTOX) product and examining the effect foreign protein production has on cell growth parameters. It also uses molecular modelling tools to generate data relating to FAD flux and MTOX production, comparable to that seen in E.coli fermentations. The MG1655 strain was chosen as it was the focus of the first K-12 complete sequencing project and closely related to the strain W3110, a second K strain that had been used to develop a number of deletion mutants which were central to the study. It presents data from shake flask and stirred tank reactor fermentations on minimal, carbon limited and complex media. Samples from these growth experiments were then analysed concentrating on biomass concentration, protein assays (both chemical and fluorimetric), high performance liquid chromatography and calculation of yield parameters. From this a baseline of growth was established with which to compare changes in growth after a shift in protein product from GFP to MTOX. An assay was also developed to measure the amount of active and inactive MTOX enzyme produced and this data compared with the level of FAD available to the cell at specific time points throughout growth. Finally modelling work is presented and in silico values compared with those generated in vitro. A discussion of the entire study concludes the work

Adaptive evolvability through direct selection instead of indirect, second‐order selection

Can evolvability itself be the product of adaptive evolution? To answer this question is challenging, because any DNA mutation that alters only evolvability is subject to indirect, “second order” selection on the future effects of this mutation. Such indirect selection is weaker than “first-order” selection on mutations that alter fitness, in the sense that it can operate only under restrictive conditions. Here I discuss a route to adaptive evolvability that overcomes this challenge. Specifically, a recent evolution experiment showed that some mutations can enhance both fitness and evolvability through a combination of direct and indirect selection. Unrelated evidence from gene duplication and the evolution of gene regulation suggests that mutations with such dual effects may not be rare. Through such mutations, evolvability may increase at least in part because it provides an adaptive advantage. These observations suggest a research program on the adaptive evolution of evolvability, which aims to identify such mutations and to disentangle their direct fitness effects from their indirect effects on evolvability. If evolvability is itself adaptive, Darwinian evolution may have created more than life's diversity. It may also have helped create the very conditions that made the success of Darwinian evolution possible

Analysis of Coevolving Residues of xCDxCDx-PHD, A Distinct type of PHD-Finger

Plant homeodomain (PHD), a Zinc-finger scaffold, is a conserved protein module in eukaryotes that typically recognizes unstructured histone tails, and thus, PHDs play a crucial role in chromatin signaling. The sequences of Zinc-fingers, in general, diversify during the course of evolution often giving rise to subtypes (e.g., RBR-RING (Dove 2017) or zf-CxxC subtypes (Long 2013) who typically contain specific sequence signatures. We recently discovered that PHD fingers also contain a distinct subtype, namely the xCDxCDx-PHD. xCDxCDx-PHD has a distinct composition of specific amino acids that coevolved (coevolving residues) in the course of evolution, and xCDxCDx-PHD also shows a unique mechanism for interacting with histone H3 (i.e., unique function). It, however, is unclear if the set of coevolving residues were selected by nature exclusively for function alone or the selection was for the maintenance of the structure, stability and/or dynamics in order to perform the specific function. Here, we systematically characterize the contributions of the evolving residues in the PHD scaffold with detailed experimentation (e.g., using NMR, CD, FASTpp and ITC) to discover that the coevolving residues of xCDxCDx-PHD contribute only towards histone recognition (i.e., function). As somatic and germline mutations of xCDxCDx-PHD are often associated with a number of cancers and inherited diseases, this investigation provides a platform to further probe the consequence of disease-causing mutations in xCDxCDx-PHD