<p>The aim of this work was to investigate and determine central parameters that can be used to control and increase the solubility, quality and productivity of recombinant proteins. These central parameters should be applicable under the constraints of high-throughput protein production in <em>Escherichia coli.</em></p><p>The present investigation shows that alternative methods exist to improve solubility, quality and productivity of the recombinant protein. The hypothesis is that by reducing the synthesis rate of the recombinant protein, a higher quality protein should be produced. The feed rate of glucose can be used to decrease the synthesis rate of the recombinant protein.</p><p>The influence of feed rate on solubility and proteolysis was investigated using the <em>lac</em>UV5-promoter and two model proteins, Zb-MalE and Zb-MalE31. Zb-MalE31 is a mutated form of Zb-MalE that contains two different amino acids. These altered amino acids greatly affect the solubility of the protein. The soluble fraction is generally twice as high using Zb-MalE compared to Zb-MalE31. Using a low feed rate compared to high benefits the formation of the full-length soluble protein. Furthermore, by using a low feed rate, the proteolysis can be decreased. One other factor that influences the solubility is the amount of inducer used. An increase from 100 µM to 300 µM IPTG only results in more inclusion bodies being formed, the fraction of soluble protein is the same.</p><p>The quality aspect of protein production was investigated for a secreted version of Zb-MalE using two different feed rates of glucose and the maltose induced promoter P<em>malK</em>. It was shown that when the protein was secreted to the periplasm, the stringent response as well as the accumulation of acetic acid (even for high feed rates) was reduced. The stringent response and accumulation of acetic acid are factors that are known to affect the quality and quantity of recombinant proteins. Transporting the protein to the periplasm results in this case on a lower burden on the cell, which leads to less degradation products being formed when the protein is secreted to the periplasm.</p><p>Seeing the feed rate as a critical parameter, the high-throughput production would benefit from a variation in the feed rate. However, since the fed-batch technique is technically complicated for small volumes another approach is needed. <em>E.coli</em> strains that have been mutated to create an internal growth limitation that simulate fed-batch were cultivated in batch and were compared to the parent strain. It was shown that the growth rate and acetic acid formation was comparable to the parent strain in fed-batch. Furthermore it was shown that a higher cell mass was reached using one of the mutants when the cells were cultivated for as long time as possible. The higher cell mass can be used to reach a higher total productivity.</p>

The aim of this work was to investigate and determine central parameters that can be used to control and increase the solubility, quality and productivity of recombinant proteins. These central parameters should be applicable under the constraints of high-throughput protein production in Escherichia coli. The present investigation shows that alternative methods exist to improve solubility, quality and productivity of the recombinant protein. The hypothesis is that by reducing the synthesis rate of the recombinant protein, a higher quality protein should be produced. The feed rate of glucose can be used to decrease the synthesis rate of the recombinant protein. The influence of feed rate on solubility and proteolysis was investigated using the lacUV5-promoter and two model proteins, Zb-MalE and Zb-MalE31. Zb-MalE31 is a mutated form of Zb-MalE that contains two different amino acids. These altered amino acids greatly affect the solubility of the protein. The soluble fraction is generally twice as high using Zb-MalE compared to Zb-MalE31. Using a low feed rate compared to high benefits the formation of the full-length soluble protein. Furthermore, by using a low feed rate, the proteolysis can be decreased. One other factor that influences the solubility is the amount of inducer used. An increase from 100 µM to 300 µM IPTG only results in more inclusion bodies being formed, the fraction of soluble protein is the same. The quality aspect of protein production was investigated for a secreted version of Zb-MalE using two different feed rates of glucose and the maltose induced promoter PmalK. It was shown that when the protein was secreted to the periplasm, the stringent response as well as the accumulation of acetic acid (even for high feed rates) was reduced. The stringent response and accumulation of acetic acid are factors that are known to affect the quality and quantity of recombinant proteins. Transporting the protein to the periplasm results in this case on a lower burden on the cell, which leads to less degradation products being formed when the protein is secreted to the periplasm. Seeing the feed rate as a critical parameter, the high-throughput production would benefit from a variation in the feed rate. However, since the fed-batch technique is technically complicated for small volumes another approach is needed. E.coli strains that have been mutated to create an internal growth limitation that simulate fed-batch were cultivated in batch and were compared to the parent strain. It was shown that the growth rate and acetic acid formation was comparable to the parent strain in fed-batch. Furthermore it was shown that a higher cell mass was reached using one of the mutants when the cells were cultivated for as long time as possible. The higher cell mass can be used to reach a higher total productivity. / QC 20101112

La capture de CO2 par adsorption sur des solides poreux (adsorbants) est une alternative prometteuse en raison de sa sélectivité et de sa faible consommation d’énergie. Nous avons étudié l'adsorption in-situ de CO2 sur des adsorbants solides en combinant la spectroscopie infrarouge par réflexion diffuse (DRIFT) avec la thermographie infrarouge afin de mieux comprendre les mécanismes d'interaction CO2-adsorbant et ainsi optimiser sa captation dans des procédés de capture en post-combustion. La thermographie IR est utilisée pour détecter la source de chaleur transitoire provenant de la surface de l’adsorbant au cours de l'adsorption de CO2. Un modèle de transfert de chaleur a été développé afin d’estimer les chaleurs d’adsorption. Un mini réacteur conçu pour la DRIFT nous a permis d’identifier les espèces adsorbées et d'étudier leur évolution sur la surface de l’adsorbant selon la température et l'atmosphère environnante. Enfin, le couplage d’informations provenant des deux approches nous a permis l’investigation haut-débit des paramètres clefs pour le choix des adsorbants les plus performants. / CO2 capture via adsorption process on porous materials (adsorbents) is a promising alternative due to its high selectivity and low energy penalties. We have investigated in-situ CO2 adsorption on solid adsorbents by combining Diffuse Reflectance Infrared Fourier Transform spectroscopy (DRIFT) with infrared thermography to better understand the mechanisms controlling CO2-adsorbent interactions and thus optimize its capture in post-combustion capture processes. Infrared thermography is used to detect the transient heat source coming from the adsorbent surface during CO2 adsorption. A heat transfer model has been developed in order to estimate the adsorption heats. A model chemical reactor designed for DRIFT allowed us to clearly evidence the adsorbed species and to study the surface species evolution according to the temperature and the surrounding atmosphere. Finally, the coupling of information coming from the two approaches allowed us a high-throughput investigation of key parameters for the selection of the most efficient adsorbents.