With the increased demands of therapeutic proteins, there is going to be a need for new purification technologies which have high throughput, high yield and high resolution. Three purification technologies were explored as potential new technology to isolate recombinant and endogenous milk proteins: Expanded bed adsorption chromatography(EBAC) combined with hydrophobic interaction chromatography(HIC), Recycle continuous flow electrophoresis(RCFE) and Free flow isoelectric focusing(FFIEF). The first process(EBAC/HIC) used with Zn2+ as a selective precipitating agent, purified recombinant human protein C(rhPC) and IgG(contaminated with less than 1% IgA) from swine milk with high resolution and high yield while processing about 10-20 grams in a single operation. The second process(RCFE) was able to isolate the active sub-populations of rhPC from major milk contaminants( - and -pig casein) as wells as from the inactive sub-populations of rhPC. RCFE was able to process 1.5g total protein per hour on a small scale and is currently being researched to process 1kg total protein per hour. The third and final purification process(FFIEF) sub-fractionated 100mg of immuno-purified rhPC into 50 fractions. The FFIEF was able to produce a linear pH gradient over the range of 3-10 using 2% ampholytes. The fractionated rhPC showed differing degrees of activity that resulted from the -carboxylated glutamic acids and the sialic acids. / Ph. D.

La synthèse de protéines recombinantes chez les microorganismes est d'un intérêt majeur pour la production de produits biopharmaceutiques, thérapeutiques et enzymatiques industriels. Cependant, la surproduction de protéines a un effet néfaste sur la physiologie cellulaire. Les ressources cellulaires (métabolites, énergie, machinerie moléculaire, espace cytosolique, etc.) sont en effet partagées entre les protéines de l'hôte et la protéine "gratuite". Cette surcharge non naturelle entraîne une croissance plus lente et des rendements en protéines plus faibles, un phénomène connu sous le nom de "burden". Dans mon projet de doctorat, il s'agissait (1) de déchiffrer les conséquences de la surproduction de protéines gratuites sur la physiologie cellulaire, (2) d'identifier le type de ressources limitantes, et (3) de surmonter cette limitation pour améliorer la production de protéines. Afin de déchiffrer les conséquences de la surproduction de protéines (1), nous avons analysé le taux de croissance, la production de protéines d'intérêt et le protéome de souches de Bacillus subtilis surproduisant divers niveaux de protéines rapportrices. Les protéines rapportrices ont été choisies de manière à être facilement quantifiables par fluorescence et par des tests d'activité (i.e. GFP, mKate2, LacZ, etc.). Pour obtenir les différents niveaux d'expression, nous avons construit des séquences synthétiques par assemblage de promoteurs constitutifs et inductibles et de régions d'initiation de traduction (TIR, RBS) variés. Nous avons ainsi montré que plus la quantité (et la taille) de la protéine produite était élevée, plus les taux de croissance étaient faibles et plus la taille des cellules était élevée. Par exemple, le taux de croissance a diminué de plus de 20 % lorsque la GFP était surproduite à plus de 5 % de la quantité totale de protéines solubles, selon des quantifications biochimiques et de fluorescence. Pour identifier le type de ressources limitantes (2), nous avons effectué une quantification relative des protéines sur les souches surproductrices de GFP et montré que certaines protéines non essentielles étaient moins abondantes dans ces souches. Nous avons ensuite dégradé spécifiquement les protéines rapportrices à l'aide d'un outil de biologie de synthèse précédemment mis au point pour B. subtilis, afin que les acides aminés puissent être recyclés dans le pool de ressources cellulaires. Avec une dégradation de 50-60% de GFP et mKate2, nous avons observé une restauration de 50% du taux de croissance. Ces résultats suggèrent que la quantité d'acides aminés (et par conséquent leur utilisation dans la synthèse des protéines) est le principal type de ressources limitantes. Pour améliorer la production de protéines (3), nous avons cherché à développer un système synthétique de recyclage des acides aminés basé sur le système de dégradation mentionné ci-dessus en surproduisant les protéases d'E. coli et B. subtilis (ClpXP) avec une protéine adaptatrice (SspB) d'E. coli. Cet outil pourrait permettre de dégrader spécifiquement des protéines non essentielles pour économiser des ressources cellulaires. Nous avons montré que la surproduction de ClpXP ou de SspB/ClpXP était suffisante pour permettre une dégradation complète des protéines produites à des niveaux bas et intermédiaires, et jusqu'à 50% des protéines fortement produites. Comme ClpXP est une protéase impliquée dans la réponse au stress, nous avons cherché à savoir si la surproduction de ClpXP pouvait avoir des conséquences négatives sur la physiologie cellulaire. Une quantification relative des protéines sur une souche surproductrice de ClpXP a montré que la surproduction de ClpXP provoque une réorganisation globale du protéome sans toutefois affecter le taux de croissance de la cellule. / Recombinant protein production in microorganisms is of great interest for the production of biopharmaceuticals, therapeutics and industrial enzymes. However, recombinant protein production has always shown a harmful effect on the microorganism cell physiology when excessively produced. Cell resources (i.e. metabolites, energy, molecular machinery, cytosolic space, etc.) are used to produce the host's proteins and the overproduced gratuitous protein. As a result, this unnatural extra load typically leads to slower growth and lower protein yields, a phenomenon known as ʻburdenʼ. This burden comes from the fact that the recombinant protein has no benefit for the microorganism, and that it only uses cell resources at the expense of the production of the endogenous essential proteins. In my PhD project, the issues were (1) to decipher the consequences of gratuitous protein overproduction on the cell physiology, (2) to identify the limiting type of resources, and (3) to overcome this limitation to improve protein production. To address the first issue (1), we analyzed growth rates, production of several proteins of interest, and genome-wide proteomes of Bacillus subtilis strains overproducing various levels of reporter proteins. The reporter proteins were chosen so that they were easily quantifiable by fluorescence and β-galactosidase activity assays (i.e. GFP, mKate2, LacZ, etc.). To obtain the various levels of expression, we built synthetic sequences made of the assembly of various constitutive and inducible promoters and translation initiation regions (TIR, RBS). Hence, we showed that higher was the amount (and size) of the protein produced, lower were the rates of growth and higher were the cell sizes. For instance, the growth rate decreased down by over 20% when GFP was overproduced above 5% of the total soluble protein amount according to both biochemical and fluorescence assays. To further identify the limiting type of resources (2), we performed a relative protein quantification on the strains overproducing GFP at different levels. Hence, we showed that some non-essential proteins were less abundant in the strains overproducing GFP. We next targeted the reporter proteins for degradation using a synthetic tool previously engineered in B. subtilis, so that amino acids can be recycled back to the pool of cell resources. Degrading the reporter gratuitous protein should also relieve the constraint on the cytosolic density by liberating intracellular space. With a degradation of 50-60% of GFP and mKate2, we observed a 50% restoration of the growth rate. This result together with the proteome analysis suggested that the amount of amino acids (and consequently their utilization in protein synthesis) was the main limiting type of resources. To overcome this limitation and improve protein production (3), we aimed at exploring a synthetic, amino acid recycling system based on the above mentioned degradation system. We decided to improve the targeted degradation system by overproducing the E. coli and B. subtilis ClpXP proteases together with an E. coli adaptor protein SspB. This tool may allow to target proteins for degradation in order to save resources and improve the production of a protein of interest. We showed that the overproduction of either ClpXP or SspB/ ClpXP were sufficient to allow a complete degradation of the proteins produced low and intermediate levels, and up to 50% of degradation of the proteins highly produced. As ClpXP is a protease involved in stress responses, we aimed to know whether the overproduction of ClpXP may have negative consequences on the cell physiology. We therefore performed relative protein quantification on a strain overproducing ClpXP. The results showed that ClpXP overproduction causes a global reorganisation on the proteome without affecting the growth rate of the cell.

Proteins containing Kunitz domain are mostly inhibitors of serine proteases. Their general characteristic is the presence of three disulfide bonds and small sizes around 6-10 kDa, although sometimes they consist of several Kunitz domains or they are part of more complex proteins. Their function is usually related to the regulation of physiological and proteolytic processes, but also to an interaction with pathogens or other defense mechanisms, such as being part of the sea anemone mucus or the venom of snakes and other invertebrates. We focused on Kunitz proteins in Eudiplozoon nipponicum, a helminth of the class Monogenea parasiting on gills of common carp (Cyprinus carpio). In the transcriptome of this parasite, several sequences with Kunitz domain have been identified based on similarities with the one already described Kunitz protein, EnKT1, suggesting that this parasite, like other bloodfeeding parasites, uses a whole set of these serine protease inhibitors with other specific functions. Several sequences with the Kunitz domain found in the transcriptome were verified by PCR and optionally supplemented by RACE-PCR. One protein, called EnKC1, was subsequently produced by recombinant expression in E. coli cells of SHuffleTM and Rosetta Gami B strains. Recombinant protein with the Kunitz domain...

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

The development of a biologic protein involves different stages and becomes a highly complex process which can be costly and time consuming to scale up for industrial production. Therefore, optimization is a necessary part of the production process development to lower the production expenses.An on-going project is working on upscaling the production of a protein derived from mussel adhesive proteins (MAPs) which has great properties to be used as a pharmaceutical drug or in medical devices. The protein is expressed in a bacterial host cell and the necessary post translational modifications (PTMs) are done in-vitro using enzymes. The work presented in this report was done to optimize both the protein production in lab scale bioreactors and the enzymatic reaction using an immobilized prolyl-4-hydroxylase (P4H) which does a post translational modification on prolyl-residues. Additionally, an interaction study was conducted to better understand the hydroxylation using the prolyl-4-hydroxylase.For the bioreactor optimization four initial trials were performed testing different growth and induction temperatures and also comparing exponential to linear feeding. From these trials it appeared that having 30 ℃ growth overnight and induction at the same temperature in combination with an exponential feeding rate gave the best results. The modifications done by the prolyl-4-hydroxylase were analysed by LC-MS and suggest that longer incubation time and more immobilized protein gives more modifications in the tested ranges and the possibilities of reusing the immobilized proteins looks promising. No conclusive data was discovered for the optimal substrate concentration. The interaction study revealed the importance of reagents used for catalysis with the enzyme to be present for interaction to occur, however more work needs to be done to discover an accurate KD for the interaction.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24487号 / 医博第4929号 / 新制||医||1063(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 上野 英樹, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

abstract: Influenza is a deadly disease that poses a major threat to global health. The surface proteins of influenza A, the type most often associated with epidemics and pandemics, mutate at a very high frequency from season to season, reducing the efficacy of seasonal influenza vaccines. However, certain regions of these proteins are conserved between strains of influenza A, making them attractive targets for the development of a ‘universal’ influenza vaccine. One of these highly conserved regions is the ectodomain of the influenza matrix 2 protein (M2e). Studies have shown that M2e is poorly immunogenic on its own, but when properly adjuvanted it can be used to induce protective immune responses against many strains of influenza A. In this thesis, M2e was fused to a pair experimental ‘vaccine platforms’: an antibody fusion protein designed to assemble into a recombinant immune complex (RIC) and the hepatitis B core antigen (HBc) that can assemble into virus-like particles (VLP). The two antigens were produced in Nicotiana benthamiana plants through the use of geminiviral vectors and were subsequently evaluated in mouse trials. Mice were administered three doses of either the VLP alone or a 1:1 combination of the VLP and the RIC, and recipients of both the VLP and RIC exhibited endpoint anti-M2e antibody titers that were 2 to 3 times higher than mice that received the VLP alone. While IgG2a:IgG1 ratios, which can suggest the type of immune response (TH1 vs TH2) an antigen will elicit, were higher in mice vaccinated solely with the VLP, the higher overall titers are encouraging and demonstrate a degree of interaction between the RIC and VLP vaccines. Further research is necessary to determine the optimal balance of VLP and RIC to maximize IgG2a:IGg1 ratios as well as whether such interaction would be observed through the use of a variety of diverse antigens, though the results of other studies conducted in this lab suggests that this is indeed the case. The results of this study demonstrate not only the successful development of a promising new universal influenza A vaccine, but also that co-delivering different types of recombinant vaccines could reduce the total number of vaccine doses needed to achieve a protective immune response. / Dissertation/Thesis / Masters Thesis Molecular and Cellular Biology 2019

Over the past 5 years, the use of titanium implants as temporary anchorage devices (TADs) has become an important tool in clinical orthodontic practices. The use of TADs have provided orthodontists a way of moving teeth against fixed objects rather than against the surrounding teeth, which tend to counteract desired motion. At present, viable attachment of TADs involves direct insertion through gingival tissue and piercing of the bone. Surface modifications such as sandblasted and acid-etched treatment or bone morphogenetic protein surface treatment, however, can be applied to the TADs to promote enhanced osseointegration, thereby allowing the TADs to serve as stable anchors while avoiding bone puncture. In this study, a comparison was made between sandblasted/acid-etched TADs and sandblasted/acid-etched/recombinant human bone morphogenetic protein-2 (rhBMP-2) treated TADs to determine whether rhBMP-2 promotes enhanced osseointegration. A total of 10 rats (4 controls and 6 treated with rhBMP-2) were used in the study, with 1 TAD placed on the skull of each rat. At the end of 6 weeks, the animals were euthanized by carbon dioxide asphyxiation, and bone blocks, each containing a TAD, were prepared for histological examination and biomechanical characterization. The results of this study showed that TADs treated with rhBMP-2 had greater bone formation at the bone-implant interface and an increase in total implant stability.

Flavonoid glycosyltransferases (GTs), enzymes integral to plant ecological responses and human pharmacology, necessitate rigorous structural elucidation to decipher their mechanistic function and substrate specificity, particularly given their role in the biotransformation of diverse pharmacological agents and natural products. This investigation delved into a comprehensive exploration of the flavonol 3-O GT from Citrus paradisi (Cp3GT), scrutinizing the impact of a c-terminal c-myc/6x histidine tag on its enzymatic activity and substrate specificity, and successfully achieving its purification to apparent homogeneity. This established a strong foundation for potential future crystallographic and other structure/function analyses. Through the strategic implementation of site-directed mutagenesis, a thrombin cleavage site was incorporated proximal to the tag, followed by cloning in Pichia pastoris, methanol-induced expression, and cobalt-affinity chromatography for initial purification stages. Notably, the recombinant tags did not exhibit a discernible influence on Cp3GT kinetics, substrate preference, pH optima, or metal interactions, maintaining its specificity towards flavonols at the 3-OH position and favoring glucosylation of quercetin and kaempferol. Subsequent purification steps, including MonoQ anion exchange and size-exclusion chromatography, yielded Cp3GT with ≥95% homogeneity. In silico molecular models of Cp3GT and its truncated variants, Cp3GTΔ80 and Cp3GTΔ10, were constructed using D-I-TASSER and COFACTOR to assess binding interactions with quercetin and kaempferol. Results indicated minimal interference of c-myc/6x-his tags with the native Cp3GT structure. This study not only lays a foundation for impending crystallographic studies, aiming to solidify the understanding of Cp3GT's stringent 3-O flavonol specificity, but also accentuates the potential of microbial expression platforms and plant metabolic engineering in producing beneficial compounds. To this end, a thorough review of four pivotal classes of plant secondary metabolites, flavonoids, alkaloids, betalains, and glucosinolates, was conducted. This will open avenues for further research and applications in biotechnological, medical, and agricultural domains.

L-asparaginase (EC 3.5.1.1), a therapeutic enzyme used in the treatment of childhood acute lymphoblastic leukemia (ALL). Hence, the goal of this work is study the expression and evaluation of hydrolysis activity of native sequence (X12746) encoding for L-asparaginase from Erwinia chrysanthemi NCPBB1125 in the popular expression system Pichia pastoris. The sequence of asn encoded for mature protein was expressed in P. pastoris SMD1168 and X33. SDS-PAGE analysis showed recombinant L-asparaginase was secreted efficiently. Stable and high hydrolysis activity of extracellular L-asparaginase in P. pastoris SMD1168 making it a potential candidate to produce recombinant protein. After purification, a specific band whose appearance approximately 45 kDa indicating the glycosylated protein with specific activity by 6.251 Umg-1 and about 3 folds purifications. / L-asparaginase (EC 3.5.1.1), một loại enzyme được sử dụng trong điều trị bệng ung thư bạch cầu mãn tính ở trẻ em. Mục tiêu của nghiên cứu này là biểu hiện và đánh giá hoạt tính thủy phân của L-asparaginase mã hóa bởi đoạn gene (X12746) tương ứng từ Erwinia chrysanthemi NCPBB1125 được biểu hiện trong nấm men Pichia pastoris. Gene đã được cắt signal peptide và biểu hiện trong P. pastoris SMD1168 and X33. Qua phân tích kết quả điện di SDS-PAGE của môi trường sau lên men, L-asparaginase tái tổ hợp được tìm thấy trong dịch ngoại bào của P. pastoris. Với khả năng sản xuất protein có hoạt tính cao hơn so với chủng P. pastoris X33, SMD1168 được lựa chọn để biểu hiện L-asparaginase tái tổ hợp. Sau khi tinh sạch, sự xuất hiện của một băng có kích khối lượng phân tử xấp xỉ 45 kDa trên điện di SDS-PAGE cho thấy protein tái tổ hợp đã bị glycosyl hóa với hoạt tính riêng 6.251 Umg-1 và đạt độ sạch 3.471 lần.