Le système baculovirus / cellules d’insectes est un outil performant pour la production de vecteurs AAV recombinant pour la thérapie génique. Cette production nécessite que, les gènes rep et cap de l’AAV ainsi que le transgène encadré par les ITR, soient codés dans le génome du baculovirus AcMNPV. L’utilisation de cassettes de recombinaison pour intégrer ces gènes provoque à grande échelle une certaine instabilité au sein des 134Kb du génome. Pour pallier à ce phénomène, une nouvelle stratégie d’intégration de gènes a été mise en place. Celle-ci doit permettre notamment d’éviter la présence de cicatrices de recombinaison et de modifier le génome du bacmid d’AcMNPV. Basée sur la technique de Gibson Assembly, nous avons tenté d’assembler de novo le génome du baculovirus d’AcMNPV à partir de fragments PCR chevauchants. Nous avons réussi à pré assembler en 4 parties la totalité du génome. Celles-ci ont permis d’insérer le gène de la GFP comme gène d’intérêt mais également d’éliminer le gène de résistance antibiotique et le Mini-F réplicon, facteur d’instabilité en cellules d’insecte. Plusieurs clones obtenus ne contiennent aucune mutation. Cette technique pourrait être appliquée à la production de vecteurs AAVr. / The baculovirus / insect cell system allows AAV vector production for gene therapy purposes. Current baculoviruses used for the production of rAAV vectors are a bottleneck for Scaling up production. Indeed the use of recombination boxes to insert the genes brings instability to the134kb genome. To avoid this, a new gene integration strategy has been implemented. In this work, we have assayed the full assembly of AcMNPV´s genome using the Gibson Assembly technic. PCR fragments covering the totality of genome and able to assemble through overlapping terminal regions have been pre-assembled in 4 segments. The finally assembly should contain the eGFP gene used has gene of interest along with replacement of the Mini-F replicon by polyhedrin gene. This feasibility study has shown that we could obtain segments without any mutation. Final assembly is still on-going. This technology should be applied next for the generation of baculovirus used for the production of rAAV vectors. This marker less combination method should solve problems of genome instability.

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Nutrition and Food Science, 1981. / Bibliography: leaves 100-104. / by Tina Talvikki Kangas. / M.S.

The ligand activated transcription factor Aryl Hydrocarbon Receptor (AHR) forms a DNA binding heterodimer with the Aryl Hydrocarbon Nuclear Translocator (ARNT) in response to planar aromatic hydrocarbons. In addition to AHR and ARNT there are at least three other proteins involved in AHR signaling. These proteins are the co-chaperone p23, Ara-9 and two molecules of Heat Shock Protein-90 (HSP-90). This study documents the production of Ara-9 and C∆418 (an ARNT deletion construct) in a modified thioredoxin fusion system. These proteins were expressed in a system that allowed for removal from the fusion partner via a thrombin recognition site as well as the incorporation of an in vitro phosphorylation site. The proteins were then expressed and column purified from E. coli. Once the proteins were expressed and purified they were cleaved from the thioredoxin fusion partner and radioloabeled. Following optimization of the proteolytic digest and radio-labeling each protein was subject to two methods of functional analysis. C∆418 function was assessed by electrophoretic mobility shift assay (EMSA) and proved to effectively form a DNA binding heterodimer with ARNT. In addition the functionality of C∆418 was assessed by co-precipitation showing that the ThioHis-produced C∆418 was indeed able to dimerize with C∆553 (an AHR deletion construct). The ThioHisproduced Ara-9 was also assessed for functionality by EMSA and showed that it was able to restore AHR/ ARNT/DRE complex formation as effectively as Ara-9 produced in a baculovirus system. In addition the function of ThioHis-Ara-9 was also assessed through Far-Western blotting for its ability to associate with renatured HSP-90. These studies involving C∆418 and Ara-9 show that these proteins can be efficiently produced in a functional manner utilizing an inexpensive bacterial system In addition this study documents the production of a plasmid (pCMV-Ara-9) for transfection into the HepG2 cell line to monitor the effects of increased cellular Ara-9 on AHR.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20450号 / 農博第2235号 / 新制||農||1050(附属図書館) / 学位論文||H29||N5071(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 杉山 淳司, 教授 髙部 圭司, 教授 梅澤 俊明 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No description available.

The methylotrophic yeast, Pichia pastoris, has been used as a protein expression system to express over 500 heterologous proteins. P. pastoris provides many advantages over other organisms that have been utilized for this purpose. In this project, we developed a new host/selectable marker and auxotrophic strains of P. pastoris based on methionine biosynthesis to increase P. pastoris's versatility as a host for homologous protein expression. This was accomplished by selecting for a yeast that is deficient in methionine biosynthesis, P. pastoris (yJC239), and gene complementation through transformation with a genomic DNA library. Bioinformatics show that the P. pastor is MET gene has 54% amino acid identity with 68% similarity to the S. cerevisiae MET2 gene, which codes for homoserine O-transacetylase. We have constructed expression vectors for intracellular and extracellular expression of proteins with the MET2 marker and have also constructed strains with various auxotrophs including me/2.

The methylotropic yeast Pichia pastoris has been used for many applications, particularly for its ability to produce and readily secrete heterologous proteins. Nonetheless, there are obstacles in making this useful yeast into a more efficient secretion system that readily secretes problem proteins. In the Lin-Cereghino lab, mutant strains were developed by the method of restriction enzyme mediated integration. These mutants have the ability to secrete β-galactosidase at higher levels in comparison to the wild type. This study focused on characterizing the specific mutant ah2 for its ability to secrete HRP, SLPI, and CALB lipase proteins, as well as using transmission electron microscopy to observe the effect of the pREMI-Z mutation on the morphology. Analysis of the Ah2 protein resulted in a comparative β-galactosidase secretion study, as well as a growth rate study, between the original pREMI-Z ah2 mutant and ah2 mutant cells that were transformed with pKanB-AH2 rescue construct. Lastly, a cell localization experiment was done to examine where Ah2p localizes. By these analyses, we gain a bit more understanding of the P. pastoris secretion pathway, while also outlining a procedure by which to characterize the other pREMI-Z mutants.

Advances in molecular biology have revolutionized the field of biotechnology and allowed the development of recombinant protein production as an alternative to harvesting proteins from their natural sources. Production of target proteins in controllable host organisms offers scalable and economic approaches to meet market needs. Current host cell expression systems vary, and each has advantages and disadvantages. Photoautotrophic organisms, like microalgae, represent alternatives to fermentative microbes with the promise of recombinant protein production from sustainable inputs like carbon dioxide as a carbon source. In this thesis, a Chlamydomonas reinhardtii strain that was recently developed for phototrophic high- density cultivation and nuclear transgene expression was used to express target recombinant proteins from its plastid genome as a demonstration of possibilities for expansion of its potential value. Here, sequences of two anti-freeze proteins, the insect Choristoneura fumiferana (CfAFP) and grass Lolium perenne ice binding protein (LpIBP), and a yellow fluorescent protein (YFP, mVenus) were adapted to algal chloroplast genome expression plasmids, transformed, and protein titers characterized under various nutrient and growth regimes in alga. Rather than antibiotic selection, transformants were selected based on photosynthesis restoration in a knock-out recipient strain. LpIBP and mVenus expression were detected by Western blot and in gel fluorescence and estimated to be expressed up to ~7.65% and ~8.41% total soluble protein, respectively, whereas expression of CfAFP was not observed in any transformant. This work forms the basis of further investigation of recombinant protein expression in C. reinhardtii in high-density antibiotic-free culture and may influence feasibility assessments of scale up processes.

As antibiotic resistance has become a major global threat, the World Health Organization has urgently called scientists for alternative strategies for control of bacterial infections. Endolysin, a protein encoded by a phage gene, can degrade bacterial peptidoglycan (PG). Currently, there are three endolysin products in the clinical phase. We, thus, are interested in exploring novel endolysins from Streptomyces phages as only a few of them have been experimentally characterized. Using bioinformatics tools, we identified nine functional domain groups from 250 Streptomyces phages putative endolysins. NootNoot gp34 (transglycosylase; Nt34lys), Nabi gp26 (amidase; Nb26lys), Tribute gp42 (PGRP; Tb42lys), and LazerLemon gp35 (CHAP; LL35lys) were selected for experimental studies. We hypothesized that (1) the proteins of interest will have the ability to degrade PG, and (2) the proteins will be potential antimicrobial agents against ESKAPE safe relatives. The results showed that LL35lys, Nb26lys and Tb42lys exhibit PG-degrading activity on zymography and hydrolysis assay. The enzymes (400 µg/mL) can reduce PG turbidity to 32-40%. The killing assay suggested that Tb42lys possess a boarder range (Escherichia coli, Pseudomonas putida, Acinetobacter baylyi and Klebsiella aerogenes). While Nb26lys can attack Gram-negative bacteria, LL35lys can only reduce the growth of the Gram-positive strains with an MIC90 of 2 µg/mL. A higher concentration (≥300 µg/mL) of Nb26lys is needed to treat P. putida and K. aerogenes. Therefore, endolysins from Streptomyces phage have potential as possible antimicrobial agents against ESKAPE bacteria.

Hemophilia B is a debilitating and life-threatening disorder caused by a deficiency in or dysfunction of factor IX (FIX), a complex plasma glycoprotein required for the formation and maintenance of blood clots. Treatment of hemophilia B involves infusion of replacement FIX currently derived from two sources: FIX purified from pools of human plasma (pd-FIX) and a single recombinant FIX product generated in genetically engineered Chinese hamster ovary (CHO) cells. Both of these FIX products are prohibitively expensive, limiting of the treatment options of hemophiliacs worldwide. As a result, a more abundant and affordable FIX product would greatly improve the life prospects for hemophiliacs. The biological activity of FIX is dependent upon its numerous post-translational modifications (PTMs), including gamma-carboxylation, proteolytic maturation, phosphorylation, sulfation, and glycosylation. Of these PTMs, those known to be vital for activity are gamma-carboxylation of multiple glutamate residues near the N-terminus and proteolytic cleavage of the FIX propeptide. When expressed at a high rate in exogenous expression systems, however, the ability of current systems to effect the necessary PTMs is severely rate limited, restricting the production of active FIX. The transgenic pig bioreactor represents a promising source for the production of large quantities biologically active FIX due to its demonstrated ability to perform the required FIX PTMs. It was the goal of this study to characterize the PTM structure and the resulting function of recombinant FIX when expressed at 1-3 mg/ml in the transgenic pig mammary epithelium (tg-FIX). It was found that the expressed tg-FIX is comprised of a heterogeneous mixture of FIX PTM isoforms. This mixture represents a spectrum of tg-FIX molecules of varying gamma-carboxyglutamic acid (Gla) and propeptide content, indicating that rate limitations in effecting these PTMs are present. A purification process was developed utilizing heparin-affinity chromatography to purify the total population of tg-FIX from pig milk, a complex multi-phase feedstock. Subsequently, a process was developed to fractionate the total population of tg-FIX into subpopulations based upon the extent of post-translational modification. Q ion-exchange chromatography was utilized to fractionate tg-FIX based upon molecular acidity which was found to be correlated to both biological activity and Gla content. The resulting biologically active tg-FIX population contained an average of 7 of the 12 Gla residues found in pd-FIX. Immuno-affinity chromatography was subsequently utilized to further fractionate tg-FIX into mature tg-FIX and propeptide-containing tg-FIX populations. The isolated FIX PTM populations were subjected to functional analysis by investigating in vitro clotting activity, activation by factor XIa, and in vivo pharmacokinetics. From this analysis it was found that mature tg-FIX with an average 7 Gla residues, representing approximately 9% of the total tg-FIX produced, exhibits wild-type in vitro clotting activity and normal activation by factor XIa. The remainder of the tg-FIX produced, characterized by either a lower Gla content or the presence of the propeptide, was found to be inactive and displayed less efficient activation by factor IXa. In an in vivo pharmacokinetic study in the hemophilia B mouse model, biologically active tg-FIX was found to possess altered circulating properties. Tg-FIX was characterized by a lower recovery, approximately one-sixth that of pd-FIX, but an extended circulation half-life. From this study it was found that the mean residence time of tg-FIX after injections is approximately twice that observed for pd-FIX. These altered pharmacokinetic properties are likely linked to the unique tg-FIX PTM structure, perhaps through altered endothelial cell binding characteristics caused by the reduced Gla content. / Ph. D.