<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>

Affibody® molecules are small therapeutic proteins which mimics antibody functionality. This is a report of several methods for increasing productivity and yield in recombinant production of Affibody® molecules. This literature study shows several steps in the production line which can be optimised, several novel methods for cultivating and harvesting cells and purication of proteins. There is also a section about validation of therapeutic protein production according to The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) are presented.

Abstract Potential lethal diarrhoea caused by enterotoxigenic Escherichia coli strains is one of the most common diseases in young pigs. It can be cured by single-chain antibody fragments (scFv), which can be produced in recombinant microorganisms. Pichia pastoris, a methylotrophic yeast, is generally considered an interesting production system candidate, as it can secrete properly folded proteins. These proteins accumulate in high concentrations during fermentation, reducing the cost for product recovery. Strong inducible AOX1 promoter, widely used in P. pastoris for fast, inexpensive production, is typically induced by methanol. The high oxygen demand of methanol metabolism makes oxygen supply a major parameter in cultivations requiring special process design strategies. In standard fed-batch cultivation, dissolved oxygen concentration inside a bioreactor is kept at a certain level by pumping air and pure oxygen into the reactor. There are safety concerns over the handling of oxygen, especially at a large scale. Therefore, there is a need to develop a production process under oxygen-limited conditions. This dissertation studies the development of a cost-efficient production process of scFv in P. pastoris. Both methanol and oxygen parameters influence the production process and the objective was to find a robust production process. Fed-batch cultivations were performed in a 10 L scale bioreactor. The effects of lower oxygen level, methanol concentration, glycerol feeding duration and specific substrate-uptake rates on product formation were studied. A P. pastoris GS115 his4 strain under an AOX1 promoter system expressing scFv was used in this study. The fed-batch fermentations were carried out in a bioreactor with basal salt media. In this doctoral dissertation, a process was developed for a single-chain antibody fragment (scFv) production in P. pastoris. The product levels of 3.5 g L-1 scFv in culture supernatant were achieved and a production process was designed without additional need of pure oxygen, thus relieving safety requirements and lowering the amount of methanol. The process developed during this research may potentially be utilised by both academia and industry having interests in expressing proteins in P. pastoris. The methanol-uptake control strategy is beneficial for those products that suffer from degradation or modification during limited feeding of methanol. / Tiivistelmä Enterotoksigeenisten E.coli kantojen aiheuttama ripuli on porsaiden tavallisimpia tauteja, joka voi johtaa jopa kuolemaan. Tautia voidaan hoitaa yhdistelmä-DNA-tekniikalla tuotetuilla vasta-ainefragmenteilla (scFv). Metylotrofista Pichia pastoris hiivaa pidetään kiinnostavana vasta-ainefragmenttien tuottoisäntänä, koska se pystyy erittämään oikealla tavalla laskostuneita proteiineja. Näitä proteiineja kertyy fermentointiprosessissa solujen ulkopuolelle korkeina pitoisuuksina, mikä vähentää tuotteiden talteenottokustannuksia. Vahva metanolilla indusoituva AOX1-promoottori on laajassa käytössä P. pastoris tuottosysteemissä tuoton nopeuden ja alhaisten kustannusten ansiosta. Metanolin aineenvaihdunta vaatii paljon happea, joten riittävän tehokas hapen liuottaminen on tärkeimpiä fermentointiparametreja ja vaatii erityisiä prosessin toteutusstrategioita. Perinteisessä fed-batch-fermentoinnissa liuenneen hapen pitoisuus bioreaktorissa pidetään halutulla tasolla lisäämällä ilmaa ja puhdasta happea reaktoriin. Koska hapen käsittelyyn liittyy turvallisuusriskejä erityisesti teollisuusmittakaavassa, happirajoitteisissa olosuhteissa toimiva tuotantoprosessi olisi hyödyllinen. Tässä väitöstutkimuksessa kehitettiin kustannustehokasta prosessia scFv-:n tuottoon P. pastoris hiivalla. Metanoliin ja happeen liittyvät parametrit ovat olennaisia prosessiin vaikuttavia tekijöitä. Tavoite oli kehittää yksinkertainen ja käytännöllinen prosessi. Työssä tutkittiin alhaisen happitason, metanolin pitoisuuden, glyserolisyötön keston ja substraattien spesifisten kulutusnopeuksien vaikutuksia tuotteen muodostumiseen 10 litran bioreaktorissa. Isäntäkantana oli P. pastoris GS115 his4, jossa scFv-ekspressiota säädeltiin AOX1 promoottorilla. Fed-batch fermentointien kasvatusalustana käytettiin Basal Salt Medium alustaa (BSM). Väitöstyössä kehitettiin tavoitteiden mukainen vasta-ainefragmenttien tuottoprosessi P.pastoris hiivalle. Menetelmällä saavutettiin tuotepitoisuus 3,5 g L-1 kasvatusliemen supernatantissa ilman puhtaan hapen lisäystarvetta, ja siten metanolin kulutus väheni ja prosessiturvallisuus parani verrattuna perinteisiin prosesseihin. Kehitetty prosessi soveltuu käytettäväksi sekä akateemisessa tutkimuksessa että teollisuudessa tuotettaessa erilaisia proteiineja P. pastoris hiivalla. Metanolin kulutuksen säätöstrategia on erityisen hyödyllinen tuotteille, joilla ongelmana on proteolyysi tai muokkautuminen metanolirajoitteisessa fermentoinnissa.

ABSTRACT The aim of this study is develop and test an inexpensive molecular tool kit to be used for diagnostic PCR for diseases such as Leber hereditary optic neuropathy (LHON) and Cystic fibrosis(CF). By developing and optimizing recombinant Taq polymerase and making a DNA size ladder from plasmids pPSU1 and pPSU2 the financial cost for the tool kit would be reduced significantly compared to the commercial components. With an inhouse method both the recombinant Taq polymerase and the pPSU1 and pPSU2 plasmids were purified from the E.coil strain DH5-α. Thereafter to analyse the components of the tool kit both conventional PCR and Real-time PCR to make sure that the tool kit would work for both types of PCRs.     The homemade Taq polymerase proved to be able to sustain in room temperature for at least 24 h and the polymerase also showed that it works with different primers such as LHON, CF and Beta-globin in both endpoint and probe base real-time PCR. The homemade size marker produced a reliable in agarose gel electrophoresis but requires optimization for continued usage for smaller PCR products.     In conclusion the homemade Taq polymerase will be used in future PCR analysis in the laboratory and the recombinant production process as well. Meanwhile the homemade size marker did not work sufficiency enough to be continuously used with gel electrophoresis in the laboratory without being further modified.

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.

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

<p>The aim of this study was to better understand the role of conditioned medium (CM) in Spodoptera frugiperda Sf9 insect cell proliferation and recombinant protein production using the baculovirus expression system.</p><p>CM was found to stimulate cell proliferation. Addition of CM and 10 kDa CM filtrate to an Sf9 culture decreased the lagphase and the maximum cell density was reached earlier than for cultures in fresh medium. The positive effect of 10 kDa CM filtrate showed that CM contains at least one small growth promoting factor. The effect was not eliminated by trypsin treatment. Addition of CM or 10 kDa CM filtrate to Sf9 cultures was found to have a negative effect on the recombinant protein production. The effect was thought to be indirect and most probably via the impact of CM on cell physiology. CM was also found to contain proteinase activity. The proteinase was identified as Sf9 cathepsin L. A proform with a molecular mass about 49 kDa and two active forms at about 39 and 22 kDa were found. The role of cathepsin L in Sf9 cultures is not yet clear. However, the knowledge of the presence of this proteinase in CM can be of great value for improving product quality and yield. Further, CM was found to have other properties as well: a concentrated fraction of CM exhibited strong antibacterial activity towards Bacillus megaterium and a weaker activity towards Escherichia coli. B. megaterium lysed rapidly after incubation in the CM fraction.</p><p>Repeated subculturing of Sf9 cells provoked a switch in growth kinetics. After 30-45 passages the cells started to proliferate earlier after inoculation and addition of CM had no longer a growth stimulating effect. However, CM still stimulated growth of a culture with low passage (LP) number (up to 45 passages). High passage cells (HP cells, over 100 passages) displayed a shorter lagphase than LP cells and the culture reached the maximum cell density 24-48 h earlier. Cell cycle analysis showed that the Sf9 cells were transiently synchronised in the G2/M phase 10 h after inoculation, before proliferation was initiated. This synchronisation was more pronounced for HP cells than for LP cells, which correlated to a higher recombinant protein production in baculovirus infected HP cells than in LP cells. Synchronisation of cells in G2/M by yeastolate-limitation before infection with baculoviruses suggested that the degree of synchronisation is connected to the cell density dependent decrease in recombinant protein production of Sf9 cultures.</p>

O vírus da dengue (DENV) e o vírus da febre amarela (YFV) (gênero Flavivirus, família Flaviviridae) representam importantes arbovírus causadores de doenças em humanos que afetam as regiões tropicais e sub-tropicais do planeta, somando milhões de infectados anualmente. Embora exista uma vacina contra o YFV, ainda são notificados muitos casos de febre amarela nas regiões endêmicas das Américas e, principalmente, da África. Recentemente, foi licenciada uma vacina contra o DENV, mas seu uso ainda é bastante restrito. Não existem agentes terapêuticos para tratamento da infecção contra nenhum desses vírus. Portanto, estudos para identificação de fármacos para combaterem a infecção pelos DENV e YFV são de suma importância. Nosso grupo descreveu a ação antiviral da fosfolipase A2 crotoxina B (PLA2-CB) da serpente Crotalus durissus terrificus, a qual tem ação diretamente sobre o envelope de DENV e YFV. A meta principal deste trabalho foi a produção das duas isoformas da PLA2-CB (CB1 e CB2) recombinantes e avaliação de suas atividades antivirais, a fim de contribuir com a prospecção de novas drogas antivirais. Para alcançar tais propósitos, as sequências codificadoras das isoformas foram otimizadas para expressão em sistema procarioto, quimicamente sintetizadas e enzimaticamente inseridas no vetor de expressão pGS-21a. Os plasmídeos gerados foram utilizados para transformar células E. coli das cepas BL21(DE3), Origami B(DE3) e ArcticExpress (DE3). As proteínas recombinantes foram expressas juntamente com uma cauda de polihistidina (6xHis) no extremo C-terminal (CB1+6xHis_opt e CB2+6xHis_opt). Ambas as proteínas foram reconhecidas por anticorpos produzidos contra a PLA2-CB in natura em ensaio de Western Blot. Considerando que as proteínas foram expressas de forma insolúvel, a purificação foi realizada em sistema de cromatografia líquida (FPLC), utilizando coluna com resina de afinidade por níquel sob condições desnaturantes, utilizando ureia como agente solubilizador. As proteínas foram renaturadas na própria coluna de níquel (on-column) durante a purificação, utilizando um gradiente decrescente de ureia (6-0M - 120ml - 0,1ml/min) e o detergente CHAPS como agente estabilizador. As proteínas CB1+6xHis_opt e CB2+6xHis_opt em ensaio colorimétrico de hidrólise de fosfatidilcolina apresentaram atividade fosfolipásica, indicando preservação do sítio catalítico. Em ensaio virucida contra o DENV-2, YFV e outros dois vírus envelopados: Vírus Chikungunya (CHIKV) e vírus Zika (ZIKV), as proteínas recombinantes reduziram a formação de placas de lise exibindo índices de seletividade na média de 0,6. As proteínas CB1+6xHis_opt e CB2+6xHis_opt podem vir a ser um importante modelo na prospecção de novas drogas antivirais e como ferramenta no estudo do mecanismo de replicação viral. / Dengue virus (DENV) and yellow fever virus (YFV) (genus Flavivirus, family Flaviviridae) are important arboviruses that cause diseases in humans affecting the tropical and subtropical regions of the planet with millions of infected annually. Although there is a vaccine against YFV, many cases of yellow fever are still reported in the endemic regions of the Americas, and especially in Africa. A vaccine against DENV has recently been licensed, but its use is still quite restricted. There are no therapeutic agents to treat the infection against any of these viruses. Therefore, studies to identify drugs to combat DENV and YFV infection are of extreme importance. Our group described the antiviral action of the phospholipase A2 crotoxin B (PLA2-CB) isolated from Crotalus durissus terrificus, which acts directly on the envelope of DENV and YFV. The aim of this study was to produce the two recombinant PLA2-CB (CB1 and CB2) isoforms and evaluate their antiviral activities in order to contribute to the prospection of new antiviral drugs. To achieve such purposes, the coding sequences of the isoforms were optimized for prokaryotic expression system, chemically synthesized and enzymatically inserted into the pGS-21a vector. The plasmids generated were used to transform E. coli cells from BL21 (DE3), Origami B (DE3) and ArcticExpress (DE3) strains. Recombinant proteins were expressed tagged with a polyhistidine (6xHis) tail at the C-terminus (CB1+6xHis_opt and CB2+6xHis_opt). Both proteins were recognized by antibodies raised against native PLA2-CB in Western blot assay. Considering that the proteins were expressed in insoluble form, purification was performed in liquid chromatography system (FPLC) using nickel resin column under denaturing conditions, using urea as the solubilizing agent. The proteins were renatured on-column during purification procedure using a decreasing gradient of urea (6-0M - 120ml - 0,1ml/min) and CHAPS as a stabilizing agent. CB1+6xHis_opt and CB2+6xHis_opt proteins showed phospholipase activity in a colorimetric assay based on phosphatidylcholine hydrolysis, indicating preservation of the catalytic site. In a virucidal assay against DENV-2, YFV and two other enveloped viruses: Chikungunya virus (CHIKV) and Zika virus (ZIKV), the recombinant proteins reduced the plaques of lysis formation exhibiting selectivity indices at the mean of 0.6. The CB1+6xHis_opt and CB2+6xHis_opt proteins could be important models for the prospection of new antiviral drugs and as tools for the study of the mechanism of viral replication.

The aim of this study was to better understand the role of conditioned medium (CM) in Spodoptera frugiperda Sf9 insect cell proliferation and recombinant protein production using the baculovirus expression system. CM was found to stimulate cell proliferation. Addition of CM and 10 kDa CM filtrate to an Sf9 culture decreased the lagphase and the maximum cell density was reached earlier than for cultures in fresh medium. The positive effect of 10 kDa CM filtrate showed that CM contains at least one small growth promoting factor. The effect was not eliminated by trypsin treatment. Addition of CM or 10 kDa CM filtrate to Sf9 cultures was found to have a negative effect on the recombinant protein production. The effect was thought to be indirect and most probably via the impact of CM on cell physiology. CM was also found to contain proteinase activity. The proteinase was identified as Sf9 cathepsin L. A proform with a molecular mass about 49 kDa and two active forms at about 39 and 22 kDa were found. The role of cathepsin L in Sf9 cultures is not yet clear. However, the knowledge of the presence of this proteinase in CM can be of great value for improving product quality and yield. Further, CM was found to have other properties as well: a concentrated fraction of CM exhibited strong antibacterial activity towards Bacillus megaterium and a weaker activity towards Escherichia coli. B. megaterium lysed rapidly after incubation in the CM fraction. Repeated subculturing of Sf9 cells provoked a switch in growth kinetics. After 30-45 passages the cells started to proliferate earlier after inoculation and addition of CM had no longer a growth stimulating effect. However, CM still stimulated growth of a culture with low passage (LP) number (up to 45 passages). High passage cells (HP cells, over 100 passages) displayed a shorter lagphase than LP cells and the culture reached the maximum cell density 24-48 h earlier. Cell cycle analysis showed that the Sf9 cells were transiently synchronised in the G2/M phase 10 h after inoculation, before proliferation was initiated. This synchronisation was more pronounced for HP cells than for LP cells, which correlated to a higher recombinant protein production in baculovirus infected HP cells than in LP cells. Synchronisation of cells in G2/M by yeastolate-limitation before infection with baculoviruses suggested that the degree of synchronisation is connected to the cell density dependent decrease in recombinant protein production of Sf9 cultures. / QC 20101222

LOPAP, proteína isolada da toxina de lagartas Lonomia obliqua, possui ação ativadora de protrombina, efeito pró-coagulante e ação citoprotetora em células do endotélio humano, em cultura. Tem cadeia única com 181 resíduos de aminoácidos e 21 kDa. Sua estrutura terciária é formada por oito folhas-b fechadas em uma extremidade, mantidas juntas por pontes de hidrogênio, em formato de barril. Está classificada como pertencente ao grupo das Lipocalinas (proteínas de transporte). Neste trabalho estudou-se o LOPAP, que foi produzido recombinante em cultivo de Pichia pastoris em biorreator e purificado. Avaliou-se sua estabilidade quanto às atividades enzimática e citoprotetora, e sua estrutura secundária. Não foi detectada ativação de protrombina para o r-LOPAP obtido, mas foi observada ação citoprotetora. Considerando estes resultados e a análise de sua estrutura secundária por dicroísmo circular, concluiu-se que a proteína foi expressa com tamanho e sequência corretos, mas sem uma estrutura terciária correta, o que é determinante para a atividade enzimática. / LOPAP, a protein isolated from the toxin of Lonomia obliqua caterpillars, has prothrombin activation action, procoagulant effect and cytoprotection action in human endothelium cells culture. It has only chain with 181 amino acid residues and 21 kDa of size. Its tertiary structure is made by eight b-sheets closed at one end, hold together by hydrogen bonds, barrel-shaped. It is classified as belonging to the Lipocalin group (proteins of transport). This work studied the LOPAP, which was produced recombinant in Pichia pastoris culture in bioreactor, was purified, and it was evaluated its stability related to enzymatic and cytoprotection activities, and its secondary structure. It was not detected prothrombin activation for the r-LOPAP obtained, but it was observed a cytoprotective effect. Regarding these results and the analysis of its secondary structure, by circular dichroism, it was concluded that the protein was expressed with correct size and sequence, but without a correct tertiary structure, which is determinant for the enzymatic activity.