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Avaliação como probióticos para frangos de corte de Pichia pastoris e Pichia pastoris recombinante contendo o gene da fosfolipase C de Clostridium perfringens / Evaluation of the probiotic properties in broilers of Pichia pastoris and a recombinant Pichia pastoris containing the Clostridium perfringens fosfolipase C geneSTORCH, Otávio Brod 17 November 2008 (has links)
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Previous issue date: 2008-11-17 / Antibiotics used for the last decades as growth promoters for animals, were banned
in several countries because of the risk of inducing the appearance of antibiotic
resistant bacteria. Probiotics, live microorganisms that benefit health promoting the
stability of the gut microbiota, are their most promising substitutes. The objective of
this research was to determine the probiotic properties of Pichia pastoris and a
recombinant P. pastoris containing the Clostridium perfringens fosfolipase C gene.
Ross® P8 female chicks were randomly distributed in four groups of ten each, in
three repeats, and fed with a commercial food devoid of antibacterials. Group 1 was
fed with un-supplemented food; group 2 was supplemented with 1x106 viable P.
pastoris strain KM71H gr-1; group 3 with 1x106 viable recombinant P. pastoris
containing the C. perfringens fosfolipase C gene gr-1 and group 4 with 1x106 viable
spores of Bacillus cereus var. Toyoi gr-1. Water and food were supplied ad libitum.
Weight gain of each animal and food conversion of each group at 49 days of age,
were estimated. Individual seroconversions against C. perfringens α toxin at days 1,
10, 20, 30 and 49 were estimated. At the end of the experiment the animals were
slaughtered and samples from different organs examined histologically. Mean live
weights at 49 days of age were 2.172, 2.228, 2.410 and 2.333 Kg for groups 1, 2, 3
and 4, respectively, different at P<0.05. Mean food conversions were 2.58, 2.41,
2.35 and 2.50 for groups 1, 2, 3 and 4, respectively. Seroconversions at 49 days of
age were 1.1, 1.4, 1.5 and 1.3 for groups 1, 2, 3 and 4, respectively, different at
P<0.05. Histological alterations were not detected. It was concluded that P. pastoris
and recombinant P. pastoris may be used as probiotics for broilers due to their
capacity of increasing food efficiency and seroconversion without undesirable
effects. / Os antibióticos utilizados como promotores de crescimento nas últimas décadas
foram banidos em vários países devido ao risco de induzir o surgimento de
bactérias resistentes. Entre as alternativas para sua substituição, os probióticos,
suplementos alimentares compostos de microrganismos vivos que beneficiam a
saúde do hospedeiro através do equilíbrio da microbiota intestinal, aparecem como
a mais plausível. Este trabalho objetivou determinar as propriedades como
probiótico da levedura Pichia pastoris e sua variante recombinante contendo o
gene da fosfolipase C de Clostridium perfringens. Frangas de um dia de idade da
linhagem Ross® P8 foram distribuídas aleatoriamente em quatro grupos de dez
animais cada, em três repetições, e alimentadas com ração comercial isenta de
antibacterianos. O grupo 1 (Controle) recebeu ração não suplementada, o grupo 2
recebeu a mesma ração suplementada com 1x106 células viáveis gr-1 de P. pastoris
cepa KM71H, o grupo 3 ração suplementada com 1x106 gr-1 de células viáveis de
P. pastoris recombinante contendo o gene da toxina α de C. perfringens, e o grupo
4 ração suplementada com 1x106 gr-1 de esporos viáveis de Bacillus cereus var.
Toyoi. Ração e água foram oferecidos ad libitum. Estimou-se o ganho de peso de
cada animal aos 49 dias de idade, e a conversão alimentar de cada grupo.
Determinou-se a soroconversão individual por ELISA, utilizando como antígeno a
toxina α padrão de C. perfringens, a partir de amostras de sangue coletadas aos 1,
10, 20, 30 e 49 dias de idade. Ao término do experimento os animais foram
abatidos e amostras de órgãos submetidos a análise histopatológica. No dia 49, os
pesos vivos médios foram 2,172, 2,228, 2,410 e 2,333 kg, significativamente
diferentes (P<0,05) ao grupo controle, para os grupos 1, 2, 3 e 4, respectivamente.
As conversões alimentares médias foram 2,58, 2,41, 2,35 e 2,5 para os grupos 1,
2, 3 e 4, respectivamente. As soroconversões ao dia 49 foram 1,1, 1,4, 1,5 e 1,3
para os grupos 1, 2, 3 e 4, respectivamente, significativamente diferentes (P<0,05)
ao grupo controle. Nos estudos histopatológicos não foram encontradas alterações.
Concluiu-se que P. pastoris e P. pastoris recombinante podem ser utilizadas como
probiótico em frangos de corte por aumentar a eficiência alimentar e a
soroconversão, sem apresentar contraindicações.
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Expressão heteróloga e utilização da proteína recombinante EMA-1 de Theileria equi como imunobiológico / Expressão heteróloga e utilização da proteína recombinante EMA-1 de Theileria equi como imunobiológicoNizoli, Leandro Quintana 18 March 2009 (has links)
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Previous issue date: 2009-03-18 / Equine theileriosis is considered to be one of the most important parasitic diseases
that affect horses, and has great economic impact on the equine industry. The
disease is caused by the etiologic agent Theileria equi, which is classified as a
hematozoan. The losses associated with equine theileriosis are related to clinical
manifestation as well as restriction to international travel to positive horses. Chronic
infected equines suffer the risk of the disease relapse which leads to losses in
reproduction performance and are potentially disseminators of the disease. In the last
years, studies on the immunologic diagnosis and vaccination against T. equi have
focused to obtain distinct antigenic proteins. On the outer membrane of this
protozoan, major surface proteins has been characterized and named as EMAs (equi
merozoite antigen). Of these, EMA-1 has been used as antigen for diagnosis due to
its conservation in diverse isolates. Its role as a potential immunogen has been well
documented due its ability to stimulate a humoral response with production of
specific antibodies in infected animals. Through this antibodies one can used as tool
for immune diagnostic of this disease. EMA-1 is also a strong candidate to be use as
a vaccine in the control of equine theileriosis. In this study we used the Pichia
pastoris yeast as expression system for the production of the EMA-1 protein of T.
equi and evaluated its antigenicity and immunogenicity. When tested for antigenicity,
the recombinant protein was recognized by antibodies form chronic T. equi infected
horses, suggesting that epitopes of the native were conserved in the recombinant
protein. Also we were able to observe that this protein was immunogenic in mice.
The data obtained in this study demonstrated that the yeast P. pastoris is an
expression system of heterologous protein suitable for the production of EMA-1 from
T. equi. / A Theileriose eqüina é considerada uma das principais doenças parasitárias que
acometem os eqüinos, acarretando grande impacto econômico na equinocultura. A
doença é causada pelo hematozoário Theileria equi. As perdas econômicas
associadas à theileriose eqüina estão relacionadas tanto aos fatores clínicos, quanto
à restrição ao trânsito internacional de animais soropositivos, já que animais
portadores crônicos são passíveis de reagudizações, gerando perda de performance
física e reprodutiva, e são potencialmente disseminadores da enfermidade. Nos
últimos anos, os estudos sobre o diagnóstico imunológico e vacinação contra T. equi
concentram-se na obtenção de frações antigênicas. Na membrana externa deste
protozoário foram caracterizadas proteínas principais de superfície denominadas de
EMAs (equi merozoite antigen). Dentre estas, a EMA-1 destaca-se como antígeno
para diagnóstico em função de sua conservação entre diversos isolados. Seu papel
também tem sido caracterizado como imunógeno por estimular forte resposta
humoral com produção de anticorpos em animais infectados, podendo ser usado
como ferramenta para imunodiagnóstico dessa doença. EMA-1 é também um
potencial candidato como antígeno vacinal no controle da theileriose equina. Neste
estudo utilizou-se o sistema eucariótico de expressão baseado na levedura
metilotrófica Pichia pastoris, para a produção da proteína EMA-1 de T. equi e a
avaliação quanto a sua antigenicidade e imunogenicidade. Quanto a sua
antigenicidade, a proteína recombinante foi reconhecida por anticorpos de animais
portadores crônicos de T. equi, sugerindo que epítopos nativos foram conservados
na proteína recombinante. Também foi observado que a proteína recombinante foi
capaz de gerar resposta imune em camundongos vacinados com esta proteína. Os
dados obtidos neste estudo demonstram que a levedura P. pastoris é um sistema de
expressão heterólogo adequado para a produção da proteína EMA-1 de T. equi,
podendo ser utilizada como imunobiológico no desenvolvimento de testes
diagnósticos e vacina recombinante.
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Production, Purification, and Characterization of wood substrates and Galactose oxidase enzyme / Produktion, rening och karakterisering av träsubstrat och galaktosoxidasenzymPongalikonnar Ranganathan, Rakhesh January 2023 (has links)
Trä är den bästa förnyelsebara källan för att producera många produkter på grund av dess biokompatibla och biologiskt nedbrytbara natur. Träets biomassa har en motstridig natur mot enzymatisk uppgradering. Det beror på olika orsaker som ligninhalt, acetylhalt i hemicellulosa och cellulosakristallinitet som blockerar enzymets bindningsställe. Denna studie kommer under BioUPGRADE, som är en samarbetsplattform för att skapa högvärdiga och mångsidiga material på ett hållbart sätt med hjälp av biokatalys. Det allmänna syftet med denna studie är att producera holocellulosa och hemicellulosasubstrat från olika träslag och producera, rena och validera galaktosoxidas som en potentiell biokatalysator för träfibermodifiering. Studien undersöker effekten av kemisk perättiksyradelignifiering på två träslag, lövträ av eukalyptus (HW), och barrträ av gran (SW), undersökta vid olika tidsintervall, där PAA framställdes exsitu med ett volymetriskt förhållande på 1:3. Med resultaten från PAA-behandlingen avlägsnades 38,53 % lignin i eukalyptus och 31,80 % i barrved. Hemicellulosautbytet ökade med 47,40 % för eukalyptus och 19,05 % för gran med en ökning av tiden för PAA-behandling. Acetylhalten i hemicellulosan minskade från 2 % till 0,6 % i lövträ och 1,96 % till 0,6 % i barrträ. Cellulosautvinningen efter delignifieringen var nästan 100 %. Galaktosoxidaset producerades i en skakkolv med användning av Pichia pastoris KM71H-stammen. Pichia pastoris KM71H-celler odlades med användning av det buffrade komplexa glycerolmediet (BMGY) och galaktosoxidas uttrycktes med användning av Pichia pastoris KM71H-stammen i buffrat komplex metanolmedium (BMMY). Det uttryckta GaOx-proteinet renades därefter med användning av AKTA-kromatografi med användning av en 5 ml Histrap FF-kolonn. För att bestämma proteinkoncentrationen utfördes bicinchoninsyra (BCA) analys och GaOx som producerades i skakkolvsodling var 286,25 mg/L. Den specifika aktiviteten hos skakkolven som produceras GaOx är 164,24 U/mg. Det kan observeras att PAA-behandling visar sig vara en effektiv metod för delignifiering eftersom cellulosautvinningen är nära 100 % och förlusten av hemicellulosa är relativt låg med det använda volymetriska förhållandet. GaOx som produceras i skakkolvsproduktion visar ett lovande utbyte med en betydande specifik aktivitet mot galaktosen som substrat och kan användas i framtiden för enzymatisk uppgradering av träets biomassa. / Wood is the best renewable source for producing many products due to its biocompatible and biodegradable nature. The wood biomass has a recalcitrance nature towards enzymatic upgrading. It is due to various reasons such as lignin content, acetyl content in hemicellulose, and cellulose crystallinity which blocks the enzyme binding site. This study comes under BioUPGRADE, which is a collaborative platform to create high-value and multipurpose materials sustainably using biocatalysis. The general aim of this study is to produce holocellulose and hemicellulose substrates from different wood species and produce, purify, and validate galactose oxidase as a potential biocatalyst for wood fiber modification. The study investigates the effect of chemical peracetic acid delignification on two wood species, eucalyptus hardwood (HW), and spruce softwood (SW) investigated at different time intervals, where the PAA was prepared ex-situ with a volumetric ratio of 1:3. With the results from the PAA treatment, 38.53% of lignin was removed in eucalyptus and 31.80% in softwood. The hemicellulose yield increased by 47.40% for eucalyptus and 19.05% for spruce with an increase in the time of PAA treatment. The acetyl content of the hemicellulose was reduced from 2% to 0.6% in hardwood and 1.96% to 0.6% in softwood. The cellulose recovery after the delignification was nearly 100%. The galactose oxidase was produced in a shake flask using the Pichia pastoris KM71H strain. Pichia pastoris KM71H cells were cultivated using the buffered complex glycerol media (BMGY) and galactose oxidase was expressed using the Pichia pastoris KM71H strain in Buffered complex methanol media (BMMY). The expressed GaOx protein was subsequently purified using AKTA chromatography using a 5ml Histrap FF column. To determine the protein concentration Bicinchoninic acid (BCA) analysis was performed and the GaOx produced in shake flask cultivation was 286.25 mg/L. The specific activity of the shake flask produced GaOx is 164.24 U/mg. It can be observed that PAA treatment proves to be an efficient method for delignification as the cellulose recovery is near 100% and the loss of hemicellulose is relatively low with the volumetric ratio used. The GaOx produced in shake flask production shows a promising yield with a significant specific activity towards the galactose as substrate and could be used in the future for the enzymatic upgrading of the wood biomass.
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Development of Pichia pastoris as a production system for HPV16 L1 virus-like particles as component to a subunit vaccineKotze, Lara 03 1900 (has links)
Human papillomavirus (HPV) is a sexually transmitted virus and known precursor to
cervical cancer, the second most lethal cancer in females across the world. Two virus-like
particle (VLP) vaccines exist that provide immunity against the main serotypes of the
disease and are produced in Saccharomyces cerevisiae (S. cerevisiae) and baculovirus
infected insect cells. Pichia pastoris (P. pastoris) was chosen as an alternative expression
system for HPV VLP production based on its history as prolific heterologous protein
producer that circumvent many of the problems associated with aforementioned
expression systems. The strongly inducible AOX promoter allows three-phase
fermentations (1.3 L bioreactors) in which high cell densities (>100gCDW.L-1) are
obtained prior to induction with methanol. During the induction phase the dissolved
oxygen concentration may be used to control addition of methanol. It is also possible to
use predetermined methanol feed rates and to adjust the amount of additional oxygen
sparged to maintain a constant dissolved oxygen level. The effects of these control
strategies, different gene constructs and multiple gene integrations were quantified
through monomer-, VLP- and mRNA production levels.
Increased biomass concentrations in the 20% dissolved oxygen control strategy led to the
highest volumetric VLP concentration (68.53 mg.L-1). VLPs were located intracellularly
in both the cytoplasm and membranes of the yeast cells. Despite lower codon adaptation
of the h-L1 gene expressed in the X33[h-L1] strain it still had higher volumetric VLP
concentrations under 40% dissolved oxygen control than the X33[Syn-L1] and
X33[SA-L1] strain containing the SA-L1 and Syn-L1 genes. This was ascribed to the
possible presence of rare codons in the Syn-hL1 and SA-L1 genes and a lower A+T
content in the h-L1 gene. Multiple gene integrations of the h-L1 gene had a negative
effect on VLP production and this conclusion was supported by lower mRNA
concentrations indicating lower transcriptional efficiency. Increased methanol induction
efficiency in the DO control strategies was indicated by higher specific L1 monomer
levels. Decreased VLP to monomer ratios in the DO control strategies indicated that a
bottleneck existed in the assembly process due to increased L1 monomer concentrations. Due to the hydrophobic region on the L1 protein, these proteins associated with the
membranes within the yeast cells especially when efficient assembly to VLPs did not
occur. HPV16 L1 VLP concentrations obtained in P. pastoris in this study are
comparable to the study by Li et al., (2003), but much lower than expression levels
obtained in baculovirus infected insect cells. Based on the expression levels of HBsAg
VLPs obtained in P. pastoris, this system, with the necessary recommended optimisation,
has the capacity for increased HPV VLP production ability.
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Pichia pastoris : a viable expression system for steroidogenic cytochrome P450 enzymesWepener, Ilse 12 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: This study describes:
I. The cloning of the CVP 19 gene and construction of the intracellular expression
vector pPIC3.5K-CYP19.
II. The transformation of the yeast, Pichia pastoris with the constructed vector.
III. The expression ofP450arom in Pichia pastoris.
IV. The determination of enzyme activity and isolation of the protein from the Pichia
pastoris cells.
V. The expression of P450c 17 in Pichia pastoris.
VI. The determination of kinetic constants for the conversion of progesterone to
170H-progesterone and 160H-progesterone by P450c17. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf:
I. Die klonering van die CVP 19 geen en die konstruksie van die intrasellulêre
uitdrukkingsplasmied, pPIC3.5K-CYPI9.
II. Die transformasie van die gis, Pichia pastoris, met die gekonstrueerde plasmied.
III. Die uitdrukking van aromatase in Pichia pastoris.
IV. Die bepaling van ensiemaktiwiteit en die isolering van die proteïen vanuit Pichia
pastoris.
V. Die uitdrukking van P450c17 in Pichia pastoris.
VI. Die bepaling van kinetiese konstantes vir die omsetting van progesteroon na
170H-progesteroon en 160H-progesteroon deur P450c17.
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Expression of the chimeric SAF gene from Human Papillomavirus in the methylotrophic yeasts Pichia pastoris and Hansenula polymorphaBurke, Arista 03 1900 (has links)
Thesis (MSc (Microbiology))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The link between infection with Human Papillomavirus (HPV) and the development of cervical
cancer has been established by several epidemiology studies. Cervical cancer is the second most
common cancer among women and it occurs at a rate of 22.8 cases per 100 000 women in South
Africa. Approximately 86% of newly reported cases of cervical cancer occur in developing
countries where limited access to medical facilities hampers efforts to prevent and screen for
HPV infection. Two commercial virus-like particle (VLP) vaccines consisting of HPV major
structural protein L1, which protect against the most common high-risk HPV-types, are currently
available. The high cost and type specificity of these commercially available vaccines have
necessitated the development of a low cost, broad-spectrum HPV vaccine. Inclusion of the minor
structural protein L2 has been shown to induce broadly cross-neutralizing antibodies and
therefore a chimera was constructed that contains an epitope of L2 inserted within the L1
sequence. This construct, renamed SAF, was shown to be highly immunogenic and thus has the
potential to be used as a prophylactic cervical cancer vaccine. Methylotrophic yeasts are known
to be excellent producers of recombinant proteins due to their strongly inducible promoters that
allow culturing of these yeasts to very high cell densities. Pichia pastoris and Hansenula
polymorpha have been employed in several studies for heterologous protein production and
levels of protein higher than 1 g/L have been reported. These yeasts also have GRAS status and
can therefore be used to manufacture products for use in humans.
In this study, the potential of H. polymorpha and P. pastoris to produce SAF intracellularly was
evaluated. The effect of increased gene dosage and peroxisomal targeting on SAF production
was examined as possible strategies to increase the yield of SAF. Peroxisomal targeting was
achieved by fusing the SAF gene at the C-terminal end with the Peroxisomal Targeting
Sequence 1 (PTS1) which consists of a short tri-peptide: –SKL. The functionality of PTS1 was
confirmed using green fluorescent protein (GFP), fluorescence microscopy and peroxisome
isolation. Peroxisomal targeting was shown to have a negative effect on SAF production levels
in both H. polymorpha and P. pastoris. An increase in gene dosage had no discernable effect on
SAF yield in H. polymorpha which is in contrast to previous research. The highest production
levels were achieved by P. pastoris KM71 (24.86 mg/L) which compares well to levels of L1 achieved by other research groups. The most significant insight emerging from this work was
that all the strains that produced SAF at detectable levels were equally efficient at the production
of SAF. Increased biomass was therefore the biggest contributor to high SAF levels (mg/L) in
the P. pastoris strains as significantly higher cell densities were achieved during culturing of
these strains. With the necessary optimisation, the methylotrophic yeasts have the potential to be
used as hosts for the production of a broad-spectrum HPV vaccine. / AFRIKAANSE OPSOMMING: Die skakel tussen infeksie met Mens Papilloomvirus (HPV) en die ontwikkeling van servikale
kanker is deur verskeie epidemiologiese studies bevestig. Servikale kanker is die tweede mees
algemene kanker onder vroue en dit kom voor teen ‘n tempo van 22.8 gevalle per 100 000 vroue
in Suid Afrika. Ongeveer 86% van alle nuwe gevalle kom voor in ontwikkelende lande waar
beperkte toegang tot mediese fasiliteite pogings om HPV infeksie te voorkom en te behandel,
belemmer. Twee pseudovirale-partikel (VLP) entstowwe teen HPV is tans op die mark
beskikbaar en hierdie entstowwe verleen immuniteit teen die mees algemene hoë-risiko HPV
tipes. Die hoë koste en nou spektrum van hierdie entstowwe het dit nodig gemaak om ‘n
goedkoop, wye-spektrum HPV entstof te ontwikkel. Navorsing het bewys dat die insluiting van
die strukturele L2 proteïen in die VLP entstof, lei tot die indusering van neutraliserende
teenliggame, wat wye spektrum antigenisiteit tot gevolg het. ‘n Chimeriese proteïen wat ‘n
epitoop van L2 binne die L1 volgorde bevat is gekonstrueer, en hierdie proteïen is benoem SAF.
SAF het hoë immunogenisiteit en kan dus potensieel as ‘n voorkomende servikale kanker entstof
gebruik word. Metielotrofiese giste is bekend vir hulle vermoë om hoë vlakke rekombinante
proteïene te produseer as gevolg van hulle induseerbare promotors wat groei tot baie hoë sel
digthede toelaat. Pichia pastoris en Hansenula polymorpha is in menigte studies gebruik om
heteroloë proteïene te produseer tot vlakke bo 1 g/L. Hierdie giste en die proteïen produkte wat
hulle vorm word algemeen aanvaar as veilig vir menslike gebruik.
In hierdie studie het ons die potensiaal van H. polymorpha en P. pastoris om SAF intrasellulêr te
produseer, geevalueer. Die effek op SAF produksie van verhoogde geen dosering asook die
teiken van SAF na die peroksisoom was ondersoek as moontlike strategieë om die opbrengs van
SAF te verhoog. Die teiken van SAF na die peroksisoom is behaal deur die Peroksisomale
Teiken Volgorde 1 (PTS1) aan die C-terminaal van SAF te heg. Die funksionaliteit van PTS1
was bevestig deur gebruik te maak van groen fluoroserende proteïen (GFP), fluoressensie
mikroskopie en isolering van peroksisome. Teiken van SAF na die peroksisoom het ‘n negatiewe
uitwerking gehad op SAF uitdrukking in beide H. polymorpha en P. pastoris. ‘n Verhoging in
geen dosering het geen onderskeibare effek gehad op SAF opbrengs in H. polymorpha nie wat in
teenstelling is met vorige navorsing. Die hoogste produksie vlakke is opgelewer deur P. pastoris KM71 (24.86 mg/L) wat goed vergelyk met vlakke van L1 wat deur ander navorsings groepe
behaal is. Die belangrikste gevolgtrekking wat gemaak kan word uit hierdie studie is dat al die
rasse wat SAF geproduseer het in meetbare hoeveelhede ewe effektief was. Verhoogde biomassa
was dus die grootste bydraende faktor tot hoë SAF vlakke (mg/L) in die P. pastoris rasse as
gevolg van die hoë sel digthede wat hierdie rasse kan bereik. Dit is duidelik dat metielotrofiese
giste, met die nodige optimisering, oor die potensiaal beskik om as gasheer sisteme te dien vir die
produksie van ‘n wye spektrum HPV entstof. / The NRF and the Department of Microbiology for financial support
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BI-VARIATE GROWTH MODEL OF PICHIA PASTORIS INCLUDING OXYGEN CONSIDERATIONS AND ITS IMPORTANCE IN RECOMBINANT PROTEIN PRODUCTIONRobert Michael Binkley (6867047) 13 August 2019 (has links)
<p>The methylotrophic yeast, <i>Pichia pastoris </i>(recently reclassified as <i>spp. Komagatella</i>) has long been regarded as a useful host organism
for the production of recombinant proteins, particularly when using the AOX
system which utilizes methanol as both the inducing agent as well as the
primary carbon source for growth and energy. Significant historical work has
shown that growth rate and protein productivity can be correlated to methanol
concentration. However, the relationship between oxygen and protein
productivity are less consistent. While with many variations models having been
developed and used for analyzing culture kinetics, these models have only been
applied to methanol concentration. Furthermore, while results for methanol are
fairly consistent, oxygen considerations have been far less consistent. </p>
<p> </p>
<p>This work presents various bi-variate models which
includes considerations for growth and inhibition for both methanol and oxygen
with this expanded model showing strong alignment to previous works to both
oxygen and methanol data. While more work is necessary to fully confirm and
validate which form of the bivariate model is most appropriate, this work
provides a framework necessary to expand analysis to include oxygen
considerations. This framework has the potential to be used to further inform
selection of feeding methodology as well as direct investigations into
metabolic studies. </p>
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Functional expression of influenza neuraminidase in Pichia pastoris. / 流行性感冒病毒神經氨酸酶於巴斯德畢赤酵母中的功能性表達 / Liu xing xing gan mao bing du shen jing an suan mei yu Baside bi chi xiao mu zhong de gong neng xing biao daJanuary 2009 (has links)
Tse, Yuk Tin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 141-149). / Abstracts in English and Chinese. / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- The influenza virus --- p.1 / Chapter 1.1.1 --- Influenza NA and its inhibitors --- p.3 / Chapter 1.1.2 --- Follow-up on the use of NAIs --- p.9 / Chapter 1.2 --- Sources of NA for experimental studies --- p.11 / Chapter 1.2.1 --- Viral sources --- p.11 / Chapter 1.2.2 --- NA isolation --- p.12 / Chapter 1.2.3 --- Recombinant NA expressed in cell lines --- p.12 / Chapter 1.2.4 --- Glycosylation on NA functionality --- p.13 / Chapter 1.2.5 --- Recombinant NA expressed in yeast --- p.15 / Chapter 1.3 --- Research objectives --- p.16 / Chapter 2 --- Cloning of Influenza Neuraminidase and Expression in P. pastoris --- p.17 / Chapter 2.1 --- Background --- p.17 / Chapter 2.1.1 --- Full-length cloning of the A/HongKong/483/97(H5N 1) NA --- p.17 / Chapter 2.1.2 --- Identification of the H274 equivalent --- p.19 / Chapter 2.1.3 --- The experiment --- p.22 / Chapter 2.2 --- Materials and methods --- p.23 / Chapter 2.2.1 --- Preparation of chemically competent Escherichia coli --- p.23 / Chapter 2.2.1.1 --- Reagents --- p.23 / Chapter 2.2.1.2 --- Reagent setup --- p.23 / Chapter 2.2.1.3 --- Equipment --- p.23 / Chapter 2.2.1.4 --- Procedure --- p.23 / Chapter 2.2.2 --- Amplification of N1 NA and EGFP genes --- p.24 / Chapter 2.2.2.1 --- Reagents --- p.24 / Chapter 2.2.2.2 --- Reagent setup --- p.25 / Chapter 2.2.2.3 --- Equipment --- p.25 / Chapter 2.2.2.4 --- Procedure --- p.26 / Chapter 2.2.2.4.1 --- Amplification of the full-length N1 NA gene from cDNA --- p.26 / Chapter 2.2.2.4.2 --- Amplification of the EGFP gene --- p.26 / Chapter 2.2.3 --- TA cloning of PCR products --- p.27 / Chapter 2.2.3.1 --- Reagents --- p.27 / Chapter 2.2.3.2 --- Reagent setup --- p.27 / Chapter 2.2.3.3 --- Equipment --- p.28 / Chapter 2.2.3.4 --- Procedure --- p.28 / Chapter 2.2.3.4.1 --- TA cloning of PCR products --- p.28 / Chapter 2.2.3.4.2 --- Site-directed mutagenesis by overlapping PCR --- p.30 / Chapter 2.2.4 --- Construction of P. pastoris expression vectors --- p.31 / Chapter 2.2.4.1 --- Reagents --- p.31 / Chapter 2.2.4.2 --- Reagent setup --- p.31 / Chapter 2.2.4.3 --- Procedure --- p.32 / Chapter 2.2.4.3.1 --- Generation of N1 NA expression vectors --- p.32 / Chapter 2.2.4.3.2 --- Generation of EGFP expression vectors --- p.34 / Chapter 2.2.5 --- Transformation of P. pastoris --- p.37 / Chapter 2.2.5.1 --- Reagents --- p.37 / Chapter 2.2.5.2 --- Reagent setup --- p.37 / Chapter 2.2.5.3 --- Equipment --- p.38 / Chapter 2.2.5.4 --- Procedure --- p.38 / Chapter 2.2.5.4.1 --- Preparation and transformation of electrocompetent P. pastoris --- p.38 / Chapter 2.2.5.4.2 --- PCR analysis of P. pastoris transformants (colony PCR) --- p.39 / Chapter 2.2.6 --- Expression of N1 NA and EGFP in P. pastoris --- p.40 / Chapter 2.2.6.1 --- Reagents --- p.40 / Chapter 2.2.6.2 --- Reagent setup --- p.40 / Chapter 2.2.6.3 --- Procedure --- p.41 / Chapter 2.2.6.3.1 --- Small-scale protein expression in P. pastoris --- p.41 / Chapter 2.2.6.3.2 --- Sequence alignment --- p.42 / Chapter 2.2.6.3.3 --- Data processing --- p.42 / Chapter 2.3 --- Results and Discussion --- p.43 / Chapter 2.3.1 --- Cloning of NA and EGFP into the pPICZB expression vector --- p.43 / Chapter 2.3.2 --- Growth of P. pastoris transformants --- p.51 / Chapter 3 --- Physical Characterization of Influenza Neuraminidase Expressed in P. pastoris --- p.53 / Chapter 3.1 --- Background --- p.53 / Chapter 3.1.1 --- Structural significance of disulphide bonds in NA --- p.53 / Chapter 3.1.2 --- Localization of recombinant N1 NA in P. pastoris --- p.55 / Chapter 3.1.3 --- The experiment --- p.56 / Chapter 3.2 --- Materials and methods --- p.57 / Chapter 3.2.1 --- Differential centrifugation --- p.57 / Chapter 3.2.1.1 --- Reagents --- p.57 / Chapter 3.2.1.2 --- Reagent setup --- p.57 / Chapter 3.2.1.3 --- Equipment --- p.57 / Chapter 3.2.1.4 --- Procedures --- p.58 / Chapter 3.2.1.4.1 --- Cell harvesting and lysis --- p.58 / Chapter 3.2.1.4.2 --- Preparation of crude membrane --- p.58 / Chapter 3.2.1.4.3 --- Preparation of plasma membrane --- p.58 / Chapter 3.2.2 --- Sodium dodecyl sulphate polyaciylamide gel electrophoresis (SDS-PAGE)… --- p.59 / Chapter 3.2.2.1 --- Reagents --- p.59 / Chapter 3.2.2.2 --- Reagent setup --- p.60 / Chapter 3.2.2.3 --- Equipment --- p.61 / Chapter 3.2.2.4 --- Procedure --- p.61 / Chapter 3.2.3 --- Immunoblotting --- p.61 / Chapter 3.2.3.1 --- Reagents --- p.61 / Chapter 3.2.3.2 --- Reagent setup --- p.62 / Chapter 3.2.3.3 --- Equipment --- p.62 / Chapter 3.2.3.4 --- Procedure --- p.62 / Chapter 3.2.3.4.1 --- Electroblotting --- p.62 / Chapter 3.2.3.4.2 --- Blocking and probing --- p.63 / Chapter 3.2.3.4.3 --- Immunodetection --- p.63 / Chapter 3.2.3.4.4 --- Molecular weight determination --- p.63 / Chapter 3.2.4 --- Confocal microscopy --- p.64 / Chapter 3.2.4.1 --- Equipment --- p.64 / Chapter 3.2.4.2 --- Procedure --- p.64 / Chapter 3.2.4.2.1 --- Image acquisition --- p.64 / Chapter 3.2.4.2.2 --- Image processing --- p.65 / Chapter 3.3 --- Results --- p.66 / Chapter 3.3.1 --- Localization of recombinant N1 NA in P. pastoris sub-cellular fractions --- p.66 / Chapter 3.3.2 --- Molecular weight determination for the N1 NA expressed in P. pastoris --- p.69 / Chapter 3.3.3 --- Cellular localization of recombinant N1 NA in P. pastoris --- p.71 / Chapter 3.4 --- Discussion --- p.77 / Chapter 3.4.1 --- Molecular weight determination for N1 NA expressed in P. pastoris --- p.77 / Chapter 3.4.2 --- Disulphide bond formation in N1 NA expressed in P. pastoris --- p.78 / Chapter 3.4.3 --- Cell-surface association of recombinant N1 NA in P. pastoris --- p.79 / Chapter 3.5 --- Conclusion --- p.81 / Chapter 4 --- Functional Characterization of Influenza Neuraminidase Expressed in P. pastor --- p.is / Chapter 4.1 --- Background --- p.82 / Chapter 4.1.1 --- Fluorometric NA activity assay --- p.82 / Chapter 4.1.2 --- Colorimetric assay of NA activity --- p.84 / Chapter 4.1.3 --- The experiment --- p.85 / Chapter 4.2 --- Materials and methods --- p.86 / Chapter 4.2.1 --- Fluorometric assay of N1 NA expressed in P. pastoris --- p.86 / Chapter 4.2.1.1 --- Reagents --- p.86 / Chapter 4.2.1.2 --- Reagent setup --- p.86 / Chapter 4.2.1.3 --- Equipment --- p.86 / Chapter 4.2.1.4 --- Procedure --- p.87 / Chapter 4.2.1.4.1 --- Calibrating cell density with viable cell counts --- p.87 / Chapter 4.2.1.4.2 --- End-point measurement of NA activity --- p.87 / Chapter 4.2.1.4.2.1 --- Determination of expression yield --- p.89 / Chapter 4.2.1.4.2.2 --- End-point assay of NAI sensitivity --- p.89 / Chapter 4.2.1.4.3 --- Kinetic measurement of NA activity --- p.90 / Chapter 4.2.1.4.3.1 --- Derivation ofV0 --- p.92 / Chapter 4.2.1.4.3.2 --- Graphical determination of KM --- p.93 / Chapter 4.2.1.4.3.3 --- Graphical determination of KI --- p.94 / Chapter 4.2.2 --- Colorimetric assay of N1 NA expressed in P. pastoris --- p.96 / Chapter 4.2.2.1 --- Reagents --- p.96 / Chapter 4.2.2.2 --- Reagent setup --- p.96 / Chapter 4.2.2.3 --- Equipment --- p.96 / Chapter 4.2.2.4 --- Procedure --- p.96 / Chapter 4.3 --- Results --- p.98 / Chapter 4.3.1 --- CFU determination --- p.98 / Chapter 4.3.2 --- Fluorescent NA activity assay for N1 NA expressed in P. pastoris --- p.98 / Chapter 4.3.2.1 --- End-point measurement of NA activity --- p.98 / Chapter 4.3.2.1.1 --- Course of N1 NA expression in P. pastoris --- p.102 / Chapter 4.3.2.1.1.1 --- NA activity per unit cell mass --- p.102 / Chapter 4.3.2.1.1.2 --- Yield of NA --- p.102 / Chapter 4.3.2.1.2 --- End-point assay for NAI sensitivity --- p.105 / Chapter 4.3.2.2 --- Kinetic measurement of NA activity and NAI sensitivity --- p.107 / Chapter 4.3.2.2.1 --- Graphical determination of KM --- p.107 / Chapter 4.3.2.2.2 --- Graphical determination of KI --- p.107 / Chapter 4.3.2.3 --- Colorimetric NA activity assay --- p.111 / Chapter 4.4 --- Discussion --- p.114 / Chapter 4.4.1 --- Fluorescent NA activity assay of N1 NA expressed in P. pastoris --- p.115 / Chapter 4.4.1.1 --- End-point measurement of NA activity --- p.115 / Chapter 4.4.1.1.1 --- Time course of expression --- p.115 / Chapter 4.4.1.1.2 --- Effect of H275Y mutation on NA activity and NAI sensitivity --- p.117 / Chapter 4.4.1.1.3 --- Effect of C-terminal tags on NA activity and NAI sensitivity --- p.117 / Chapter 4.4.1.2 --- Kinetic measurement of NA activity --- p.118 / Chapter 4.4.1.2.1 --- Graphical determination of KM --- p.119 / Chapter 4.4.1.2.2 --- Graphical determination of KI --- p.120 / Chapter 4.4.1.3 --- Comparison of fluorometric NA activity assays for use with whole P pastoris cells --- p.122 / Chapter 4.4.2 --- Colorimetric NA activity assay --- p.124 / Chapter 4.5 --- Conclusion --- p.126 / Chapter 5 --- Conclusions and Discussions --- p.127 / Chapter 5.1 --- General conclusions --- p.127 / Chapter 5.2 --- Follow-up --- p.127 / Chapter 5.2.1 --- Studies of influenza NA with enhanced activity --- p.128 / Chapter 5.2.2 --- NAI screening using yeast-expressed NA --- p.132 / Appendix --- p.134 / References --- p.141
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Expressão e caracterização das proteínas VP1 e VP2 de parvovírus humano B19 em Pichia pastoris. / Expression and characterization of VP1 and VP2 proteins of the human parvovirus B19 in Pichia pastoris.Silva Filho, Claudionor Gomes da 10 December 2007 (has links)
O parvovírus B19 é o agente causador de eritemas infecciosos em crianças, hidropsia fetal em mulheres gestantes, esse vírus pode causar anemia crônica e crise aplástica transitória respectivamente. A levedura P. pastoris é um sistema de expressão usado na produção de várias proteínas heterólogas. O objetivo deste trabalho foi expressar as proteínas VP1 e VP2 do parvovírus humano B19 em levedura P. pastoris. As seqüências gênicas VP1 e VP2 foram amplificadas por PCR, usando DNA do vírus B19, os produtos obtidos foram inicialmente subclonados no vetor pGEM-TEasy. Os fragmentos de DNA foram digeridos com enzima de restrição EcoRI e NotI , purificados e inseridos no vetor de expressão e excreção pPIC9K de P. pastoris, entre os sítios EcoRI e NotI. Para expressão das proteínas recombinantes VP1 e VP2 de parvovírus humano B19, os transformantes foram crescidos em glicerol e induzidos pela adição de metanol. As expressões dos antígenos recombinantes foram analisadas por SDS-PAGE e atividade biológica foram confirmadas pelos ensaios imunológicos ELISA, Dot-Blot e Western Blot. / Human Parvovirus B19 is the causative agent of erythema infectiosum in children, hydrops fetalis in pregnant women, B19 may cause chronic anemia and aplastic crisis, respectively. The yeast P. pastoris expression system is being used for the production of various recombinant heterologous proteins. The objective of this work was to express the VP1 and VP2 proteins of the human parvovirus B19 in the yeast Pichia pastoris. The coding sequence of VP1 and VP2 were amplified by PCR, using DNA virus of B19. PCR-products were initially subcloned in the vector pGEM-TEasy. The DNA fragment EcoRI and NotI was excised, purified, and inserted between the sites EcoRI and NotI of P. pastoris expression-secretion vector pPIC9K.For heterologous expression of the proteins VP1 and VP2 Human parvovirus B19, the transformants were growth on glycerol and induced by the addition of methanol. The expressed recombinant antigens VP1 and VP2 were analyzed by SDS-PAGE and its biological activity were confirmed through Enzyme immunoassay EIA, Dot-Blot e Western Blot.
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Characterization of SBIP68: A Putative Tobacco Glucosyltransferase Protein and Its Role in Plant Defense MechanismsOdesina, Abdulkareem O 01 December 2015 (has links)
Plant secondary metabolites are essential for normal growth and development in plants ultimately affecting crop yield. They play roles ranging from appearance of the plants to defending against pathogen attack and herbivory. They have been used by humans for medicinal and recreational purposes amongst others. Glycosyltransferases catalyze the transfer of sugars from donor substrates to acceptors. Glucosyltransferases are a specific type of glycosyltransferases known to transfer glucose molecules from a glucose donor to a glucose acceptor (aglycone) producing the corresponding glucose secondary metabolite or glycone, in this case glucosides. It was hypothesized that SBIP68, a tobacco putative glucosyltransferase-like protein glucosylated salicylic acid. Salicylic acid is an essential plant defense secondary metabolite. SBIP68 was cloned and heterologously expressed in both prokaryotic and eukaryotic systems. Results from activity screening suggest that SBIP68 is a UDP-glucose flavonoid glucosyltransferase with broad substrate specificity. Further studies are required to fully characterize SBIP68.
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