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Avalia??o de dois clones de Escherichia coli recombinante quanto ao crescimento e express?o de ant?genos de Leishmania chagasi (kmp11 e P36)Chaves, Roberta Viana Ara?jo 14 December 2009 (has links)
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Previous issue date: 2009-12-14 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Visceral leishmaniosis caused by Leishmania chagasi, also known as calazar, presented, in the period from 1990 to 2005, tax of incidence in Brazil varying between 1 and 3 cases for 100 000 inhabitants. The Northeast region that up to the year of 2000 contributed with almost 90% of the registered cases is reducing his participation in the current decade, reaching 56% in 2005. Conventional leishmaniasis treatment is costly and it shows high
toxicity, demanding more research for alternative treatments, with special interest in development of vaccines and diagnosis kits which include production of recombinant antigens by host cells. Escherichia coli has been the microorganism most studied and used as a host for recombinant protein production. Therefore, the aim of this work was to study the influence of
induction on cellular growth and to verify the type of Leishmania chagasi antigens expression (intra or extracellular) during two recombinant E. coli clones (kmp11 and P36) cultivation in rotary incubator (shaker) using three different media (2xTY, TB, FASS+EL). For that, tests were carried out using conditions established in the literature for E. coli (37?C and 200 rpm) and media supplemented with antibiotics to guarantee that only competent cells grows. First, tests were carried out without induction in order to verify the two microorganisms kinetic behavior (growth and substrate consumption) in different media. Next, the induction was
carried out through the addition of IPTG (1mM as final concentration), at the first hour of cultivation. It was observed that protein expression were intracellular for all clones and media
tested, however the highest level of expression was clearly observed by the electrophoresis band density (intensity) for 2xTY medium and kmp11 protein. Although it contains the lowest
substrate concentration, consequently, a reduced cellular concentration when compared to other media, it appeared that this medium and clone combination is the most indicated for
recombinant protein production. Therefore, the objective of this work was achieved, since the interested proteins were produced. Consequently, this result motivates new studies for
production optimization using different cultivation strategies / A leishmaniose visceral, causada pela esp?cie Leishmania chagasi, tamb?m conhecida como calazar, apresentou, no per?odo de 1990 a 2005, taxa de incid?ncia no Brasil variando entre 1 e 3 casos por 100 mil habitantes. A regi?o Nordeste, que at? o ano de 2000 contribuiu com quase 90% dos casos registrados, est? reduzindo sua participa??o na d?cada atual, chegando a 56% em 2005. O alto custo e toxicidade das drogas usadas no tratamento convencional dessa leishmaniose levaram a busca de tratamentos alternativos, com interesse especial na pesquisa e produ??o de ant?genos por microrganismos recombinantes para desenvolvimento de vacinas e kits de diagn?stico. A bact?ria Escherichia coli tem sido o microrganismo mais estudado e usado como hospedeiro para produ??o de prote?nas recombinantes. Nesse contexto, este trabalho tem como objetivo estudar a influ?ncia da indu??o no crescimento celular e a verifica??o do tipo de express?o (intra ou extracelular) de ant?genos de Leishmania chagasi atrav?s de cultivo de dois clones de E. coli recombinante (kmp11 e P36) em incubador rotativo (shaker) usando tr?s meios diferentes (2xTY, TB, FASS+EL). Para isso, foram realizados ensaios em condi??es estabelecidas na literatura para E. coli (37?C ? 200 rpm) e os meios suplementados com antibi?ticos para garantir somente o
crescimento de c?lulas competentes. Na primeira etapa, foram realizados ensaios sem indu??o a fim de se verificar o comportamento cin?tico dos dois clones (crescimento e consumo de substrato) nos diferentes meios. Na segunda etapa, a indu??o foi realizada atrav?s da adi??o de IPTG (concentra??o final de 1mM), na primeira hora de cultivo. Foi observada que a express?o das prote?nas foi intracelular para todos os clones e meios testados, entretanto o maior n?vel foi verificado nitidamente pela densidade (intensidade) da banda nos g?is de eletroforese para o meio 2xTY e prote?na kmp11. Apesar de o meio 2xTY conter uma menor concentra??o de substratos, conseq?entemente, uma concentra??o celular reduzida quando comparada aos outros meios, pareceu que esta combina??o de meio e clone ? mais indicada para produ??o das prote?nas recombinantes testadas neste trabalho. O objetivo desse trabalho foi alcan?ado j? que a prote?na de interesse foi produzida. Conseq?entemente, este resultado motiva novos estudos para otimiza??o da produ??o usando diferentes estrat?gias de cultivo
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Development of recombinant Saccharomyces cerevisiae for improved D-xylose utilisationDe Villiers, Gillian K. 04 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Plant biomass is potentially an inexhaustible source of bioenergy. To be more useful in an
industrialised context, conversion to liquid biofuel is necessary, which could provide the
motor vehicle market with energy. To enable fermentation of both hexose and pentose
sugars present in plant biomass, many researchers have introduced eukaryotic D-xylose
utilisation metabolic pathways into S. cerevisiae as these yeasts cannot utilise D-xylose.
The aim of this study was to increase D-xylose utilisation and lower the xylitol production
found with the eukaryotic pathway, thus redirecting carbon to the increased production of
ethanol.
In order to reduce xylitol yield a two-fold approach was followed. Firstly S. cerevisiae
transformed with eukaryotic XR and XDH genes were subjected to random mutagenesis
and selection for improved D-xylose utilisation. Unfortunately no mutant superior to the
parental strain with respect to D-xylose utilisation, lowered xylitol production and improved
ethanol production was obtained.
Subsequently a bacterial xylose isomerase (XI) gene was introduced into S. cerevisiae.
Bacterial xylose isomerase converts D-xylose to xylulose in a single step, while eukaryotic
pathways produce the intermediate xylitol. The chosen gene encodes for a putative xylose
isomerase gene (xylA) from the bacterium Bacteroides thetaiotaomicron, which has not
previously been transformed into yeast. When the native xylA was expressed in E. coli
and S. cerevisiae no XI activity was found, nor growth on D-xylose sustained. Lack of
activity was surmised to be due to an amino acid modification, or possibly due to a vastly
different codon bias in yeast compared to the Bacteroides strain. Northern analysis
revealed that no D-xylose transcript was formed. A synthetic D-xylose isomerase gene
(SXI) based on the B. thetaiotaomicron XI amino acid sequence, but optimised for
S. cerevisiae codon bias, was designed and manufactured. S. cerevisiae transformed with
the synthetic gene showed sustained, non-pseudohyphal growth on D-xylose as sole
carbon source, both on solid and liquid medium. This ability to utilise D-xylose represents
a significant step for recombinant S. cerevisiae to potentially ferment D-xylose for
bioethanol. / AFRIKAANSE OPSOMMING: Plant biomassa is potensieel ‘n onuitputlike bron van bio-energie. Om in die huidige
industriële konteks van groter nut te wees, en die motor-industrie met energie te voorsien,
is omskakeling na ‘n vloeistof-energievorm nodig. Om die fermentasie van beide
heksoses en pentoses teenwoordig in plantbiomassa te bewerkstellig, het verskillende
navorsingspanne eukariotiese D-xilose-afbraak metabolise weë na S. cerevisiae oorgedra
om dié gis die vermoë te gee om D-xilose af te breek. Die doel van hierdie studie was om
D-xilose-verbruik in geneties gemodifiseerde S. cerevisiae te verhoog en die hoeveelheid
xilitol wat met die eukariotiese sisteem verkry word, te verminder om ‘n hoë etanol
opbrengs te handhaaf.
Twee moontlikhede is ondersoek om die xilitol opbrengs te verminder. Eerstens is ‘n
rekombinante S. cerevisiae met die xilose reduktase (XR) en xilitol dehidrogenase (XDH)
gene aan nie-spesifieke mutagenese onderwerp en vir verbeterde D-xilose verbruik
geselekteer. Ongelukkig kon geen mutante wat beter as die oorspronklike ras D-xilose
kon gebruik, en etanol produseer met relatief min xilitol opbrengs, gevind word nie.
Daarna is ‘n bakteriese D-xilose-afbraak geen na S. cerevisiae oorgedra. Bakteriese
xilose isomerases skakel D-xilose om na xilulose in ‘n enkele stap, terwyl die eukariotiese
paaie die tussenganger xilitol produseer. Die gekose xylA geen wat vir xilose isomerase
(XI) van die bakterium Bacteriodes thetaotaomicron kodeer, is vir die eerste keer in gis
getransformeer. Toe die natuurlike xylA geen In E. coli en S. cerevisiae uitgedruk is, is
geen XI-aktiwiteit of volhoubare groei op D-xilose waargeneem nie. Die tekort aan
aktiwiteit is aan 'n aminosuurverandering, of aan die groot verskil tussen kodonkeuse
(“codon bias”) in gis teenoor die Bacteroides ras toegeskryf. Noordkladanaliese het
bepaal dat geen mRNA spesifiek tot die XI-geen geproduseer is nie. Die xilose isomerase
geen van B. thetaiomicron is toe sinteties ontwerp, met die DNA-volgorde vir die
S. cerevisiae kodonkeuse geoptimiseer. S. cerevisiae wat met die sintetiese geen (SXI)
getransformeer is, het aanhoudende, nie-pseudohife groei op D-xilose as enigste
koolstofbron op beide soliede en in vloeibare medium getoon. Die vermoë om D-xilose te
verbruik verteenwoordig ‘n betekenisvolle stap tot die fermentasie van D-xilose na etanol
met geneties gemodifiseerde S. cerevisiae.
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Molecular characterization of non-subtype C and recombinant HIV-1 viruses from Cape Town, South AfricaWilkinson, Eduan 03 1900 (has links)
Thesis (MScMedSc (Pathology. Medical Virology))--University of Stellenbosch, 2009. / Submitted in fulfilment for the degree
MSc in BioMedical Science
at
Stellenbosch University. / ENGLISH ABSTRACT: HIV-1 was first diagnosed within South Africa in 1982. In the 1980’s homosexual transmission dominated the HIV-1 epidemic within the country. In the late 1980’s the second HIV-1 epidemic was recognized amongst heterosexual individuals. Today heterosexual transmission of HIV-1 dominates the epidemic in South Africa. Subtype C HIV-1 is responsible for the overwhelming majority of heterosexual infections. An estimated 95% of all infections in the country are thought to be subtype C related. To date only a few papers have been published on non-subtype C HIV within the country. This study characterized subgenomic and near full-length sequences of non-subtype C HIV-1 viruses from the Cape Town area.
The gag p24, pol-integrase, and env gp41 regions of 11 of the 12 samples were characterized by amplification and direct sequencing. Phylogenetic analysis of the sequenced data, with online subtyping tools (REGA and jpHMM) and the drawing of NJ-trees revealed the presence of subtype A1, B, F1 and recombinant viral forms such as AD, AG and AC. One of the isolates was classified as a subtype C and was included for control purposes.
Near full-length characterization of four of the samples were attempted, through full genome PCR amplification and sequencing. Analysis of sequenced data with the use of subtyping-, recombination identification, and tree drawing tools revealed a subtype B, and A1 isolate. The other two isolates were identified as possible AC and AD recombinants.
The data that was generated will greatly improve our knowledge of non-subtype C isolates circulating within South Africa. Due to the possible impact that the high degree of genetic variation that HIV may have on vaccine design and development and ARV treatment and HIV diagnosis, ongoing research of the epidemiology and spread of HIV within South Africa are needed. / AFRIKAANSE OPSOMMING: MIV was in 1982 vir die eerste keer in Suid Afrika gediagnoseer en was hoofsaaklik deur homoseksuele kontak oorgedra. Aan die begin van die 1990’s is `n tweede MIV epidemie gewaar onder heteroseksuele individue. Heteroseksuele oordrag van die virus domineer tans die MIV epidemie in Suid Afrika en is meestal subtipe C verwant. Subtipe C, MIV-1 is verantwoordelik vir 95 persent van alle infeksies in die land. Tot hede is slegs `n paar publikasies oor die nie-subtipe C epidemie in die land gepubliseer. Die huidige studie was gemik op die karakterisering van subgenomiese en vollengte genome van nie-subtipe C MIV isolate van die Kaapstad omgewing.
Die gag p24, pol-integrase en env gp41 subgenomiese fragmente van 12 monsters was gekarakteriseer deur amplifikasie en DNS nukleotied volgorde bepaling. Filogenetiese analise deur middel van subtipering (REGA en jpHMM aanlyn subtiperings programme) asook NJ-filogenetiese bome van die data het die teenwoordigheid van subtipe A1, B, en F1, asook verskeie rekombinante viruse insluitende AG, AD en AC vorme aangedui. Een van die isolate was geklassifiseer as `n subtipe C maar is in die studie ingevoeg vir kontrole doeleindes.
Vollengte karakterisering van 4 uit die 12 isolate was ook gedoen deur vollengte genoom amplifikasie en DNS nukleotied volgorde bepaling. Tydens die analisering van die DNS volgorde data, deur middel van aanlyn subtipering, rekombinasie identifikasie (Simplot en RIP), en filogenetiese boom konstruksie programme is twee isolate geidentifiseer as subtipe B en A1 MIV-1 viruse. Die ander twee isolate was as moontlike AC en AD rekombinante geklassifiseer.
Die data van nie-subtipe C MIV isolate sal ons kennis van die nie-subtipe C epidemie in Suid Afrika versterk. As gevolg van die impak wat die hoë graad van genetisie variasie van MIV op die ontwikkeling van entstowwe, sowel as die diagnose en behandeling van pasiente kan hê, is verdere navorsing in die epidemiologie van die MI-virus in Suid Afrika nodig.
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The evaluation of heterologous eukaryotic expression systems for the production of biocatalytic enzymesRoth, Robyn Lindsay 03 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Heterologous gene expression is of considerable interest for the production of proteins
of therapeutic and industrial importance. As the nature of recombinant proteins has
become more complex and as transformation systems have been established in more
species, so the variety of hosts available for expression has increased. Every system
available has both advantages and disadvantages. The research presented here
highlights the advantages of selecting the most appropriate expression system for
different recombinant proteins. Expression of different biocatalytically-relevant
enzymes, epoxide hydrolases, halohydrin dehalogenase, laccase and mannanase, in
different host systems is undertaken, and expression levels and activity are compared.
The development of Yarrowia lipolytica as a whole-cell biocatalyst is described.
Y. lipolytica is used for the functional expression of epoxide hydrolases (EHs) and
halohydrin dehalogenases. EHs are hydrolytic enzymes that convert epoxides to
vicinal diols by ring-opening. Two new fungal EHs from Rhodosporidium toruloides
NCYC 3181 and NCYC 3158 (a putative Cryptococcus curvatus strain) were
identified and cloned. Additional EHs from different sources, including bacteria,
yeasts, fungi and plants, were chosen for expression in Y. lipolytica, in order to
determine its suitability as the expression system of choice for the production of EHs.
Multi-copy integrants were developed, with the genes under control of the growthphase
dependent hp4d promoter. A Saccharomyces cerevisiae strain was developed,
expressing the EH from Rhodotorula araucariae1, to compare as a whole-cell
biocatalyst with Y. lipolytica. This strain proved to be an exceptionally poor wholecell
biocatalyst. All the Y. lipolytica strains developed showed varying levels of
activity towards different classes of epoxides. Some strains displayed opposite
enantioselectivities, allowing for potential complete conversions of racemic epoxides
to the desired enantiomeric product.
Halohydrins can be considered direct precursors of epoxides. Halohydrin
dehalogenases catalyse the nucleophilic displacement of a halogen ion in halohydrins by a vicinal hydroxyl group, yielding an epoxide, a proton and a halide ion. The
HheC gene from Agrobacterium radiobacter AD1, codon-optimised to match the
codon usage of Y. lipolytica, was over-expressed in Y. lipolytica by generation of
multi-copy integrants, further expanding the use of this organism as a host strain for
heterologous production of enzymes. Expression levels were maximised by creating
tandem repeats of the introduced HheC gene. The ring-closure activity with 2-chloro-
1-phenylethanol as substrate was demonstrated to be broadly dose-dependent.
The b-mannanase gene (man1) from Aspergillus aculeatus MRC11624 was expressed
in Y. lipolytica with effective secretion in the presence of its native secretion signal,
using the hp4d promoter. The same gene was expressed in Aspergillus niger2 under
control of the A. niger glyceraldehyde-3-phosphate dehydrogenase promoter (gpdP)
and the Aspergillus awamori glucoamylase terminator (glaT). Following optimisation
with copy numbers and culture conditions, maximal activity levels of 26,140 nkat.ml-1
for Y. lipolytica, and 16,596 nkat.ml-1 for A. niger were obtained.
Laccases are important enzymes for bioremediation, and the best characterised
enzymes are from the fungus Trametes versicolor. The objective of this research was
to optimise expression of T. versicolor laccases (lcc1 and lcc2) in A. niger D15 and
Pichia pastoris3. The Lcc1 enzyme was less active than Lcc2 in both hosts. P.
pastoris secreted 0.4 U.L-1 Lcc1 and 2.8 U.L-1 Lcc2, compared to 2,700 U.L-1
produced by A. niger. The Lcc2 enzyme from recombinant A. niger was subsequently
purified and characterised in terms of molecular weight and glycosylation, and
compared to the wild-type enzyme purified from T. versicolor.
The work presented underscores the requirement for experimentation before finalising
the choice of an expression system for the optimal production of the desired protein.
Every system available has both advantages and disadvantages, and when considering
which system to use for producing a recombinant protein, various factors must be
taken into consideration. However, the choice is broad and each decision needs to be
made empirically. 1 The construction of the S. cerevisiae epoxide hydrolase production strain was carried out by Dr
Neeresh Rohitlall of CSIR Biosciences. The Y. lipolytica epoxide hydrolase strains were constructed
by the author. 2 The construction of Man1-producing A. niger strain was done by Dr Shaunita Rose of the University
of Stellenbosch. The construction of Y. lipolytica Man1 production strains was done by the author.
3 The expression of T. versicolor laccases in P. pastoris was done by Christina Bohlin of Karlstad
University. A niger laccase production strains were created by the author. / AFRIKAANSE OPSOMMING: Heteroloë geen uitdrukking is van groot belang vir die produksie van proteïene wat
van terapeutiese en industriele belang is. Soos die aard van rekombinante proteïene
meer ingewikkeld raak en getransformasie-sisteme vir verskeie spesies gevestig raak,
is daar ’n groter verskeidenheid van gashere beskikbaar vir geenuitdrukking. Elke
sisteem het beide sy voor- en nadele. Hierdie navorsing beklemtoon die voordele
wanneer die mees gepaste uitdrukkingssiteem gekies word. Die uitdrukking van
verskeie ensieme van biokatalities belang, epoksiedhidrolases, halohidrien
dehalogenase, lakkase en mannanase in verskillende gasheersisteme is onderneem en
die uitdrukkingsvlakke en aktiwiteite vergelyk.
Die ontwikkeling van Yarrowia lipolytica as ’n heelsel biokatalis word beskryf.
Y. lipolytica word gebruik vir die funksionele uitdrukking van epoksiedhidrolases
(EHs) en halohidrien dehalogenases. EHs is hidroliseringsensieme wat die epoksiede
omskakel na aangrensende diole deur middel van ring-opening. Twee nuwe fungi
EHs vanaf Rhodosporidiom toruloides NCYC 3181 en NCYC 3158 (’n moontlike
Cryptococcus curvatus) is geïdentifiseer en gekloneer. Verdere EHs van verskillende
bronne, insluitend bakterieë, giste, fungi en plante, is gekies vir uitdrukking in
Y. lipolytica ten einde sy geskiktheid vir die produksie van EHs te bepaal. Multikopie
integrante is ook ontwikkel met gene onder beheer van die groei-fase afhanklike hp4d
promotor. ’n Saccharomyces cerevisiae ras is ook ontwikkel vir die uitdrukking van
die EH van Rhodotorula araucariae4 sodat dit met Y. lipolytica as ’n heelsel
biokatalis vergelyk kan word. Hierdie ras was ‘n buitengewone swak heelsel biokatalis. Al die Y. lipolytica rasse wat ontwikkel is het wisselende aktiwiteitsvlakke
teenoor verskillende klasse van epoksiede getoon. Sommige rasse het teenoorgestelde
enantio-selektiwiteit getoon en het die potensiaal om rasemiese epoksiede volledig na
die gewensde enantiomeriese produk om te skakeling.
Halohidriene kan as direkte voorgangers van epoksiede beskou word. Halohidrien
dehalogenases kataliseer die nukleofiliese vervanging van ’n halogeen-ioon in
halohidriene deur ’n aangrensende hidroksiel groep, wat ’n epoksied, ’n proton en ’n
halied-ioon lewer. Die HheC geen van Agrobacterium radiobacter AD1 is kodon–
geöptimiseer om te pas by die kodon gebruik van Y. lipolytica en was uitgedruk in
Y. lipolytica deur die skep van mulitkopie integrante, ’n verdere verbreeding van die
toepaslikheid van die organisme as gasheerras vir die heteroloë produksie van
ensieme. Maksimum uitdrukkingsvlakke is bereik deur die skep van opeenvolgende
herhalings van die ingevoegde HheC-geen. Daar is ook gewys dat die ring-sluitingsaktiwiteit
met 2-chloro-1-pheniel-etanol as substraat meestal dosis-afhanklik is.
Die -mannanase geen (man1) van Aspergillus aculeatus MRC11624 is uitgedruk en
effektief in Y. lipolytica mbv sy eie uitskeidings sein uitgeskei, met die gebruik van
die groei-fase afhanklike hp4d promotor. Dieselfde geen is uitgedruk in Aspergillus
niger5 onder beheer van die A. niger gliseraldehied-3-fosfaat dehidrogenase promotor
(gpdp) en die Aspergillus awamori glikoamilase termineerder (glaT). Verdere
optimisering van kopiegetal en voedingskondisies het gelei tot maksimum
aktiwiteitsvlakke van 26,140 nkat.ml-1 vir Y. lipolytica en 16,596 nkat.ml-1 vir
A. niger. Lakkases is belangrike ensieme vir bio-remediëring, en die ensieme van die fungus
Trametes versicolor is die beste gekarateriseer. Die doelwit van hierdie navorsing
was die optimisering van die uitdrukking van T. versicolor lakkases (lcc1 en lcc2) in
A. niger en Pichia pastoris6. Die Lcc1 ensiem was minder aktief as Lcc2 in altwee
die gashere. P. pastoris het 0.4 U.L-1 Lcc1 en 2.8 U.L-1 Lcc2 onderskeidelik
uitgeskei, in vergelyking met 2,700 U.L-1 Lcc2 wat deur A. niger geproduseer is. Die
Lcc2 ensiem afkomstig van die rekombinante A. niger is vervolgens gesuiwer en
gekarakteriseer met betrekking tot molekulêre massa en glikosilering, en daarna
vergelyk met die wilde-tipe ensiem wat deur T. versicolor geproduseer word.
Die werk wat hier aangebied word, beklemtoon die vereistes vir eksperimentering
voor die finale keuse met betrekking tot ’n gepaste uitdrukkingsisteem gemaak kan
word vir die optimale produksie van die gewensde proteïen. Elke sisteem het beide
voordele en nadele, en wanneer ’n sisteem oorweeg word is daar verskeie faktore wat
in ag geneem moet word. ’n wye verskeidenheid van keuses is beskikbaar en elke
besluit moet empiries gemaak word. 4 Die konstruksie van die S. cerevisiae epoksiedhidrolase-produserende ras is deur Dr Neeresh Rohitlall
van CSIR Biosciences gedoen. Die Y. lipolytica epoxied hydrolase rasse is deur die outeur gemaak. 5 Die konstruksie van die Man1-produserende A. niger ras is deur Dr Shaunita Rose van die
Universiteit van Stellenbosch gemaak. Die Y. lipolytica Man1 ras is gemaak deur die outeur. 6 Die uitdrukking van T. versicolor lakkases in P. pastoris is gedoen deur Christina Bohlin van Karlstad
University. Die A niger lakkase produksie ras is geskep deur die outeur.
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CLONING OF BACILLUS SUBTILIS DNA: EXPRESSION IN B. SUBTILIS AND ESCHERICHIA COLI.ZUKOWSKI, MARK MICHAEL. January 1982 (has links)
Bacillus subtilis DNA was cloned by ligating restriction endonuclease-generated fragments to plasmid vectors. The plasmid pUB110 was the vehicle in the construction of eight recombinant plasmids, pNM1 through pNM8. Each bears one or more EcoRI fragment(s) of B. subtilis chromosomal DNA. Recovery of the plasmids from host cells demonstrated that recombinant plasmids that bear some homology to the B, subtilis chromosome may be maintained outside of the chromosome in recombination-proficient hosts. The mean size of cloned fragments was 0.78 megadaltons (Mdal). The recombinant plasmid pNM1 interferes with the mechanism that blocks chromosomal recombination in B. subtilis cells that carry the recE4 mutation. Low-level chromosomal recombination at several loci was demonstrated when chromosomal DNA was accompanied by pNM1 in the transformation of recE4 recipient cells. The recombinant plasmid does not appear to code for recE gene products nor does it produce novel proteins when assayed in minicells of B. subtilis. An alternative approach to cloning B. subtilis DNA was successfully accomplished with the vector plasmid pHV33. The vector functions in both B. subtilis and E. coli hosts. B. subtilis chromosomal DNA was digested with Bg1II, then ligated to the unique BamHI site of pHV33. Ligation products were introduced into E. coli by transformation. Plasmid DNAs were isolated from transformants, pooled into several lots, then used to transform auxotrophic B. subtilis recipient cells. The procedure resulted in the construction of two new recombinant plasmids, pNM1055 and pNM1326. B. subtilis cells with the aroD120 mutation restored their ability to synthesize aromatic amino acids when pNM1055 was introduced. The same effect was observed in E. coli recipient cells that had the equivalent mutation. E. coli cells that carried pNM1326 produced granular colonies characteristic of the extraordinary filamentous growth exhibited by individual cells. The pNM1326 plasmid coded for a 16,000 dalton polypeptide produced in abundant quantities in E. coli hosts. A deletion derivative of pNM1326 did not produce the polypeptide, nor was filamentous growth of host cells exhibited. A plasmid-borne fragment of B. subtilis DNA affects cells growth and division of E. coli hosts.
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Expression & Purification of Recombinant Plasmodium falciparum Erythrocyte-binding LigandsCofie, Seward Joann 29 April 2010 (has links)
Plasmodium falciparum, the most virulent malarial parasite, is capable of invading all known human blood types. Erythrocyte invasion depends on specific parasite ligand and erythrocyte receptor interactions. These interactions are mediated by Region II of the P. falciparum erythrocyte binding ligands. Although invasion does not seem dependent upon a singular ligand, their individual contributions to the invasion process are yet to be explained. In this study, Region II of P. falciparum binding ligands BAEBL and JESEBL were transiently expressed as hexahistidyl recombinant proteins in COS-1 cells. Purification by column chromatography yielded 0.52 mg of BAEBL and 0.433 mg of JESEBL. The production and purification of these recombinant hexahistidyl proteins can allow for future binding affinity and kinetic analysis that may eventually define the contributive roles of each ligand during erythrocyte invasion.
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Structural Analysis of the TOL pDK1 xylGFJQK Region and Partial Characterization of the xylF and xylG Gene ProductsPoulter, Melinda D. 12 1900 (has links)
TOL plasmids encode enzymes responsible for utilization of toluene and related aromatic compounds by Pseudomonas putida, ultimately converting them to central metabolic intermediates. The nucleotide sequence for the 5.6 kb xylGFJQK region of the pDK1 TOL meta operon was determined. DNA sequence analysis revealed the presence of five open reading frames corresponding to xylG (1458 bp), xylF (846 bp), xylJ (783 bp), xylQ (936 bp) and xylK (1047 bp), encoding predicted protein products of 51.6, 31.3, 27.8, 32.8, and 36.6 kDa in size, respectively. The average G+C content of the xylLTEGFJQK region was 65.7%, somewhat higher than the 58.9% seen in the immediately upstream xylXYZ region and substantially more than the 50% G+C content reported for the upper TOL operon of this plasmid. Homology comparisons were made with genes and proteins of related catabolic plasmids. The dmpCDEFG and pWWO xylGFJQK regions exhibit consistently high levels of nucleotide and amino acid homology to pDK1 xylGFJQK throughout the entire region. In contrast, although the nucleotide sequence homology of the Acinetobacter atdCDE region to xylGFJ is high, the homology of atdFG to xylQK is markedly less. Such radical changes in homology between corresponding regions of different operons, combined with variable base and codon usage patterns within and between operons, provides additional support for the idea that the upper and lower operons encoding enzymes of aromatic pathways have evolved independently of one another and that these operons have continued to exchange genetic material with homologous expression units through a series of recombination events. Recombinant plasmids were constructed for individual expression of each of the xylGFJQK genes. HMSD (XylG) and HMSH (XylF) were partially purified and characterized with respect to substrate specificity and kinetic mechanism. Evidence was obtained suggesting that the HMSD reaction occurs via a steady state ordered mechanism or a random mechanism where binding of the first substrate effects the enzyme's affinity for the second substrate.
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Élaboration d'un vaccin contre HPV16 (cancer du col de l'utérus)Falconi, Sarah January 2002 (has links)
Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.
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Rekombinantní exprese a purifikace nitrilasy z Neurospora crassa / Recombinant expresion and purification of nitrilase from Neurospora crassaZawadová, Dorota January 2014 (has links)
Nitrilases are enzymes able to convert toxic nitriles to corresponding carboxylic acids or amides. Thus they might be used in the detoxification of dyes, herbicides and pharmaceutical intermediates and byproducts. They can be used also for enzymatic syntheses of carboxylic acids not available by standard procedures. The aim of this diploma thesis is a recombinant expression of nitrilases from Neurospora crassa and the optimization of their purification. Cells of E. coli (BL 21 Gold) were utilized as an expression system. The purification was performed by ion-exchange chromatography, chelation chromatography and gel filtration - all under reducing conditions. Purified enzymes were studied by sedimentation analysis in an analytical ultracentrifuge. They were also used for searching of optimal conditions for their crystallization. Keywords: nitrilase, Neurospora crassa, recombinant expression
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The production, expression, and characterisation of insulin and GAD65 recombinant FAB for use inPadoa, Carolyn Jane 14 October 2009 (has links)
Ph.D., Faculty of Health Sciences, University of the Witwatersrand, 2006. / Objectives. Autoantibodies to the 65kDa isoform of glutamic acid decarboxylase
(GAD65Abs) are accepted markers for type 1 diabetes and, together with
autoantibodies to insulin (IAA) and a protein tyrosine phosphatase-like islet cell
antigen (IA-2), predict the disease. IAA are often the first autoantibodies detected
in type 1 diabetics and can be present before the onset of clinical diabetes. These
autoantibodies and their epitopes are however not well characterized. We
explored the use of monoclonal antibodies and their recombinant Fab (rFab) as
reagents for epitope analysis.
Methods and Results. Four rFab specific for insulin were cloned from murine
monoclonal antibodies (mAbs) 1E2, HB-126, HB-123, HB-127, and one rFab
specific for GAD65 was cloned from human mAb IgG antibody DP-D (derived from
autoimmune disease patients), to characterise insulin and GAD65 autoantibodies
present in the sera of patients with type 1 diabetes. Only rFab 126 and DP-D
showed insulin and GAD65 specific binding, respectively in radiobinding assays.
In competition experiments with sera positive for autoantibodies to insulin the rFab
126 significantly reduced the binding to 125I-insulin by sera of type 1 (n=35) and
type 1.5 diabetes (or LADA) (n=14) patients (p<0.0001). There was no difference
in the competition pattern in IAA positive type 1 diabetes patients (n=35) and IAA
positive type 1.5 diabetes patients (n=14). The insulin epitope that the rFab binds
to was mapped using competitive radiobinding assays with two monoclonal
antibodies (mAb 1 and mAb 125) whose epitopes are on the B chain and A chain
loop of insulin, respectively. We found the epitope of this recombinant antibody to
be located on the A chain loop of the insulin molecule. The 3-dimensional
structure of rFab 123, 126 and DP-D were determined using an automated
homology modelling programme. Using the computer programme ‘PatchDock’ we
attempted to further map the epitope that rFab 126 binds to on insulin. Of the
three models generated, only one supported our findings that rFab 126 binds to
the A chain loop of insulin.
The binding of GAD65Ab in 61 type 1 diabetes patients to GAD65 was analyzed
by competitive radioimmunoassays with rFab DP-D to ascertain disease-specific
GAD65Ab binding specificities. The median binding was reduced significantly by
rFab DP-D (80%) (p<0.0001). The competition pattern in type 1 diabetes patients
was different from that in GAD65Ab-positive type 1.5 diabetes patients (n=44), first
degree relatives (n=38), and healthy individuals (n=14) (Padoa et al., 2003).
Conclusions. We have shown that rFab specific for insulin and GAD65 can be
generated using PCR technology and that such agents can be used to determine
the insulin/GAD65 epitopes recognized by autoantibodies from type 1 and 1.5
diabetics. These novel findings with GAD65- and insulin-specific rFab support the
view that type 1 diabetes is associated with disease- and epitope-specific GAD65-
and insulin-autoantibodies and supports the notion that the middle epitope of
GAD65 is disease-specific. These GAD65-specific rFab should prove useful in
predicting type 1 diabetes. Furthermore, rFabs may be a novel method for
blocking autoimmune responses against β cell autoantigens in type 1 diabetics.
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