Dissertation (PhD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Since the advent of recombinant DNA technology in the 1970s, various
microbial hosts have been employed for the efficient high-level heterologous
production of a variety of proteins, ranging from enzymes and reagents to
therapeutics and vaccines. More recent microbial hosts to be employed for
these purposes are filamentous fungi, and particularly the genus Aspergillus.
Aspergilli have been associated with industrial processes for many years and
are used in the production of antibiotics, enzymes, citric acid and Oriental foods
and beverages, and thus strains such as Aspergillus niger and Aspergillus
oryzae have been afforded GRAS (Generally Regarded 8s ~afe) status. They
also secrete copious amounts of homologous and heterologous proteins and
are able to perform post-translational modifications effectively. Various proteins
of pharmaceutical interest have been successfully expressed in Aspergillus, but
the potential of these fungi to produce heterologous viral proteins has not been
explored extensively.
In this study, we evaluated the potential of the filamentous fungi A. niger and
Aspergillus awamori as alternative hosts for the heterologous production of
hepatitis B viral proteins. Hepatitis B is a serious, potentially lethal liver disease
that affects 2000 million people world-wide and has a high endemicity in
Southern Africa. Currently there is no effective treatment for viral hepatitis and
thus a mass vaccination strategy is the only solution to curb the spread of the
disease. This kind of vaccination strategy requires a cheap, safe and effective
vaccine and these objectives have been achieved in the development of
recombinant subunit vaccines from yeasts such as Saccharomyces cerevisiae,
Hansenula polymorpha and Pichia pastoris that are commercially available.
The hepatitis B virus envelope consists of a membrane fraction and three
proteins, namely the major (S) protein (encoded by the S gene), the middle (M)
protein (encoded by the preS2S gene) and the large (L) protein (encoded by the
preS1preS2S gene). When produced in the above-mentioned yeasts, the S
protein was shown to spontaneously assemble into pseudoviral particles devoid
of viral DNA, which were then purified and used as vaccine. In the present study the Sand preS1preS2S genes from a local isolate of
hepatitis B subtype adw2 were placed under transcriptional control of the
constitutive Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase
(gpdA) promoter and the inducible A. niger glucoamylase (glaA) promoter. The
respective viral genes were also fused to the region encoding the catalytic
domain of the highly expressed and secreted A. niger glucoamylase, which
served as a carrier moiety to possibly facilitate viral protein secretion. The
various gene constructs were subsequently transformed to laboratory strains of
A. niger and A. awamori and numerous transformants were obtained. One
A. niger transformant carrying the S gene under control of the gpdA promoter
contained approximately 7 integrated copies of the expression cassette and
produced hepatitis B pseudoviral particles intracellularly at levels of 0.4 mg/I
culture. These levels are approximately ten-fold higher than those initially
obtained from the yeast S.cerevisiae, which showed yields of 0.01 to
0.025 mg/I. None of the other transformants could be shown to produce
recombinant S or L protein and no secretion of viral protein could be
demonstrated. This could be attributed to numerous factors, including vector
copy number, site of integration or proteolytic activity. The most important
insight emerging from this work regarding secretion of heterologous viral protein
was that the addition of a carrier protein hampered, rather than enhanced
secretion of the viral envelope protein, due to the inherent properties of viral
protein assembly.
This work also serves as a "proof of principle", showing that Aspergillus is
indeed a viable alternative host for the production of hepatitis B pseudoviral
particles, and could be investigated further for its potential as host for the
heterologous expression of other viral proteins. / AFRIKAANSE OPSOMMING: Sedert die ontwikkeling van rekombinante DNA tegnologie in die sewentigerjare
is verskeie mikroorganismes reeds gebruik vir die doeltreffende produksie van
'n verskeidenheid proteïne teen hoë vlakke; onder andere ensieme, reagense,
terapeutiese middels en vaksiene. Onlangs is filamentagtige swamme, veral
van die genus Aspergillus, ontwikkel vir heteroloë proteïenproduksie. Aspergilli
word al vir baie jare in nywerheidsprosesse gebruik, onder andere in die
vervaardiging van antibiotika, ensieme, sitroensuur en sekere Oosterse
voedsel- en drankprodukte. As gevolg van hierdie jarelange gebruik van rasse
soos Aspergillus niger en Aspergillus oryzae, word hulle algemeen aanvaar as
veilig vir menslike gebruik. Hierdie swamme besit veral die vermoë om hoë
vlakke van homoloë en heteroloë proteïene uit te skei en die na-translasiemodifisering
van proteïene korrek uit te voer. Verskeie proteïene van
farmaseutiese belang is al suksesvol in Aspergillus uitgedruk, maar die
potensiaal van hierdie swamme om virale proteïene te vervaardig is nog nie
deeglik ondersoek nie.
Hierdie studie ondersoek die geskiktheid van die filamentagtige swamme
A. niger en Aspergillus awamori om as alternatiewe gashere vir die heteroloë
produksie van hepatitis B proteïene te dien. Hepatitis B is 'n ernstige en selfs
dodelike lewersiekte. Omtrent 2000 miljoen mense wêreld-wyd is met die virus
geïnfekteer en dit is veral endemies in Suiderlike Afrika. Daar is tans geen
doeltreffende behandeling vir virale hepatitis en dus is wêreld-wye
inentingsprogramme die enigste oplossing om die verspreiding van die siekte te
bekamp. Hierdie inentingsstrategie vereis die beskikbaarheid van 'n
bekostigbare, veilige en doeltreffende vaksien. Die rekombinante subeenheidvaksiene
wat ontwikkel is deur van gashere soos Saccharomyces cerevisiae,
Hansenula polymorpha en Pichia pastoris gebruik te maak, voldoen aan hierdie
vereistes en is kommersieel beskikbaar. Die omhulsel van die hepatitis B virus
bestaan uit 'n membraangedeelte en drie proteïene, naamlik die hoofproteïen
(S) (gekodeer deur die S-geen), die middelproteïen (M) (gekodeer deur die
preS2S-geen) en die grootproteïen (L) (gekodeer deur die preS1preS2S-geen). Wanneer die S-proteïen in bo-genoemde giste uitgedruk word, vorm dit
spontaan pseudovirale partikels wat nie virale DNA bevat nie. Hierdie partikels
word dan gesuiwer en as vaksien gebruik.
In hierdie studie is die S- en preS1preS2S-gene, vanaf 'n plaaslike isolaat van
hepatitis B subtipe adw2, onder transkripsionele beheer van die konstitutiewe
Aspergillus nidulans gliseraldehied-3-fosfaat-dehidrogenasepromoter (gpdA) en
die induseerbare A. niger glukoamilasepromoter (glaA) geplaas. Die
onderskeie virale gene is ook aan die koderende gedeelte vir die katalitiese
domein van A. niger glukoamilase gelas om fusieproteïene te vorm.
Glukoamilase word teen hoë vlakke deur Aspergillus vervaardig en uitgeskei en
kan dus moontlik dien as draerproteïen om sekresie van die proteïne te
bevorder. Transformasie van die geenkonstrukte na laboratoriumrasse van
A. niger en A. awamori het verskeie transformante gelewer. Een A. niger
transformant bevattende die S-geen onder transkripsionele beheer van die
gpdA promoter het minstens sewe kopieë van die uitdrukkingskaset in sy
genoom geïntegreer en het hepatitis B pseudovirale partikels intrasellulêr teen
vlakke van 0.4 mg/I swamkultuur vervaardig. Hierdie vlakke is omtrent tienvoudig
hoër as die vlakke van 0.01 - 0.025 mg/I wat S.cerevisiae oorspronklik
opgelewer het. Nie een van die ander transformante het rekombinante S of L
proteïene vervaardig nie en sekresie van virale proteïen kon nie getoon word
nie. Hierdie verskynsel mag te wyte wees aan verskeie faktore insluitende
vektor-kopiegetal, setel van integrasie en proteolitiese aktiwiteit. Die
belangrikste insig uit hierdie studie aangaande sekresie van heteroloë virale
proteïene is dat die koppeling van die virale omhulsel-proteïen aan 'n
draerproteïen sekresie benadeel het, eerder as om dit te bevorder. Hierdie
verskynsel is te wyte aan die inherente geneigdheid van virale omhulselproteïene
om 'n kompleks te vorm.
Die studie dien ook as "bewys van beginsel" dat Aspergillus wel 'n werkbare
alternatiewe gasheer vir die produksie van hepatitis B pseudovirale partikels is,
en dat dit verder ondersoek sou kon word as potensiële gasheer vir die
heteroloë uitdrukking van ander virale proteïene.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52834 |
| Date | 12 1900 |
| Creators | Pluddemann, Annette, 1972- |
| Contributors | Van Zyl, W. H., Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 227 p. : ill. |
| Rights | Stellenbosch University |
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