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.

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.

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.

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.

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.

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.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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

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.

A leptospirose é uma zoonose de distribuição global causada por bactérias patogênicas do gênero Leptospira. A doença possui um quadro de manifestações clínicas muito variadas em humanos, que pode apresentar febre, dores de cabeça e muscular, até um quadro clínico mais severo, conhecido como síndrome de Weil, caracterizado por hemorragias, icterícia, falência renal e hepática. Até o presente momento, os mecanismos de patogenicidade da leptospira não são totalmente claros e não existe uma vacina eficaz contra a doença. O sequenciamento de genomas de leptospiras patogênicas possibilitou a identificação de proteínas da membrana externa conservadas entre cepas patogênicas, que são os principais alvos de pesquisa para elucidar os mecanismos de patogenicidade e possivelmente o desenvolvimento de uma vacina. Nesse sentido, foram selecionados por bioinformática os genes LIC11711 e LIC12587 a partir do genoma sequenciado de Leptospira interrogans sorovar Copenhageni, os quais codificam para duas preditas lipoproteínas hipotéticas de funções desconhecidas. Os genes foram amplificados por PCR a partir do DNA genômico da bactéria e clonados no vetor de expressão pAE. As proteínas recombinantes foram expressas em E. coli BL21 DE3 Star pLysS e purificadas por cromatografia de afinidade a metal. As proteínas recombinantes foram inoculadas em camundongos para avaliação da sua atividade imunogênica e obtenção de soros imunes. Ambas as proteínas recombinantes se mostraram reativas com anticorpos em soros de pacientes diagnosticados com a doença, apresentando uma sensibilidade maior em relação ao teste de aglutinação microscópica (MAT), e alta especificidade, diferenciando anticorpos presentes em soros de pacientes diagnosticados com outras doenças não relacionadas. Os resultados de ELISA de bactéria intacta, proteólise por proteinase K e imunofluorescência sugerem que ambas as proteínas estão localizadas na membrana externa bactéria. Com base no ensaio de conservação por western blotting, as proteínas nativas estão presentes em diferentes cepas patogênicas de Leptospira, e ausentes na espécie saprófita L. biflexa. Em ensaios de adesão in vitro, as proteínas recombinantes interagiram com os componentes humanos laminina, e-caderina, plasminogênio, fibrinogênio, fibronectina plasmática, vitronectina, C7, C8 e C9 de forma dose-dependentes, porém com valores de afinidade baixos. Estes resultados sugerem que as proteínas codificadas pelos genes LIC11711 e LIC12587 são expressas durante a patogênese e podem desempenhar múltiplas funções a partir da interação com diferentes componentes, e deste modo auxiliam nas etapas de invasão, disseminação e evasão do sistema imune, auxiliando as leptospiras no processo de infecção. / Leptospirosis is a zoonosis of global distribution caused by pathogenic bacteria of the genus Leptospira. The disease has a wide range of clinical manifestations in humans, which can present with fever, headaches and muscular pain, to a more severe clinical condition, known as Weil\'s syndrome, characterized by hemorrhages, jaundice, renal and hepatic failure. So far, the pathogenicity mechanisms of leptospira are not entirely clear and there is no effective vaccine against a disease. Sequencing of pathogenic leptospires genomes has enabled the identification of conserved outer membrane proteins among pathogenic strains, which are the main research focus to understand the mechanism of pathogenicity and possibly identify a vaccine candidate. In this sense, the genes LIC11711 and LIC12587 were selected from the genome sequence of Leptospira interrogans serovar Copenhageni by bioinformatics, which encode two hypothetical predicted lipoproteins of unknown functions. The genes were amplified by PCR from the genomic DNA of the bacteria and cloned into pAE expression vector. The recombinant proteins were expressed in E. coli BL21 DE3 Star pLysS and purified by metal affinity chromatography. Recombinant proteins were inoculated in mice to evaluate their immunogenic activity and to obtain immune sera. Both recombinant proteins are reactive with antibodies in sera from patients diagnosed with a disease, presenting higher sensitivity than the microscopic agglutination test (MAT), high specificity, differentiating antibodies present in sera from patients diagnosed with other non-specific diseases related. The results of intact bacterial ELISA, proteinase K proteolysis and Immunofluorescence suggest that both proteins are located in the surface of the bacteria. Based on western blotting conservation assay, the coding sequences are present in different pathogenic strains of Leptospira, and absent in the nom-pathogenic L. biflexa. In in vitro adhesion assays, recombinant proteins showed interaction with the human components laminina, e-cadherin, plasminogen, fibrinogen, plasma fibronectina, vitronectin, C7, C8 and C9, in dose-dependent manner, but with low affinity values. These results suggest that the proteins encoded by the genes LIC11711 and LIC12587 are expressed during the infection and may perform multiple tasks and thus assist in the steps of invasion, dissemination and evasion of the immune system, helping leptospires during the establishment of the disease.