Directed evolution is a powerful method for engineering of specific affinity proteins such as antibodies and alternative scaffold proteins. For selections from combinatorial protein libraries, robust and high-throughput selection platforms are needed. An attractive technology for this purpose is cell surface display, offering many advantages, such as the quantitative isolation of high-affinity library members using flow-cytometric cell sorting. This thesis describes the development, evaluation and use of bacterial display technologies for the engineering of affinity proteins. Affinity proteins used in therapeutic and diagnostic applications commonly aim to specifically bind to disease-related drug targets. Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a critical process in various types of cancer and vascular eye disorders. Vascular Growth Factor Receptor 2 (VEGFR2) is one of the main regulators of angiogenesis. The first two studies presented in this thesis describe the engineering of a biparatopic Affibody molecule targeting VEGFR2, intended for therapeutic and in vivo imaging applications. Monomeric VEGFR2-specific Affibody molecules were generated by combining phage and staphylococcal display technologies, and the engineering of two Affibody molecules, targeting distinct epitopes on VEGFR2 into a biparatopic construct, resulted in a dramatic increase in affinity. The biparatopic construct was able to block the ligand VEGF-A from binding to VEGFR2-expressing cells, resulting in an efficient inhibition of VEGFR2 phosphorylation and angiogenesis-like tube formation in vitro. In the third study, the staphylococcal display system was evaluated for the selection from a single-domain antibody library. This was the first demonstration of successful selection from an antibody-based library on Gram-positive bacteria. A direct comparison to the selection from the same library displayed on phage resulted in different sets of binders, and higher affinities among the clones selected by staphylococcal display. These results highlight the importance of choosing a display system that is suitable for the intended application. The last study describes the development and evaluation of an autotransporter-based display system intended for display of Affibody libraries on E. coli. A dual-purpose expression vector was designed, allowing efficient display of Affibody molecules, as well as small-scale protein production and purification of selected candidates without the need for sub-cloning. The use of E. coli would allow the display of large Affibody libraries due to a high transformation frequency. In combination with the facilitated means for protein production, this system has potential to improve the throughput of the engineering process of Affibody molecules. In summary, this thesis describes the development, evaluation and use of bacterial display systems for engineering of affinity proteins. The results demonstrate great potential of these display systems and the generated affinity proteins for future biotechnological and therapeutic use. / <p>QC 20141203</p>

Beyond their physiological roles, nuclear receptors have been exploited for their ability to act as intracellular sensors of small molecules. Accordingly, yeast two- and three-hybrid systems have been developed, exploiting them to control reporter gene expression. These systems may be used to identify nuclear receptor ligand interaction, or for protein engineering applications, particularly of the nuclear receptor ligand binding domain. In this work, the use of estrogen receptors as sensors for enzyme catalysis is explored, where expression of a reporter gene is induced in the presence of the product from an enzymatic reaction. This system, which we have called enzyme-activated growth, has applications for the engineering of biocatalysts. Biocatalytic routes are currently being explored in industrial applications since they often have financial and environmental benefits over traditional heterogeneous catalysis. Enzyme-activated growth is designed to serve as a system to select for engineered enzymes capable of catalyzing the desired reaction. For this work, a new yeast two-hybrid strain has been developed and characterized to allow for detection of both agonist and antagonist compounds. To increase the sensitivity of this assay, a variant of the estrogen receptor was created through random mutation, which responded to ligand concentrations an order of magnitude lower than the wild type receptor. The five mutations identified in the best variant were previously unknown in the literature and the roles of each of these are investigated, as is the mechanism by which they alter ligand sensitivity. As a proof-of-principle, the enzymatic production of genistein, an estrogenic metabolite from plants, using the enzyme isoflavone synthase, as well as the production of estrogen from testosterone, is explored. Synthesis of genistein from the starting material naringenin in vivo was detected in the yeast two-hybrid strain; however, attempts at pairing this with estrogen receptor activation and cell growth were met with limited success. Lastly, targeting the estrogen receptor with a series of novel anti-cancer therapeutics is explored. These compounds were designed to both bind and (in)activate the estrogen receptor while inhibiting histone deacetylase activity. The (anti-)estrogenic properties were analyzed as well as their potency as histone deacetylase inhibitors. These properties were compared to their anti-proliferative effects against various cancerous and healthy cell lines to determine their potential as selective anti-cancer therapeutics.

I denna avhandling presenteras arbete utfört med enzymet humant karboanhydras II (HCAII). Enzymer är en typ av proteiner som accelererar (katalyserar) kemiska reaktioner, vilket är nödvändigt för allt levande. Den naturliga funktionen för HCAII är att katalysera omvandlingen av gasen koldioxid till vätekarbonat, som är löslig i vätska. Detta är viktigt bl.a. för att koldioxid som bildas i kroppen, och fraktas i blodet i form av vätekarbonat, skall hinna över till utandningsluften under den korta tid blodet är i lungorna. Proteiner består av aminosyror som länkats samman i en lång kedja, där varje aminosyra är en av de 20 naturliga aminosyratyperna. Ett proteins struktur och egenskaper bestäms av aminosyrasekvensen, som i sin tur bestäms av genen för just det proteinet. Med genteknik kan ett proteins gen ändras (muteras), så att aminosyrasekvensen ändras, och det har här utnyttjats för att förändra HCAIIs katalytiska egenskaper. Förutom dess naturliga funktion kan HCAII även klyva (hydrolysera) vissa estrar. Mutationer gjordes så att en ’ficka’ i HCAIIs struktur, där molekylerna (substraten) som skall klyvas binder, fick en större volym. På så sätt skapades varianter med en kraftigt ökad kapacitet för att hydrolysera långa estersubstrat jämfört med icke-muterat HCAII. Som en utveckling av detta projekt skapades en mutant av HCAII, som kan hydrolysera ett än mer skrymmande substrat. I ett annat projekt har en ny katalytisk aktivitet skapats i HCAII, som inte utnyttjar enzymets naturliga katalytiska förmåga. Ett nytt estersubstrat konstruerades, med en del som binder kraftigt till HCAII, så att en stark substratbindning erhölls. Sedan muterades vissa aminosyror till en reaktiv aminosyra som heter histidin. Valet av positioner för mutation baserades på en datormodell av enzymet med bundet substrat. Eftersom histidin kan delta i hydrolysreaktioner, får det muterade enzymet möjlighet att klyva substratet. Flera olika mutanter testades, och den effektivaste innehöll ett nära kopplat par av histidiner. Denna mutant undersöktes mer noggrannt, vilket gav viss information om den katalytiska mekanismen. Det långsiktiga målet med detta arbete är att konstruera muterade enzymer som kan klyva giftiga ämnen, eller användas vid framställning av kemikalier. Det finns behov av nya enzymer för olika typer av substrat, och att med rationella metoder skapa nya katalytiska aktiviteter i proteiner är ett svårt vetenskapligt problem som ännu är i ett tidigt utvecklingsskede. / The main part of this thesis describes results from protein engineering experiments, in which the catalytic activity of the enzyme human carbonic anhydrase II (HCAII) is engineered by mutagenesis. This enzyme, which catalyzes the interconversion between CO2 and HCO3- in the body, also has the ability to hydrolyze ester bonds. In one project, the specificity of HCAII towards a panel of para-nitrophenyl ester substrates, with acyl chain lengths ranging from one to five carbon atoms, was changed by enlarging the substrate binding hydrophobic pocket. A variant was identified that has highly increased specificity towards substrates with long acyl chains. The mutant V121A/V143A hydrolyzes pNPV, which has four carbon atoms in the acyl chain, with an efficiency that is increased by a factor of 3000 compared to HCAII. Further, transition state analogues (TSAs) were docked to HCAII and mutant variants, and the results were correlated to the results from kinetic measurements. This indicated that automated docking could be used to some extent to construct HCAII variants with a designed specificity. Using this approach, a HCAII mutant that can hydrolyze a model benzoate ester was created. Interestingly, the resulting variant V121A/V143A/T200A was found to be highly active with other ester substrates as well. For pNPA, a kcat/KM of 1*105 M-1s-1 was achieved, which is the highest efficiency for hydrolysis of carboxylic acid esters reported for any HCAII variant. In another project, the strong affinity between the active site zinc ion and sulfonamide was used to achieve binding of a designed substrate. Thus, the natural Zn-OH- site of HCAII was not used for catalysis, but for substrate binding. The substrate contains a benzenesulfonamide part in one end, with a para-nitrophenyl ester connected via a linker. The linker was chosen to ensure that the scissile bond is positioned close to His-64 and histidine residues introduced by mutagenesis in other positions. Using this approach, an enzyme was designed with a distinctly new two-histidine catalytic site for ester hydrolysis. The mutant, F131H/V135H, has a kcat/KM of approximately 14000 M-1s-1, which corresponds to a rate enhancement of 107 compared to a histidine mimic. Finally, results are reported on a project aimed at cloning and producing a putative carbonic anhydrase from the malaria parasite Plasmodium falciparum. The gene was cloned by PCR and the construct was overexpressed in E. coli. However, the resulting protein was not soluble, and initial attempts to refold it are also reported.

The growing understanding of tumor biology and the identification of tumor specificgenetic and molecular alterations, such as the overexpression of human epidermal growthfactor receptor 2 (HER2), opens up for personalization of patient management using targeted therapies. However, this puts stringent demands on the diagnostic tools usedto identify patients that are likely to respond to a particular treatment. Radionuclide molecular imaging is a promising non-invasive method to visualize and characterize the expression of such targets. This thesis, based on five papers, is focused on the development of radiolabeled Affibody molecules for imaging of HER2-expression in malignant tumors. Affibody molecules, which represent a rather novel class of affinity proteins developed by combinatorial protein engineering of the protein A derived Z-domain, display manyfeatures that make them promising tracers for molecular imaging applications. The aim of the work presented here was to further develop the tracer format for improved in vivo properties and flexibility in the choice of radionuclide. In paper I, the development of an assay that enables quantitative studies of the internalization rate and cellular processing of high affinity Affibody molecules is described. The assay was applied to a HER2-binding Affibody variant that was efficiently retained by HER2-expressing cells, although characterized by a slow internalization rate. This may have implications for the choice of label for Affibody molecules since high affinity to the target may be equally, or more, important for good imaging quality than residualizing properties of the radiolabel. In paper II, a HER2-binding Affibody molecule and the monoclonal antibody trastuzumab were labeled with positron emitting 124I, for a head-to-head in vivocomparison of the two tracer formats. The effects of tracer size and presence of an Fc region on the biodistribution profile were investigated. In paper III, a HER2-binding Affibody molecule was site-specifically labeled with radiocobalt and evaluated in vitro and in vivo.A head-to-head in vivo comparison with the well-studied 111In-labeled counterpart was performed, revealing promising potential for the cobalt-labeled molecule as a PET-tracerfor visualization of HER2. Paper IV describes the in vitro and in vivo evaluation of a panel of Affibody molecules with different C-terminal peptide-based chelators for the coordination of 99mTc. Even small changes in the C-terminal sequence had appreciable impact on the biodistribution of the Affibody molecules and by optimizing the design of the chelator, the kidney uptake of 99mTc could be significantly reduced. Finally, in paper V we describe the development of a HER2-targeting Affibody variant equipped with a Sel-tag for site-specific labeling with the short-lived positron emitter 11C. This novel Affibody tracer could be used to image HER2-expressing tumors in vivo within one hour after injection. Taken together, Affibody molecules show great promise as targeting tracers for radionuclide molecular imaging of HER2. Careful design and optimization of the tracer protein is important and can be used to improve the biodistribution and targeting properties of Affibody molecules. / QC 20110922

The recent development of methods for constructing andhandling large collections (libraries) of proteins, from whichvariants with desired traits can be isolated, hasrevolutionized the field of protein engineering. Key elementsof such methods are the various ways in which the genotypes(the genes) and the phenotypes (the encoded proteins) arephysically linked during the process. In one section of thework underlying this thesis, one such technique (phagedisplay), was used to isolateand identify protein librarymembers based on their catalytic or target molecule-bindingproperties. In a first study, phage display libraries of the lipolyticenzyme Lipolase from Thermomyces lanuginosa were constructed,the objective being to identify variants with improvedcatalytic efficiency in the presence of detergents. Toconstruct the libraries, nine positions were targeted for codonrandomization, all of which are thought to be involved in theconformational change-dependent enzyme activation that occursat water-lipid interfaces. The aim was to introduce two tothree amino acid mutations at these positions per lipase gene.After confirming that the wt enzyme could be functionallydisplayed on phage, selections with the library were performedutilizing a mechanism-based biotinylated inhibitor in thepresence of a detergent formulation. According to rhodamineB-based activity assays, the fraction of active clonesincreased from 0.2 to 90 % over three rounds of selection.Although none of the variants selected using this approachshowed increased activity, in either the presence or absence ofdetergent compared to the wild type enzyme, the resultsdemonstrated the possibility of selecting variants of theenzyme based on catalytic activity. In the following work, phage libraries of the StaphylococcalProtein A (SPA)-derived Z-domain, constructed by randomizationof 13 surface-located positions, were used to isolate Z domainvariants (affibodies) with novel binding specificities. Astargets for selections, the parental SPA domains as well as twopreviously selected affibodies directed against two unrelatedtarget proteins were used. Binders of all three targets wereisolated with affinities (KD) in the range of 2-0.5 µM.One SPA binding affibody (ZSPA-1) was shown to bind to each of the fivehomologous native IgG-binding domains of SPA, as well as theZdomain used as the scaffold for library constructions.Furthermore, the ZSPA-1affibody was shown to compete with one of thenative domains of SPA for binding to the Fc part of humanantibodies, suggesting that the ZSPA-1affibody bound to the Fc-binding surface ofthe Z domain. The majority of the affibodies isolated in theother two selections using two different affibodies as targets,showed very little or no binding to unrelated affibodies,indicating that the binding was directed to the randomizedsurface of their respective targets, analogously toanti-idiotypic antibodies. The structure of the wild type Z domain/ZSPA-1affibody co-complex was determined by x-raycrystallography, which confirmed the earlier findings in thatthe affibody ZSPA-1affibody was shown to bind to the Fc bindingsurface of the Z domain. Further, both the Z domain and the ZSPA-1affibody had very similar three helix-bundletopologies, and the interaction surface involved ten out of thethirteen randomized residues, with a central hydrophobic patchsurrounded by polar residues. In addition, the interactionsurface showed a surprisingly high shape complementarity, giventhe limited size of the library used for selections. The ZSPA-1affibody was further investigated for use invarious biotechnological applications. In one study, the ZSPA-1affibody was successfully recruited as a novelaffinity gene fusion partner for production, purification anddetection of cDNA-encoded recombinant proteins using anSPA-based medium for affinity chromatography. Further, the SPAbinding capability of the ZSPA-1affibody was employed for site-specific andreversible docking of ZSPA-1affibody-tagged reporter proteins onto an SPAfusion protein anchored to a cellulose surface via acellulose-binding moiety. These generated protein complexesresembles the architecture of so-called cellulosomes observedin cellulolytic bacteria. The results suggest it may bepossible to use anti-idiotypic affibody-binding protein pairsas modules to build other self-assembling types of proteinnetworks. Keywords:phage display, selection, mechanism-basedinhibitor, affinity domains, crystal structure, Staphylococcusaureus protein A, affinity chromatography, anti-idiotypicbinding pairs, affibody, combinatorial, protein engineering,lipase, cellulosome, assembly.

Thesis (M.Eng.(Computer Engineering))--University of Pretoria, 2008. / Summaries in Afrikaans and English. Includes bibliographical references (leaves 150-160).

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 139-158).

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 115-125).

No description available.

Thesis (D.Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 xi, 209 leaves / Protein engineering technologies, such as directed evolution and DNA recombination, are often used to modify enzymes on a genetic level for the creation of useful industrial catalysts. Pre-treatment of paper pulps with xylanases have been shown to decrease the amounts of toxic chlorine dioxide used to bleach pulp. This study was undertaken to improve the thermal and alkaline stabilities of the xylanase from the fungus Thermomyces lanuginosus using ep-PCR and DNA shuffling.