Polyadenylation is an essential post-transcriptional modification resulting in a mature mRNA in eukaryotes. Three cis-elements the Far Upstream Element (FUE), Near Upstream Element (NUE), and Cleavage Site (CS) - guide the process of cleavage and polyadenylation with the help of multi-subunit protein complexes cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF) along with cleavage factors and poly(A) polymerase. Protein-protein interactions play an important role in the cleavage and polyadenylation process. WD repeat proteins play an important role in protein-protein interactions and have diverse functions in plant system. In the present study WD repeat proteins AtCstF50 and AtFY were studied for their role in polyadenylation process. Mammalian CstF50 is a WD repeat protein that is one of the subunit of CstF that aids in the cleavage step by associating with CPSF and cleavage factors. AtCstF50 was functionally characterized using T-DNA knock-out lines and by identifying the proteins that interacts with it in the process. Results shows that AtCstF50 is essential and was identified as part of CPSF complex, which is different from its mammalian counter part. CPSF was known to interact with Fip (factor interacting with PAP), Poly(A) polymerase and Poly(A) binding protein and AtCstF50 also interacts with these complexes. AtFY is a 3’ end processing factor which contains WD repeats is one of the subunits of the CPSF complex in Arabidopsis polyadenylation machinery. The AtFY interacts with FCA and promotes the alternative polyadenylation and also plays a role in polyadenylation site choice of FCA mRNA. We characterized the FY expression and localization of FY in the cell by fusing with RFP reporter. Results show that FY accumulates in the nucleus while FY with deleted calmodulin binding domain localizes both to the nucleus and outside the nucleus. The individual N-terminal and C-terminal domains also localized in the nucleus suggesting that they are multiple nuclear localization signals in FY and calmodulin might play a direct or indirect role in FY localization. Using a tethering assay we proved that AtFY is able to recruit the 3’ end processing complex in the proximal polyadenylation site choice of the reporter mRNA.

Many applications in biomedicine, research, and industry require recognition agents with specificity and selectivity for their target. Protein engineering enables the design of scaffolds that can bind targets of interest while increasing their stability, and expanding the scope of applications in which these scaffolds will be useful. Repeat proteins are instrumental in a wide variety of biological processes, including the recognition of pathogen-associated molecular patterns by the immune system. A number of successes using alternative immune system repeat protein scaffolds have expanded the scope of recognition agents available for targeting glycans and glycoproteins in particular. We have analyzed the innate immune genes of a freshwater polyp and found that they contained particularly long contiguous domains with high sequence similarity between repeats in these domains. We undertook statistical design to create a binding protein based on the H. magnipapillata innate immune TPR proteins. My second research project focused on creating a protein to bind cellulose, as it is the most abundant and inexpensive source of biomass and therefore is widely considered a possible source for liquid fuel. However, processing costs have kept lignocellulosic fuels from competing commercially with starch-based biofuels. In recent years a strategy to protect processing enzymes with synergistic proteins emerged to reduce the amount of enzyme necessary for lignocellulosic biofuel production. Simultaneously, protein engineering approaches have been developed to optimize proteins for function and stability enabling the use of proteins under non-native conditions and the unique conditions required for any necessary application. We designed a consensus protein based on the carbohydrate-binding protein domain CBM1 that will bind to cellulosic materials. The resulting designed protein is a stable monomeric protein that binds to both microcrystalline cellulose and amorphous regenerated cellulose thin films. By studying small changes to the binding site, we can better understand how these proteins bind to different cellulose-based materials in nature and how to apply their use to industrial applications such as enhancing the saccharification of lignocellulosic feedstock for biofuel production. Biomaterials made from natural human hair keratin have mechanical and biochemical properties that make them ideal scaffolds for tissue engineering and wound healing. However, the extraction process leads to protein degradation and brings with it byproducts from hair, which can cause unfavorable immune responses. Recombinant keratin biomaterials are free from these disadvantages, while heterologous expression of these proteins allows us to manipulate the primary sequence. We endeavored to add an RGD sequence to facilitate cell adhesion to the recombinant keratin proteins, to demonstrate an example of useful sequence modification. / Doctor of Philosophy / Many applications in medicine and research require molecular sensors that bind their target tightly and selectively, even in complex mixtures. Mammalian antibodies are the best-studied examples of these sensors, but problems with the stability, expense, and selectivity of these antibodies have led to the development of alternatives. In the search for better sensors, repeat proteins have emerged as one promising class, as repeat proteins are relatively simple to design while being able to bind specifically and selectively to their targets. However, a drawback of commonly used designed repeat proteins is that their targets are typically restricted to proteins, while many targets of biomedical interest are sugars, such as those that are responsible for blood types. Repeat proteins from the immune system, on the other hand, bind targets of many different types. We looked at the unusual immune system of a freshwater polyp as inspiration to design a new repeat protein to recognize nonprotein targets. My second research project focused on binding cellulose, as it is the most abundant and inexpensive source of biological matter and therefore is widely considered a possible source for liquid fuel. However, processing costs have kept cellulose-based fuels from competing commercially with biofuel made from corn and other starchy plants. One strategy to lower costs relies on using helper proteins to reduce the amount of enzyme needed to break down the cellulose, as enzymes are the most expensive part of processing. We designed such a protein for this function to be more stable than natural proteins currently used. The resulting designed protein binds to multiple cellulose structures. Designing a protein from scratch also allows us to study small changes to the binding site, allowing us to better understand how these proteins bind to different cellulose-based materials in nature and how to apply their use to industrial applications. Biomaterials made from natural human hair keratin have mechanical and biochemical properties that make them ideal for tissue engineering and wound healing applications. However, the process by which these proteins are extracted from hair leads to some protein degradation and brings with it byproducts from hair, which can cause unfavorable immune responses. Making these proteins synthetically allows us to have pure starting material, and lets us add new features to the proteins, which translates into materials better tailored for their applications. We discuss here one example, in which we added a cell-binding motif to a keratin protein sequence.

Au cours de notre programme de thèse, nous avons isolé et caractérisé des molécules protéiques à activité antivirale intracellulaire dirigée contre le VIH-1. Ces protéines, appelées aRep, ont été obtenues par criblage d'une banque de protéines artificielles de nouvelle génération, construites de façon combinatoire à partir de protéines naturelles constituées de motifs alpha-hélicoidaux répétés. La cible virale, ou "appât", utilisé pour ce criblage a été une région de la polyprotéine Gag du VIH-1 identifiée comme une cible privilégiée de nouvelles thérapeutiques antivirales, car essentielle à l'assemblage viral, l'empaquetage du génome viral et le clivage de maturation de Gag aboutissant à la formation de virions infectieux. Deux molécules d'aRep à affinité élevée pour la cible virale, l'aRep4E3 (32 kDa; 7 motifs répétés) et l'aRep9A8 (28 kDa; 6 motifs répétés) ont ainsi été identifiées, isolées et clonées. L'étude de l'activité anti-VIH intracellulaire de ces aRep a été réalisée dans différents systèmes d'expression cellulaire, nécessitant la construction de lignées stables de cellules d'insecte et de cellules épithéliales humaines, et leur infection par différents types de vecteurs viraux recombinants, baculovirus ou lentivirus, porteurs du gène rapporteur luciférase. Mais surtout, cette étude a été menée sur des cellules lymphocytaires-T (SupT1), cibles naturelles du virus, exprimant ces aRep et infectées par du VIH-1 naturel infectieux. Nos résultats ont montré que l'aRep4E3 et l'aRep9A8 ont toutes deux un effet négatif significatif sur le cycle réplicatif du VIH-1, mais ciblent des fonctions virales différentes. L'aRep4E3 bloque l'empaquetage du génome viral, tandis que l'aRep9A8 inhibe la maturation et diminue l'infectivité virale. De plus, l'aRep9A8, exprimée de façon constitutive dans les cellules SupT1, leur confère une résistance au VIH: une lignée de SupT1 chroniquement infectée par le VIH-1 a pu être ainsi isolée et maintenue en culture pendant plusieurs semaines, sans effet cytopathique viro-induit apparent. Ces nouvelles données auront des implications non-négligeables dans le choix et la conduite de futures stratégies de thérapie cellulaire anti-VIH / HIV-1 infection is a long-term disease which required a long-life treatment. Besides the standard HAART regiment, HIV gene therapy is a promising alternative strategy which give rise to hope for the better HIV-1 treatment. Protein therapeutics is one another technique that represent high impact results in curing various types of disease. It is already become a significant part of current medical treatments. In this study we first designed aRep, a non-immunoglobulin scaffold protein which target two domains of HIV-1 Pr55Gag, SP1-NC and investigated their roles as an intracellular therapeutic agents. Phage display technology was used for the specific isolation of aRep against a critical C-terminal region of the HIV-1 Pr55Gag precursor from a large and diverse library. The antiviral activity of these two Pr55Gag binders was investigated using different cell systems. Two aRep scaffolds aRep4E3 and aRep9A8 were isolated and characterized. aRep4E3 contains 7 repeat motifs (32 kDa), meanwhile aRep9A8 has 6 repeat motifs (28 kDa). These two scaffold molecules found to be able to display antiviral effects on the late stage of HIV-1 replication, by reducing and delaying the viral progeny production. The difference in the molecular mechanism was observed between these two aRep proteins: aRep4E3 mainly interferes with the packaging of the viral genome, meanwhile aRep9A8 interferes with the proteolytic processing of Gag, and performs as a protease inhibitor to prevent the PR cleavage required for the production of newly infectious mature virus. Interestingly, aRep9A8 is able to survive in the chronical HIV-1 infected cells up to D38 pi with the low level of HIV-1 replication. Taken together, results suggested that aRep, a new type of scaffold protein could serve as a promising alternative agent in protein therapy, not only the HIV-1 infection but also the others pathogens or disorders

Les protéines PentatricoPeptide-Repeats (PPR) représentent la plus grande famille de protéines de liaison à l’ARN connue. Elles sont caractérisées par la présence de motifs répétés en tandem d’environ 35 résidus ayant une structure hélice-tour-hélice. Depuis les premières études sur l’organisme modèle Arabidopsis thaliana, les protéines PPR ont aussi été découvertes chez d’autres espèces non-plantes, incluant les levures et l’humain. Cependant, la détection des protéines PPR en dehors des plantes est compliquée par le fait que les outils de recherche sont tous conçus pour les protéines de plantes. Récemment, une étude réalisée chez les levures a rapporté une méthode itérative semi-automatisée d’identification de PPR utilisant des profils Hidden Markov Models (HMM). Inspirés par cette approche, nous visons ici à développer une méthode complètement automatisée plus généralisable et sensible qui ne dépend pas du protéome de départ. Comme preuve de concept, nous avons choisi une espèce non reliée aux plantes possédant le plus grand nombre de protéines PPR en-dehors des plantes – le protiste marin unicellulaire Diplonema papillatum. Il s’agit d’un modèle émergent ayant reçu beaucoup d’intérêt pour l’excentricité de l’expression de son génome mitochondrial, pour lequel il a été suggéré que les protéines PPR jouent un rôle clé. Nous avons ici développé une approche itérative pour identifier et cataloguer les protéines PPR chez D. papillatum. Les fonctionnalités particulières de notre algorithme incluent l’inspection des intervalles de 30 à 40 résidus entre les motifs classiques déjà identifiés et l’utilisation des structures secondaires caractéristiques des motifs PPR pour valider les motifs candidats nouvellement identifiés. Au final, nous avons identifié près de 800 motifs PPR chez D.papillatum, dont plusieurs motifs « déviants » identifiés dans les espaces entre les motifs. La validation expérimentale des motifs candidats les plus prometteurs est en attente. / PentatricoPeptide-Repeat (PPR) proteins represent the largest family of RNA-binding proteins known. They are defined by containing tandemly arranged, ~35-residue long motifs assuming a helix-turn-helix structure, which are referred to as PPR motifs. Since the seminal studies undertaken in the model organism Arabidopsis, a few PPR proteins have been also discovered outside plants, including yeast and human. However, the detection of PPR proteins in non-plant eukaryotes is complicated by the fact that current search tools are tailored toward plants. Recently, a semi-automated method has been reported for identifying PPR motifs in yeast using iterative searches with profile Hidden Markov models (HMMs). Inspired by this work, we aimed to develop a fully automated, sensitive approach that can be used for detecting PPR proteins in any species, when using the corresponding proteome as input. For a proof of concept, we used a species that contains the largest number of PPR genes outside the plant kingdom –the unicellular protist Diplonema papillatum. This emerging model system has garnered much interest for the eccentricities of its mitochondrial gene expression, in which PPR proteins are posited to play a key role. Here, we have developed an iterative HMM-search method that comprehensively catalogues and classifies PPR motifs in D. papillatum. Particular features of our algorithm are that it inspects closely 30 to 40 residue-long intervals between readily identified (classical) motifs, makes use of the characteristic secondary structure of PPR motifs to validate newly detected candidate motifs. In total, we have identified around 800 PPR motifs in D. papillatum. Including several deviant candidates detected in ”gaps”. High ranking representatives of both classical and deviant motifs await experimental validation.

Both technological and experimental advancements in the field of biotechnology have allowed scientists to make leaps in areas such nucleic acid, antibody, and recombinant protein technologies. Here we focus on the use of recombinant proteins as molecular recognition motifs, wound healing biomaterials, and agents for cell cycle pathway elucidation are discussed. The author's primary project is described in chapters 2 and 3, and is focused on designed leucine-rich repeat proteins which offer increased stability, modularity, and surface area for binding interactions. These proteins bind at least two muramyl dipeptide ligands with picomolar to nanomolar affinity (Kd1 = 0.04 – 3.5 nM); as measured by fluorescence quenching experiments and ITC. The longest designed repeat, CLRR8, has a Kd app value of 1.0 nM which is comparable to full length native NOD2 protein. Molecular docking simulations revealed the locations of two potential binding sites and their respective interactions. The series of proteins represents a foundation for a high affinity and highly specific molecular recognition scaffold that has the potential to bind a variety of ligands. Previously the author contributed to the design of recombinant keratin proteins, and the work in Chapter 4 builds on the original design to allow for controlled degradation in wound healing systems. Site-directed mutagenesis was utilized to introduce these degradation sites, and modified keratin proteins were expressed with no differences to native recombinant keratin proteins. Success in engineering a variation of native keratin protein with no issues in expression lay the foundation for further engineering of native keratin or other relevant proteins for improved functionality. Chapter 5 describes steps towards producing human Aurora borealis (Bora) protein, an important substrate in cell cycle regulation, by in vitro transcription-translation with locked Ser–Pro analogues. This will allow for the elucidation of the active isomerization form to ensure proper cell division. Site-directed mutagenesis successfully introduced the amber codon to relevant Ser-Pro sites at positions 274 and 278. These mutated Bora genes along with modified ribosomes and aminoacyl tRNA will allow for the incorporation of locked dipeptide analogues. Expression of native Bora was carried out as a control, and appeared to express in dimeric form. The experiments carried out in Chapter 5 describe and outline all the molecular biology work completed and to be completed for this novel method of studying cis-trans isomerization in living cells. / Doctor of Philosophy / Sequencing of the human genome and the rapid development of gene editing and recombinant DNA technologies paved the way for a massive shift in the pharmaceutical industry. The first pharmaceutical companies in the 19th century started as fine chemicals businesses. The discovery of penicillin introduced antibiotics, and improved synthetic techniques led to the giants we know as big pharma today. Today, in the 21st century both computing and biotechnology has allowed for great leaps forward in precision medicine. Biotechnology refers to the manipulation of living organisms or their components to produce useful commercial products. In the pharmaceutical industry this refers to genetic engineering for novel pharmaceuticals. Here, we focus on the use of recombinant technology to create proteins for use in biomedical applications. Recombinant proteins are proteins formed by laboratory methods of molecular cloning. Through this technology, we are able to elucidate sequence-structure-function relationships of proteins, and determine their specific functions. Additionally, recombinant methods allow us to fine tune or modify the sequences of natural proteins to be more effective scaffolds or reagents. Chapter 3 focuses on the development of synthetic proteins for medical diagnostics. We designed a protein scaffold, based on natural innate immunity proteins, to detect bacteria cell wall components. Chapter 4 focuses on the engineering of keratin protein with applications in wound healing. We introduce controlled degradation of the biomaterial for use in potential drug delivery systems at the wound site. Chapter 5 focuses on the use of recombinant technologies aiding in the elucidation of a regulatory protein's function in cell division.

<p>Ankyrin repeats (ARs) are polypeptide motifs identified in thousands of proteins. Many AR proteins play a function as scaffolds in protein-protein interactions which may require specific mechanical properties. Also, a number of AR proteins have been proposed to mediate mechanotransduction in a variety of different functional settings. The folding and stability of a number of AR proteins have been studied in detail by chemical and temperature denaturation experiments, yet the mechanic of AR proteins remain largely unknown. In this dissertation, we have researched the mechanical properties of AR proteins by using protein engineering and a combination of atomic force microscopy (AFM)-based single-molecule force spectroscopy and steered molecular dynamics (SMD) simulations. Three kinds of AR proteins were investigated: NI6C (synthetic AR protein), D34 (of ankyrin-R) and gankyrin (oncoprotein). While the main focus of this research was to characterize the response of AR proteins to mechanical forces, our results extended beyond the protein nanomechanics to the understanding of protein folding mechanisms.</p> / Dissertation

Chlorophyll spielt eine unabdingbare Rolle für die lichtabhängige Reaktion der Photosynthese. Die adäquate Versorgung mit Chlorophyll wird dabei durch die Tetrapyrrolbiosynthese (TBS) gewährleistet. In den letzten Jahrzehnten wurde eine Vielzahl von Proteinen identifiziert, welche an der Anpassung der TBS an wechselnde (a)biotische Wachstumsbedingungen der Pflanze beteiligt sind. Allerdings konnte bislang nicht zweifelsfrei geklärt werden, wie die TBS mit der Integration von Chlorophyllen in die Photosysteme koordiniert wird. Vor einigen Jahren wurde ein Interaktionspartner der Protochlorophyllid-Oxidoreduktase (POR) in Synechocystis identifiziert, welcher als potenzieller Faktor dieser Koordination in Frage kommt. Das POR-INTERACTING TPR-Protein (Pitt) stabilisiert POR an der Thylakoidmembran und interagiert auch mit dem Vorstufenprotein des D1. Pitt gehört zur Familie der tetratricopeptide repeat (TPR) Proteine, deren Vertreter vorrangig für die Vermittlung von Protein-Protein-Interaktionen zuständig sind. Aus diesem Grund war, neben der Identifikation des potenziellen Pitt-Homologs im Modelorganismus Arabidopsis thaliana, die Analyse von anderen Vertretern dieser Proteinklasse ein vielversprechender Ansatz bei der Identifikation von weiteren Regulatoren der TBS oder Photosynthese. Von den fünf ausgewählten TPR-Proteinen aus Arabidopsis thaliana mit einer hohen Sequenzähnlichkeit zu Pitt waren vier in der Lage, physisch mit POR zu interagieren. Von diesen vier Kandidaten ist das durch das Gen At1g78915 kodierte, membranintegrale TPR-Protein (TPR1) der beste Kandidat des putativen Pitt-Homologs in Arabidopsis. Vergleichbar zu Pitt interagiert TPR1 mit POR und stabilisiert das Enzym an den plastidären Membranen. Die Stabilisierung von POR durch TPR1 spielt eine entscheidende Rolle während der Etiolierung und Ergrünung von Keimlingen. Darüber hinaus steht TPR1 im Zusammenhang mit der schnellen Inaktivierung der 5-Aminolävulinsäuresynthese. / Chlorophyll plays an indispensable role in the light reaction of the photosynthesis. The adequate supply of chlorophyll is ensured by tetrapyrrole biosynthesis (TBS). Within the last decades, multiple proteins were identified, which are involved in adjusting the TBS-pathway to changing (a)biotic plant growth conditions. Nevertheless, it is not fully understood how the TBS-pathway is coordinated parallel to the assembly of the photosystems and the integration of chlorophylls into the pigment-binding subunits of the photosystems. Several years ago, an interaction partner of the protochlorophyllide-oxidoreductase (POR) was identified in Synechocystis which was proposed to be involved in the coordination of these mechanisms. The POR-INTERACTING TPR-Protein (Pitt) binds and stabilizes POR at the thylakoid membranes and interacts with the precursor protein of D1. Therefore, Pitt could facilitate the incorporation of chlorophylls into the plastid-encoded nascent photosynthetic subunits. Pitt belongs to the tetratricopeptide repeat (TPR) protein family, whose members mediate protein-protein-interactions. Besides the identification of the potential Pitt-homolog in the model organism Arabidopsis thaliana, analysis of additional members of the TPR-protein superfamily was a promising approach for the identification of further posttranslational regulators of TBS and photosynthesis. Five Arabidopsis thaliana TPR-proteins with a high sequence similarity to Pitt were selected. Four of those proteins are able to interact physically with POR. Among them, the TPR-protein encoded by the gene At1g78915 (TPR1) was the best candidate to represent a putative Pitt homolog in Arabidopsis. Similar to Pitt, TPR1 is a plastid-localized integral membrane protein, which interacts with POR at the thylakoid membranes. The stabilizing effect of TPR1 on POR is especially needed during etioliation and greening. Additionally, TPR1 is required for a inactivation of the 5'-aminolevulinic acid synthesis.

Hypoxia-inducible factor (HIF) is the master regulator of genes involved in adaptation to hypoxia. The stability and transcriptional activity of HIF are regulated by post-translational hydroxylations: prolyl hydroxylation by the prolyl hydroxylase domain-containing enzymes PHD1 – 3 earmarks HIF for proteasomal degradation, whilst asparaginyl hydroxylation by factor inhibiting HIF (FIH) blocks the interaction of HIF with the transcriptional coactivators p300/CBP. The PHDs and FIH hydroxylate HIF directly from molecular oxygen and are therefore oxygen sensors. Recent literature shows that FIH also hydroxylates a number of proteins containing an ankyrin-repeat domain (ARD). Together with reports suggesting that the PHDs are involved in HIF-independent pathways, this suggests that the HIF hydroxylases may have a wide range of non-HIF targets. This thesis describes my investigations into novel substrates of the HIF hydroxylases. This work has characterized the FIH-dependent hydroxylation of the ARD-containing protein Notch1, and defined a consensus sequence for hydroxylation that corresponds to the ankyrin-repeat consensus. Using this consensus potential sites of hydroxylation in a novel ARD FIH substrate, myosin phosphatase targeting subunit 1 (MYPT1), were identified then subsequently confirmed and characterized. Notch1 competes with HIF for FIH hydroxylation. My experiments show that this occurs because Notch1 is a more efficient substrate than HIF, whilst studies on MYPT1 and other proteins indicate that competitive inhibition of FIH may be a general property of ARDs. There are more than 300 ARD proteins in the human genome, and this thesis demonstrates that FIH may hydroxylate a significant percentage of these. In addition to the analysis of ARD hydroxylation a proteomic investigation into novel PHD3 substrates has identified two candidate proteins, suggesting that the PHDs may also have multiple targets. These results have important implications for oxygen sensing, and indicate that post-translational hydroxylation is likely to be a widespread modification in cell biology.

Designade ankyrinupprepningsproteiner (DARPiner) är små, mycket stabila antikroppsmimetiska proteiner. I det här projektet användes anti-EpCAM-DARPiner tillsammans med mikrofluidik för att avgära om de kunde fånga upp HCT116-celler mer effektivt än anti-EpCAM-antikroppar. Ytorna på insidan av mikroffluidikkanaler förändrades genom bindning av N-γ-maleimidobutyryl-oxysuccinimidester (GMBS) och merkaptopropyltrietoxysilan (MPTES) för anti-EpCAM-antikroppar och GMBS och (3-aminopropyl)trietoxysilan (APTES) för DARPiner. Båda kanaltyperna testades genom inflöde av cancerceller och helblod blandat med cancerceller. Ingen effektiv och konsekvent celluppfångst åstadkoms trots att det visades att antikropparna och DARPinerna kunde binda till cellerna direkt och att test med fluorescenta DARPiner och antikroppar visade att ytförändringskemin var fungerande. Slutsatsen blev att de mest troliga orsakerna till misslyckandena var att ytförändringskemin påverkade proteinernas bindningsförmåga negativt eller att proteinerna bands till kanalernas yta i fel riktning. DARPiner är fortfarande intressanta för tillämpningar inom mikrofluidik, men vidare förbättring av det experimentella protokollet behövs. / Designed ankyrin repeat proteins (DARPins) are small and highly stable antibody mimetics. In this project, anti-EpCAM DARPins were used in conjunction with microfluidics to determine if they could capture HCT116 cells more effectively than anti-EpCAM antibodies. The inside surfaces of microfluidic chips were modified using N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS) and mercaptopropyltriethoxysilane (MPTES) for anti-EpCAM antibodies, and surface modifications for anti-EpCAM DARPins were made using GMBS and (3-aminopropyl)triethoxysilane (APTES). Both chip types were tested using cancer cells and whole blood mixed with cancer cells. No effective and consistent cell capture was achieved, despite the antibodies and DARPins being shown to be able to bind to the cells directly and tests with fluorescently labelled DARPins and antibodies showing that the surface modification chemistry used was functional. It was concluded that the most likely causes of the failures were surface modifications interfering with the binding ability of the proteins, or improper orientation of the bound proteins. The DARPin remains a protein of interest for microfluidic applications, but further changes and optimisation of the experimental protocol is necessary.