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Selective Control of Protein Kinases and PhosphatasesCamacho-Soto, Karla January 2015 (has links)
The reversible phosphorylation of proteins plays a key role in nearly every aspect of cell life. This essential post-translational modification controls a myriad of cellular events from cell survival, differentiation, and migration to apoptosis. Two classes of enzymes, kinases and phosphatases, tightly control all phosphorylation events. Perturbation in the activity of any member of these classes of enzymes has been linked to numerous diseases including cancer, metabolic disorders, immune disorders and neurological disorders. Therefore, there is a great interest among the scientific community to develop methods to selectively modulate the activity of kinases and phosphatases not only for therapeutic purposes but also to understand the fundamental role of these enzymes in signaling events. The more than 500 kinases encoded in the human genome share a common catalytic fold and most inhibitors target the ATP binding site. Therefore selective targeting of a single kinase by an inhibitor at the highly conserved ATP binding site is one of the main concerns for designing probes or drugs. Our group has taken advantage of the potency and possible selectivity imparted by bivalent inhibitors and developed an in vitro selection approach to discover bivalent ligands. The strategy involves the use of an ATP-competitive small molecule warhead and a library of cyclic peptides displayed on phage that interact with the kinase of interest in a dynamic selection. The selection for a kinase binding peptide is carried out until consensus peptides are found and bivalent ligands are constructed by linking the selected cyclic peptide with the small molecule warhead through a synthetic linker. Using this approach a potent and selective bivalent inhibitor was found for PKA, a serine/threonine kinase. To interrogate the generality of this approach, a kinase closely related to PKA (PRKX) was used for which a very potent and selective bivalent ligand was found. The same selection strategy was further extended to the two kinases Lyn and Brk, which belong to the tyrosine kinase family. Though peptides were isolated that bound the desired kinase, potent bivalent inhibitors were not discovered. More generally, these experiments in sum are building a library of information regarding how to best design selections of potent and selective bivalent inhibitors. We further explored modulation of the activity of kinases and phosphatases by employing a ligand-gated split-protein approach. The small molecule gated spatial and temporal control of these enzymes should allow the study of signaling events in a controlled manner. The strategy employed consists in the identification of possible fragmentation sites within the catalytic domain of kinases and phosphatases by a sequence dissimilarity approach. Loop insertion mutants at the selected sites were tested for catalytic activity. Successful insertion mutants were further split into two catalytically inactive fragments, which were appended to two conditionally interacting protein domains. Upon addition of a small molecule, the two conditionally interacting domains reassemble the catalytic domain of the enzyme and restore catalytic activity. Using this approach we were able to modulate the activity of the tyrosine kinases Lyn, Fak and Src and the AGC kinase PKA. We also extended the approach to gate the activity of tyrosine phosphatases PTP1B, SHP1 and PTPH1. Finally, these ligand-gated split-kinases and phosphatases were validated in-cellulo. Thus, this work resulted in a new method for designing split-proteins and provided a palette of kinases and phosphatases that can be turned-on by small molecules. In total, these efforts describe two alternative routes that can be used to modulate phosphorylation events in a selective and controlled manner.
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Non-Covalent Selection Methodologies Utilizing Phage DisplayMeyer, Scott C. January 2007 (has links)
In nature, non-covalent interactions are as important and dynamic as they are elusive. As such, the study of non-covalent interactions both in vivo and in vitro has proven to be challenging. Given the potential benefits of elucidating protein-protein, ligand-receptor, and other biologically relevant interactions, the development of methodologies for the study of non-covalent interactions is an attractive goal.Biologically encoded protein and peptide libraries that connect the genotypic information with the expressed phenotype have emerged in recent years as powerful methods for studying non-covalent interactions. One of the quintessential platforms for the creation of such libraries is phage display. In phage display, the connection between genetic information and the corresponding protein allows for the iterative isolation and amplification of library members that possess a desired function. Hence, an in vitro selection can be used to isolate epitopes that bind to desired targets or display specific attributes.We have sought to develop novel phage display methodologies that have the potential to expand the scope of this in vitro selection platform. Specifically, we developed a method for the non-covalent attachment of a small molecule ligand to a cyclic peptide library. This system localizes the phage display library to the ligand binding site, thus allowing for the translation of the selected cyclic peptides to a covalently tethered bivalent inhibitor.The first class of biological molecules that we chose to target with our methodology is the biologically and therapeutically important class of enzymes called protein kinases. In the first demonstration of this strategy, we were able to isolate cyclic peptide ligands for the model kinase PKA (cAMP-dependent protein kinase), which were subsequently translated to a bivalent inhibitor. This inhibitor showed both increased affinity and selectivity for PKA in relation to other protein kinases.In a separate project, we sought to develop a method for the isolation of small molecule-responsive mutants of a well-characterized protein scaffold from a phage display library. During these investigations, we discovered interesting homologous single-point mutations of the protein that resulted in large spherical oligomers that may mimic species relevant to the study of protein misfolding diseases such as Alzheimer's.
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A Phage Display System to Profile the DNA-binding Specificities of C2H2 Zinc FingersLam, Kathy 07 January 2011 (has links)
Knowing the sequence specificities of transcription factors allows us to surmise their functions and establish their regulatory roles in genomes. The most common DNA-binding domain among eukaryotic transcription factors is the Cys2His2 zinc finger domain; however, despite their prevalence, the specificities of the majority of Cys2His2 zinc finger proteins remain unknown due to the difficulty in assaying them. My objective was to develop a new phage displayed-based assay, in which individual Cys2His2 domains are displayed on phage in an otherwise constant three-finger protein scaffold. In Chapter 2, I discuss evidence for the modularity of the Cys2His2 domain, since my assay requires that zinc fingers be modular. In Chapter 3, I describe my results on the development of this phage display-based assay. This work provides support for a new strategy to determine the specificities of individual zinc fingers, which can be used to infer specificities for multi-finger Cys2His2 proteins.
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A Phage Display System to Profile the DNA-binding Specificities of C2H2 Zinc FingersLam, Kathy 07 January 2011 (has links)
Knowing the sequence specificities of transcription factors allows us to surmise their functions and establish their regulatory roles in genomes. The most common DNA-binding domain among eukaryotic transcription factors is the Cys2His2 zinc finger domain; however, despite their prevalence, the specificities of the majority of Cys2His2 zinc finger proteins remain unknown due to the difficulty in assaying them. My objective was to develop a new phage displayed-based assay, in which individual Cys2His2 domains are displayed on phage in an otherwise constant three-finger protein scaffold. In Chapter 2, I discuss evidence for the modularity of the Cys2His2 domain, since my assay requires that zinc fingers be modular. In Chapter 3, I describe my results on the development of this phage display-based assay. This work provides support for a new strategy to determine the specificities of individual zinc fingers, which can be used to infer specificities for multi-finger Cys2His2 proteins.
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Seleção e caracterização de peptídeos recombinantes miméticos de antígenos do vírus da dengue por "PHAGE DISPLAY"Santos, Paula de Souza 28 February 2006 (has links)
Dengue fever is caused by an arbovirus of the Flaviridae family, transmitted from
person to another through an intermediate fly vector, Aedes aegypti. It is a tropical
and subtropical infectious disease characterized by fever and strong pain in joints,
which could also lead to bleeding in its hemorrhagic form. In this investigation,
Phage Display technology was used to identify recombinant peptides with affinity
to polyclonal antibodies (IgY) raised in immunized chickens with total proteins of
the DENV-3 culture. Animal sera was purified in a HiTrap column and
concentrated through dialysis. The IgY s were immunoreactive against DENV
cultures, but were not type specific. Phage clones were selected from a random
peptide library (PhD-7) in four cycles of biopanning against IgY. Selected phages
were amplified in deepwell microtiter plates and submitted to ELISA tests.
Immunoreactive clones against IgY were sequenced, translated and analysed
through bioinformatics. Fourteen distinct clones were selected and aligned against
viral proteome sequences and among themselves. Three consensus sequences
among phage clones were detected: VLRN, APP and LPP. The peptide search in
BLASTp showed similarity to the following viral proteins: polyprotein, envelop, and
the nonstructural proteins NS1, NS2a, NS3 and NS5. All of them have matched
with DENV-1, -2, -3, and/or -4 sequences, corroborating with the lack of type
specificity of the raised IgY. Considering the VLRN motif, the analyses of
antigenicity indexes of similar peptides demonstrated that its antigenicity is highly
influenced by neighboring residues. Three-dimensional analysis of the DENV-2
capsid protein, with the alignment of the VLRN motif, have identified two target
sequences, NRVSTVQQL and EIGRMLNILNRR, that are present in the
polyprotein of all four viral types, which may contain the two possible domains
VxRN and LRN, respectively. Six selected phages have presented known protein
domains, and five of them presented specific phosphorylation and glycosylation
sites, similar to known eukaryotic viruses; however, they may not be
physiologically active sites in the dengue virus. Finally, the peptides were used to
detect human IgG and IgM. ELISA tests were performed in two patients with
isolated infections of DENV-1 or DENV-3. The reactivity of the 14 clones was
superior to total antigens obtained from cultures, but they were not type specific. / RESUMO - GERAL
A dengue é uma doença infecciosa febril aguda, transmitida de uma
pessoa doente a uma pessoa sadia pela picada da fêmea contaminada de um
mosquito Aedes aegypti. A doença é causada por um arbovírus, membro da
família Flaviviridae e do gênero flavivirus. Existem quatro tipos de vírus da
dengue: Dengue 1, Dengue 2, Dengue 3 e Dengue 4, e há um grande espectro de
manifestações clínicas da doença, sendo as duas principais, a dengue clássica e
a dengue hemorrágica. O diagnóstico geralmente é baseado em sintomas, e
laboratorialmente é tardio, no entanto vários testes específicos para cada
sorotipo.
A tecnologia do Phage Display (exposição de biomoléculas em fagos) tem
sido amplamente utilizada no mapeamento de epítopos de diversos antígenos,
constituintes de vários agentes causadores de doenças.
Com o objetivo de selecionar peptídeos recombinantes expressos no
capsídeo de bacteriófagos produziu-se soro policlonal em galinhas previamente
sensibilizadas com proteínas totais do vírus da dengue tipo 3. Após 4 ciclos de
seleção de uma biblioteca de fagos com peptídeos recombinantes lineares de 7
resíduos contra a IgY policlonal, 14 fagos imunorreativos foram selecionados e
suas seqüências de ácidos nucléicos foram posteriormente analisadas por
bioinformática. Foi possível identificar domínios protéicos comuns entre os
peptídeos, sendo que o principal domínio mapeado foi VLRN.
Testes subseqüentes se fazem necessários para a melhor caracterização
destes peptídeos, incluindo possíveis aplicações diagnósticas e vacinais dos
peptídeos selecionados.
RESUMO - CAPÍTULO I
A dengue é causada por um arbovírus da família Flaviridae, transmitido de uma
pessoa à outra através de um hospedeiro intermediário, o mosquito Aedes
aegypti. É uma doença infecciosa tropical e subtropical caracterizada por febre e
dor intensa nas articulações, além de sangramentos intensos quando na sua
forma hemorrágica. Neste trabalho utilizou-se da tecnologia de exposição de
peptídeos randômicos em fagos, phage display, no intuito de identificar peptídeos
recombinantes com afinidade a anticorpos policlonais (IgY) gerados em galinhas
imunizadas com proteínas totais do vírus da dengue tipo 3. As IgYs dos animais
foram purificadas em coluna HiTrap e concentradas por diálise. As IgYs foram
imunorreativas contra todas as culturas de DENV, mas não foram tipo específico.
Os clones foram selecionados de uma biblioteca de fagos randômicos (PhD-7) por
quatro ciclos de seleção contra as IgYs. Os fagos selecionados foram
amplificados em placas deepwell e submetidos a testes ELISA. Clones
imunorreativos contra IgY foram seqüenciados, traduzidos e analisados por
bioinformática. Quatorze clones distintos foram selecionados e alinhados contra a
seqüência de dados do proteoma viral e entre todos eles. Três seqüências
consenso entre os clones foram detectadas: VLRN, APP e LPP. A procura por
prováveis seqüências protéicas do vírus da dengue no BLAST mostrou
similaridade a diversos sítios protéicos virais: poliproteína, envelope e as
proteínas não-estruturais NS1, NS2a, NS3 e NS5. Todas elas se alinharam com
as seqüências dos tipos DENV-1, -2, -3, e/ou -4, corroborando com a falta de
especificidade das IgYs. Considerando o motivo VLRN, as análises dos índices de
antigenicidade dos peptídeos similares demonstraram que estes índices são
altamente influenciados pelos resíduos vizinhos. Análise 3-D da proteína do
capsídeo viral do tipo DENV-2, com superposição do motivo VLRN, identificou
duas seqüências alvos, NRVSTVQQL e EIGRMLNILNRR, que estavam presentes
na poliproteína dos quatro tipos virais, os quais podem conter dois prováveis
domínios, VxRN e LRN, respectivamente. Domínios de fosforilação e glicosilação,
encontrados em vírus eucarióticos, foram similares às seqüências presentes em
cinco fagos recombinantes selecionados, o que não implica que estes motivos
sejam fisiologicamente ativos no vírus da dengue. Finalmente, os peptídeos foram
usados para detectar IgG e IgM humana. Testes ELISA foram realizados em dois
pacientes com infecções isoladas de DENV-1 ou DENV-3. A reatividade dos 14
clones foi superior àquela observada para antígenos totais obtidos das culturas,
embora não tenha sido específica. / Mestre em Genética e Bioquímica
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Domain Antibody Fragment Phage Display as a Biomarker Discovery Tool for Traumatic Brain InjuryJanuary 2020 (has links)
abstract: Traumatic brain injury (TBI) affects an estimated 1.7 million people in the United States each year and is a leading cause of death and disability for children and young adults in industrialized countries. Unfortunately, the molecular and cellular mechanisms of injury progression have yet to be fully elucidated. Consequently, this complexity impacts the development of accurate diagnosis and treatment options. Biomarkers, objective signatures of injury, can inform and facilitate development of sensitive and specific theranostic devices. Discovery techniques that take advantage of mining the temporal complexity of TBI are critical for the identification of high specificity biomarkers.
Domain antibody fragment (dAb) phage display, a powerful screening technique to uncover protein-protein interactions, has been applied to biomarker discovery in various cancers and more recently, neurological conditions such as Alzheimer’s Disease and stroke. The small size of dAbs (12-15 kDa) and ability to screen against brain vasculature make them ideal for interacting with the neural milieu in vivo. Despite these characteristics, implementation of dAb phage display to elucidate temporal mechanisms of TBI has yet to reach its full potential.
My dissertation employs a unique target identification pipeline that entails in vivo dAb phage display and next generation sequencing (NGS) analysis to screen for temporal biomarkers of TBI. Using a mouse model of controlled cortical impact (CCI) injury, targeting motifs were designed based on the heavy complementarity determining region (HCDR3) structure of dAbs with preferential binding to acute (1 day) and subacute (7 days) post-injury timepoints. Bioreactivity for these two constructs was validated via immunohistochemistry. Further, immunoprecipitation-mass spectrometry analysis identified temporally distinct candidate biological targets in brain tissue lysate.
The pipeline of phage display followed by NGS analysis demonstrated a unique approach to discover motifs that are sensitive to the heterogeneous and diverse pathology caused by neural injury. This strategy successfully achieves 1) target motif identification for TBI at distinct timepoints and 2) characterization of their spatiotemporal specificity. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2020
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Proteoliposome-based selection of a recombinant antibody fragment against the human M2 muscarinic acetylcholine receptor / ヒトM2ムスカリン性アセチルコリン受容体に対する組換え型抗体フラグメントの効率的選抜法の確立Suharni 23 January 2015 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18675号 / 医博第3947号 / 新制||医||1007(附属図書館) / 31608 / 京都大学大学院医学研究科医学専攻 / (主査)教授 清水 章, 教授 渡邉 大, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
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Purification of Phage-Displayed HSA-Specific Peptide for Biosensor ProductionHuber, Alexander Domenico 05 June 2019 (has links)
No description available.
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Development of a Live Cell Phage Display Screening Protocol:Sisko, Sandra January 2022 (has links)
Thesis advisor: Jianmin Gao / Protein-protein interactions (PPIs) are essential for all biological functions. Developing peptides that disrupt these PPIs is an avid research effort, as peptides possess several advantages over small molecules and monoclonal antibodies. Peptide phage display is a useful tool in identifying peptides for targeting PPIs. This technology displays up to 10^10 unique polypeptides on the surface of bacteriophage, which after several rounds of panning enriches high affinity peptide sequences towards a target protein. Phage display is classically done on immobilized discrete protein; however, we propose to use this technology to identify peptides ligands for overexpressed oncogenic proteins on live cells in-vitro. This is a more accurate representation of the therapeutic target landscape and resembles how the peptide will interact with the receptor in-vivo. Several groups have explored live cell panning, such as Ruoslahti et al. and Cieslewicz et al., and while they demonstrate the capabilities of in-vitro style phage display, there are areas for improvement. We intend to improve on this previous work by 1. Identifying a peptide ligand against specific receptor/protein, and 2. By incorporating the use of covalent phage libraries to elucidate a high affinity binder. This work will be accomplished using the mammalian epidermal oncogenic cell line, A431, that is known to overexpress epidermal growth factor receptor (EGFR). Epidermal growth factor receptor (EGFR) is responsible for cellular proliferation, survival, differentiation and metastasis, which makes it an attractive target to inhibit oncogenic proliferation. Despite successfully marketed monoclonal antibodies and tyrosine kinase inhibitors, EGFR can mutate and develop resistance as diseases progress; this phenomenon, in addition to the benefits of peptides as therapeutics, are driving factors for pursuing this project. Despite our best efforts using non-covalent phage libraries to identify a viable ligand, screening against EGFR extracellular domain (ECD) has proven to be more difficult than anticipated. We hypothesize that non-covalent phage libraries do not possess any sequences with a high enough binding affinity for this protein, and that the use of covalent libraries will be needed to pull out a positive hit. Due to these findings, we have successfully constructed two phage libraries, a ACX7C and a ACX7C-TEV, where the latter introduced a TEV protease cleavage site on the C’-terminal side of the randomized amino acids suitable for covalent warhead modification and screening.
Further, we have begun work on constructing an EGF-displaying phage construct to aid in optimizing a live cell panning protocol. In the future, we plan to evaluate ligand affinity and protein density, as well as determine the optimal covalent warhead/peptide combination for live cell screenings. With this information, we intend to apply this to other oncogenic cell lines, such as MCF-10CA1a, to identify potent peptide ligands for overexpressed oncogenic proteins. / Thesis (MS) — Boston College, 2022. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.
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Discovering Peptide Inhibitors of the Spike Protein and Human ACE2 Receptor Interaction via Competitive Elution in Phage DisplayWei, Nicole January 2023 (has links)
Thesis advisor: Jianman Gao / The interaction between the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the human angiotensin-converting enzyme 2 (hACE2) receptor is an advantageous target for the development of therapies for COVID-19. We used an anti spike receptor binding domain (S RBD) antibody (AM122) to competitively elute phage binding to the S RBD in phage display screening to identify a novel peptide that binds the S protein and hACE2 interaction. We identified a peptide sequence (P1: CPLEYHTC) as a possible hit, and the KD was determined to be 2.667 μM, indicating the potential of this peptide sequence as a therapeutic agent. However, we found no inhibition of the spike protein and hACE2 receptor interaction, suggesting that the peptide may not directly bind to the hACE2 binding site on S RBD. Although further studies are needed, the competitive elution method in phage display screening appears to be an effective method for elucidating onsite peptide sequences that target protein-protein interactions (PPIs). / Thesis (BA) — Boston College, 2023. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Chemistry.
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