The need to high quality therapeutic proteins has grown significantly in the past 30 years. Recombinant proteins are often produced from vectors inserted into E. coli cell lines for large scale production. However, over-expression of the protein within the cell can lead to the formation of large, insoluble aggregates known as inclusion bodies. Native monomer protein can be isolated from inclusion bodies through a refolding process. This entails disruption of the aggregate structure with high concentrations of denaturant and renaturation in native-promoting solution. Our work characterises protein-protein interactions and aggregation between partially unfolded proteins during the refolding process. The protein-protein interactions are characterized in terms of the osmotic second virial coefficient (B22). A positive value indicates repulsive interactions while a negative value indicates attractive interactions. Measurements are carried out for lysozyme, ribonuclease A and preproinsulin as a function of pH, ionic strength and denaturant concentration, alongside a range of known refolding excipients. Past studies (Ho and Middelberg, 2004; Ho et al., 2003) have shown a link between higher B22 values in denaturant solutions and reduced aggregation during refolding. Our experiments have focused on the effects of urea and GdmHCl upon protein-protein interactions, alongside how ionic strength and refolding additives influence interactions between partially-folded states. At low ionic strength, solutions of urea increase net repulsive interactions compared to GdmHCl solutions through an attenuation of short-range attractive interactions. Electrostatic repulsive interactions are screened in solutions of GdmHCl due to the increased ionic strength of the solution; however short-range attractive interactions are also attenuated in a similar fashion to urea solutions. Protein-protein interactions in low and high concentration denaturant solutions have been shown to be highly sensitive to ionic strength and refolding experiments have shown that this correlates with increased aggregation during refolding. The solubilising additive Arg HCl has been shown to reduce short-range attraction between proteins in urea solutions, while the folding-promotor additives sucrose and hexylene glycol have been shown to have a more complex effect on protein-protein interactions in urea solutions dependent on denaturant concentration. Within the wider context of the field of protein aggregation and refolding, the work conducted here will contribute towards the understanding of how denaturants and solutes influence attractive protein-protein interactions and aggregation behaviour between unfolded or partially folded proteins.

No description available.

Proteins and nanoparticles (NPs) provide a promising platform for supramolecular interaction. We are currently exploring both fundamental and applied aspects of this interaction. On the fundamental side, we have fabricated a series of water-soluble anionic and cationic NPs to interact with cationic and anionic proteins respectively. A Varity of studies such as, activity assay, fluorescence titration, isothermal titration calorimetry etc. were carried out to quantify the binding properties of these functional NPs with those proteins. Those studies reveal the prospect of tuning the affinity between the nanoparticles and proteins by the surface modification. On the application side, we have used this protein-nanoparticle interaction in protein refolding where we successfully refolded the thermally denatured proteins toward its native structure. We have also applied this particle-protein recognition to create a biocompatible protein sensor using a protein-NP conjugate. Green fluorescent protein and a series of cationic NPs were used for a protein sensor for the identification of protein analytes through displacement process. We have extended this application even in sensing the proteins in human serum.

Previous studies in Dr. Yang’s laboratory have established a grafting, design, and subdomain approach in order to investigate the properties behind Ca2+-binding sites located in Ca2+-binding proteins by employing engineered proteins. These approaches have not only enabled us to isolate Ca2+-binding sites and obtain their Ca2+-binding affinities, but also to investigate conformational changes and cooperativity effects upon Ca2+ binding. The focus of my thesis pertains to optimizing the expression and purification of engineered proteins with tailored functions. Proteins were expressed in E. coli using different cell strains, vectors, temperatures, and inducer concentrations. After rigorous expression optimization procedures, proteins were further purified using chromatographic and/or refolding techniques. Expression and purification optimization of proteins is essential for further analyses, since the techniques used for these studies require high protein concentrations and purity. Evaluated proteins had yields between 5-70 mg/L and purities of 80-90% as confirmed by SDS-PAGE electrophoresis.

CD16 is a physiologically essential Fc and #947; receptor III as either a single- pass transmembrane protein (CD16A) or as a glycosylated phosphatidylinositol (GPI) anchored protein (CD16B) on the surface of immune cells that have been implicated in many autoimmune and immune complex-mediated diseases. Its functions include binding and clearing antibody (IgG) coated foreign pathogens, receptor-mediated phagocytosis, and triggering antibody dependent cellular cytotoxicity. It is well established that these functions depend on protein-protein interaction between CD16 and the Fc domain of IgG. However, the molecular details of CD16-IgG interactions are less well defined, but are essential to developing therapeutic compounds to treat many autoimmune and IC diseases. Stable mammalian cell lines expressing wild-type CD16 isoforms and site-specific mutants, including extracellular soluble fragments of CD16 have been established. Soluble forms of wild type CD16A and these CD16 mutants were expressed in a bacterial pathway in order to amass sufficient quantities for x-ray crystallographic studies. The soluble portions of wild-type CD16A and several site-specific CD16A and CD16B mutants were constructed by PCR amplification and ligation with a pET vector. The proteins were expressed in a prokaryotic pathway, BL21 AI, for 8-10 hours and lysed to obtain inclusion bodies. A hand-held sonicator was used to wash the inclusion bodies, while a Urea solution separated and dissolved the proteins. The target proteins were then refolded by rapid dilution, concentrated with a stir cell, and purified. Wild type sCD16A and four site specific mutants were constructed with good sequencing, while wild type sCD16A, sCD16A F176V, and sCD16A G147D were expressed and refolded to optimal levels. X-ray crystallographic data has been collected from sCD16A F176V as a result of these studies and crystals are currently being grown from wild type sCD16A and sCD16A G147D.

El premio Ig-Nobel de Química de este año 2015 fue entregado al profesor Tom Yuan y su equipo de colaboradores por crear una receta para “deshervir un huevo” de forma sencilla. Aunque no lo aparente, este trabajo es de vital importancia, pues ofrece la posibilidad de regresar proteínas desnaturalizadas y agregadas a su estado natural, lo que podría beneficiar a la industria actual evitando la pérdida de miles de millones de dólares. / Professor Tom Yuan and his team were awarded the Ig-Nobel Prize in Chemistry 2015 for creating a recipe to “unboil” an egg. Although it may seem unimportant, this work has crucial importance, as it offers the possibility of refolding denatured proteins and getting them back to its natural state. This research might have an impact on industry, as it could prevent the loss of billions of dollars.

<p>Interstrand cross-linking (ICL) damage to DNA is cytotoxic as it blocks replication and transcription. This cytotoxicity is exploited in anti-cancer therapies, but increased ICL repair limits the efficacy of these chemotherapies. SNM1A (sensitive to nitrogen mustard 1A), of the beta-CASP family of nucleases, has been shown to participate in the initiation of one of the ICL repair processes. Biochemical studies of SNM1A have been limited due to insolubility and instability of SNM1A in bacteria and insect cell lines and toxicity in human cell lines. Work reported in this thesis describes a novel and efficient method of generating active protein from inclusion body expression of the beta-CASP domain of SNM1A. This refolded beta-CASP domain shows 5’ exonuclease activity on single stranded and double stranded DNA in vitro. Nevertheless, this domain alone is unable to complement <em>pso2</em> null ICL repair defects in<em> S. cerevisiae</em> after exposure to ICL agents. These functional studies of the beta-CASP domain of SNM1A will be helpful in directing future research on its role in ICL repair. Additionally, this will aid future structural and inhibitor studies of this essential interstrand cross-link repair protein, SNM1A.</p> / Master of Science (MSc)

The serpin antiplasmin (APL) is the primary inhibitor of plasmin, a proteinase that digests fibrin, the main component of blood clots. Most serpins are serine protease inhibitors, which undergo dramatic conformational change in forming a tight covalent complex with the target protease. Plasmin has been shown to be angiogenic through its protease activity, but it is also angiostatic, being the source of angiostatin, which inhibits angiogenesis. The main objective of our study is to obtain antiplasmin in large amounts, for crystallization and structure determination of APL and of its complex with plasmin, and for solution studies of the complex. Bacterially expressed APL will not be glycosylated, an advantage in crystallization trials.Bacterial expression of rAPL has been problematic. We have found that it can be greatly enhanced through the use of host E.coli cells that carry extra copies of genes for tRNAs coding for rarely used codons in E.coli that occur in high frequency in eukaryotic genes. Several vectors were screened for rAPL expression (pET19b, pET20b and pET28b). rAPL is expressed in high yield from a pET28b construct in host BL-21 RIPL codon plus cells. rAPL thus expressed accumulates as inclusion bodies, but can be solubilized using N-lauroyl sarcosine at pH11. Refolding and purification of rAPL is achieved by using a sizing column followed by a Nickel His-tag affinity column with an imidazole gradient. rAPL fractions thus obtained are stable at 4°C in the presence of EDTA. However, no inhibitor activity of this rAPL towards trypsin was observed, nor did it form inhibition complex with trypsin. The presence of trace protease and/or failure to fold correctly may be preventing recovery of inhibitory activity. A screen of various refolding buffers failed to yield soluble, stable APL.

The contribution of Cys17 and surface-exposed histidine residues in rhG-CSF interaction with Cu(II), Ni(II) and Hg(II) ions chelated by Light Resistant Yellow 2KT-polyethylene glycol derivative was evaluated in aqueous two-phase systems composed of polyethylene glycol (PEG) and dextran. It was determined that His43, His52, His156 and His170 residues are involved in protein interaction with chelated Cu(II) ions. Protein interaction with chelated Ni(II) is governed by His52 and His170 residues, though Cys17 is also involved. The contribution of Cys17 side chain is dominant in the interaction between rhG-CSF and chelated Hg(II) ions. The direct interaction between chelated Hg(II) ions and the –SH group of protein was determined for the first time. Based on the study of the interaction between rhG-CSF and chelated metal ions, rhG-CSF was successfully refolded from inclusion bodies in aqueous two-phase systems PEG-dextran containing chelated Ni(II) or Hg(II) ions for the first time. The refolding of rhG-CSF (C17S) in these systems was more effective compared to that of intact rhG-CSF. The dependence of refolding efficiency of rhG-CSF (C17S) in two-phase systems containing chelated metal ions on the number of histidine mutations was evaluated. It was determined that the refolding efficiency of protein in the systems containing chelated Ni(II) is inversely proportional to the number of histidine mutations. The affinity of purified rhG-CSF (C17S) and its histidine mutants for... [to full text]

Orientador: Ana Carolina de Mattos Zeri / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T00:57:37Z (GMT). No. of bitstreams: 1 Nogueira_MariaLuizaCaldas_M.pdf: 8261820 bytes, checksum: 7c9ed2193fd77623b3a8beea794eb743 (MD5) Previous issue date: 2012 / Resumo: A mTOR é uma via de sinalização muito conservada que controla o crescimento celular em resposta à presença de nutrientes e fatores de crescimento. A desregulação dessa via em humanos está relacionada a doenças como câncer e diabetes. A quinase TOR é ativada na presença de aminoácidos e recentemente descobriu-se que as pequenas GTPases da família Rag são mediadoras da sinalização por Leucina. Essas GTPases são ancoradas na superfície do lisossomo por meio da interação com um complexo de três proteínas denominado Ragulator. Esse complexo também ancora um braço da via das MAPKs (MEK-ERK) aos lisossomos. O entendimento deste complexo pode nos ajudar a elucidar doenças em que a via da mTOR se encontra desregulada. Neste trabalho obtivemos o complexo Ragulator, através da expressão da proteína p18 em corpos de inclusão e sua renaturação através da adição de suas parceiras Mp1/p14 à diálise. Foram realizados estudos biofísicos com a intenção de caracterização do complexo, entretanto o alto grau de dissociação do mesmo resultou em certa dificuldade para caracterizá-lo completamente. Neste trabalho caracterizamos os agregados formados pela p18 e conseguimos reduzir sua formação através de diálise contendo agente redutor e suas proteínas parceiras. A renaturação da p18 na presença de MP1/p14 favoreceu seu rendimento, indicando a interação entre estas proteínas, porém não foi possível estabilizar o complexo Ragulator O estudo da divisão bacteriana é centralmente dependente de FtsZ, um homólogo procariótico das tubulinas. FtsZ desencadeia a divisão ao formar o "anel Z", uma estrutura supramolecular constituída por polímeros de FtsZ que circunda o interior da célula e funciona como arcabouço do aparato de divisão. A formação do anel Z é regulada por moduladores, proteínas que afetam tanto negativamente como positivamente a capacidade de FtsZ polimerizar-se. A proteína MinC é um inibidor da polimerização de FtsZ, recrutada por MinD para a face interna da membrana plasmática, onde o complexo MinCD exerce sua função. MinCD representa um inibidor sítio-específico da polimerização da FtsZ, previnindo a formação do anel Z nos pólos das células mas permitindo que isto aconteça na região central. A elucidação deste processo seria de grande valia para o desenho racional de inibidores da divisão bacteriana. Neste trabalho, comprovamos a interação entre MinC e FtsZ por Ressonância Magnética Nuclear. Estes proteínas não se encontravam em sua forma monomérica e o alto peso molecular do complexo impossibilitou a identificação dos aminoácidos envolvidos nesta interação, devido a limites da técnica 15NHSQC. No momento, a proteína MinC está sendo expressa em presença de deutério, o que aumenta significativamente o limite da técnica de 15NHSQC. Foram realizados ainda estudos biofísicos com intuito de caracterização da interação / Abstract: The mTOR signaling pathway is a very well conserved pathway that controls cell growth in response to the presence of nutrients and growth factors. Deregulation of this pathway in humans is related to diseases like cancer and diabetes. The TOR kinase is activated in the presence of amino acids and it was recently discovered that the Rag small GTPases family are mediators of signaling by Leucine. These GTPases are anchored on the surface of the lysosome through interactions with a complex of three proteins called Ragulator. This complex also anchors an arm of the pathway of MAPKs (MEK-ERK) to lysosomes. Understanding this can help us to elucidate complex diseases in which the mTOR pathway is upregulated. In this work, the Ragulator complex was obtained through the expression of p18 protein in inclusion bodies and their refolding by adding their partners MP1/p14 to dialysis. Biophysical studies were conducted with the intention of characterizing the complex, however its high degree of dissociation resulted in some difficulty to characterize it completely. In this work we characterized the aggregates formed by p18 and managed reduce its formation by dialysis containing reducing agent and its partner proteins. The p18 renatuation with MP1/p14 improve its yield, indicating interaction among these proteins, however the Ragulator complex wasn't stabilized. The study of bacterial division is centrally dependent on FtsZ, a prokaryotic homologue of tubulin. FtsZ triggers the division to form the "Z ring", a supramolecular structure consisting in FtsZ polymers that surrounds the cell and acts as a frame of the division apparatus. The formation of the Z ring is regulated by modulators, proteins that affect both negatively and positively the ability of FtsZ to polymerize. The MinC protein is an inhibitor of FtsZ polymerization, recruits MinD to the inner surface of the plasma membrane, where the complex MinCD exerts its function. MinCD is an inhibitor of site-specific polymerization of FtsZ, preventing the formation of the Z ring at the poles of the cells but allowing this to happen in the central region. The elucidation of this process would be invaluable for the rational design of bacterial division inhibitors. In this work, we confirmed the interaction between MinC and FtsZ by Nuclear Magnetic Resonance. These proteins were not in their monomeric form and the high molecular weight of the complex prevented the identification of the amino acids involved in this interaction, due to limitations of the 15NHSQC technology. At present, the MinC protein is being expressed in the presence of deuterium, which significantly increases the limit of this technique 15NHSQC. Biophysical studies were also performed with the aim of characterizing the interaction / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular