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.

El receptor Neuroquinin-1 (NK1R) és un GPCR que es troba en el sistema nerviós central i perifèric dels vertebrats i és responsable dels processos fisiològics com la transmissió de dolor, secreció endocrina i exocrina, vasodilatació, modulació de la proliferació cel·lular i molts altres. Els antagonistes del NK1R poden ser potencials analgèsics i antidepressius i també poden ser utilitzats per al tractament del trastorn bipolar, l'alcoholisme, càncer, malalties del sistema immune i algunes infeccions. Les tècniques espectroscòpiques i les estructurals d'alta resolució com NMR i la cristal·lografia requereixen quantitats de l'ordre de mil·ligrams del receptor actiu purificat. Una de les estratègies que permet produir els GPCRs recombinants per estudis estructurals és el sistema d'expressió en E.coli. No obstant, molts GPCRs degut al seu efecte tòxic per a la cèl·lula bacteriana s'expressen en forma dels cossos d'inclusió i han de ser sotmesos al procés de renaturalització. La renaturalització dels GPCRs és una tasca complexa que implica complexos ajustaments dels tampons. La primera part d'aquest treball s'ha centrat en la renaturalització de las formes truncades hNK1R-366 i hNK1R-311 del receptor expressades en cossos d'inclusió d'E.coli. Per a l'obtenció del receptor ben plegat hem establert un original protocol de la renaturalització en columna. Hem utilitzat diferents tècniques espectroscòpiques per a estudiar el receptor renaturalitzat. Els resultats de CD han demostrat que el hNK1R-366 i el hNK1R-311 renaturalitzats en DDM presenten un percentatge d'hèlix-α similar al de la rodopsina extreta de retines bovines i solubilitzada en DDM. En els estudis de fluorescència intrínseca de triptòfans a baixes concentracions del GuHCl hem pogut observar el desplaçament cap el blau en l'espectre d'emissió, típic de triptòfans que es troben en un entorn hidrofòbic. A més a més, els espectres d'emissió del hNK1R-366 expressat en cèl·lules COS-1 i solubilitzat en DDM presenten el màxim d'emissió a 335 nm, molt similar al del receptor renaturalitzat a partir dels cossos d'inclusió, indicant la seva correcte renaturalització. El hNK1R-366, renaturalitzat en tampó fisiològic presenta agregació en 24 hores. No obstant, la presència de 0.05% DDM és capaç d'estabilitzar el receptor. Els assajos de radioligand binding de saturació del hNK1R-366 renaturalitzat indiquen que el receptor actiu constitueix l’1% de la proteïna total de la mostra. No obstant, la unió de la SP al receptor en el rang de nanomols és significatiu i és un resultat important, donat que, per primera vegada s’ha obtingut NK1 funcional a partir de E.coli. Per altre costat, no hem pogut obtenir una corba de saturació del hNK1R-311, degut possiblement al plegament defectuós del receptor per falta dels darrers 96 residus. La segona part d'aquest estudi està centrada en l'expressió, purificació i caracterització estructural del C-terminal del receptor hNK1. El domini C-terminal és important per l'acoblament de la proteïna G i la β-arrestina, i també és essencial per a la dessensibilització, internalització i reciclatge del receptor. No obstant, el paper d'aquest domini ha estat infravalorat pels investigadors durant molt temps i existeix poca informació sobre la seva estructura. Els estudis espectroscòpics de UV i de fluorescència posen de manifest anomalies en l'absorbància a 292 nm i en l'emissió intrínseca de les tirosines a 345 nm, atribuïdes a formes ionitzades de l’aminoàcid, degut a la seva proximitat a grups carboxil de residus glutàmics o aspàrtics. A partir de la predicció de l'estructura secundària i terciària i dels resultats dels estudis espectroscòpics hem proposat un model tridimensional pel C-terminal del hNK1 que conté: 25% d’hèlix-α, 27% d’estructura desordenada i 48% de fulles-β i girs-β. En conjunt, els resultat obtinguts, indiquen que el C-terminal del hNK1R no és un domini desordenat, sinó que té una estructura secundària i terciària clarament definides que poden relacionar amb les seves funcions. / Neurokinin-1 receptor (NK1R) is a GPCR found in the central and peripheral nervous system of vertebrates, responsible for such physiological processes as pain transmission, exocrine and endocrine secretion, vasodilatation, modulation of cell proliferation and many others. NK1R antagonists could be potential analgesics and anti-depressants and may also be used for treatment of bipolar disorder, alcoholism, cancer, immune system diseases and selected infections. Spectroscopic studies and high resolution structural techniques, as NMR and crystallography, require milligram amounts of active purified receptor. One of the strategies to produce recombinant GPCRs for structural studies is an E.coli expression system. However, many GPCRs due to their toxic effect for bacterial host cell are expressed in form of inclusion bodies and require refolding. The refolding of GPCRs is a complicated task that requires screening and adjustment of buffer conditions. The first part of this work was centered on the refolding of hNK1R-366 and hNK1R-311 truncated forms expressed in E.coli inclusion bodies. To obtain properly folded receptor, we established an original on-column refolding protocol. Different spectroscopic techniques were applied to study the refolded receptor. The results obtained from CD measurements showed that hNK1R-366 refolded in DDM presents similar α-helical content as rhodopsin extracted from bovine retinas and solubilized in non-denaturing DDM micelles. In the intrinsic tryptophan fluorescence studies, at low concentrations of GuHCl we observed a blue-shift in the emission spectrum peak, typical for tryptophan in hydrophobic environment. Furthermore, the emission spectra of hNK1R-366 expressed in COS-1 cells and solubilized in DDM micelles show very similar emission maximum around 335 nm to that of the receptor refolded from the inclusion bodies, which may be indicative of proper protein refolding. The refolded in physiological buffer hNK1R-366 was prone to aggregate in about 24 hours, however, the presence of 0.05% DDM was found to stabilize the receptor. Saturation radioligand-binding assays for the refolded hNK1R-366 showed that the amount of the active receptor is about 1% of the total protein the sample. However, the binding of SP to the refolded hNK1R-366 in nanomolar range is significant and can be considered as a promising result, since until now the intents to produce any detectable amounts of functional NK1 receptor in E. coli were unsuccessful. On the other hand, we were unable to get any saturation binding curve for hNK1R-311 truncated form of the receptor, which could be explained by incorrect folding caused by the lack of 96 residues of the C-terminus of the receptor. The second part of the present study is centered on hNK1R C-terminus expression, purification and characterization to elucidate its structural properties. The C-terminus domain seems to be essential for the coupling to corresponding G protein and β-arrestin, and is essential for receptor desensitization, internalization and recycling. However, the role of this domain was underestimated by researchers for a long time and as a result very little information is known about its structure. UV and fluorescence spectroscopic studies revealed abnormal tyrosine red-shifted absorbance band at 292 nm and intrinsic tyrosine emission at 345 nm which could be attributed to ionized form of tyrosine and possibly arises from the proximity of one or more tyrosines to carboxyl groups of glutamic o aspartic residues. Based on secondary and tertiary structure prediction as well as on the results of spectroscopic studies we propose a 3D-model for hNK1R C-terminus. Th following assignment of the secondary structure was made: 25% α-helix, 27% unordered structure, 48% β-sheets and β-turns. The obtained ressults give evidence that hNK1R C-terminus is not an unordered region but has clearly defined secondary and tertiary structures which certainly are tightly related to its multiple functions.

Two cysteine and two serine protease inhibitors from a tick Ixodes ricinus saliva were overexpressed using a prokaryotic overexpression system and refolded to their native state. Both cysteine protease inhibitors were tested as potential antigens for an anti-tick vaccine showing no effect on tick feeding or reproduction. Various immunological methods were employed to test the potential immunomodulatory function of these proteins without success.

No description available.

Colorado potato beetle (Leptinotarsa decemlineata) is an economically important herbivorous pest. Cathepsin D-like aspartic peptidase (LdCD) plays an important role during protein degradation in the midgut of Colorado potato beetle. This work describes the preparation of two expression systems, namely in Escherichia coli and Pichia pastoris, for the production of recombinant LdCD. The protocol for refolding of denatured LdCD was designed and optimized. Activation of the inactive LdCD zymogen and cleavage of the propetide (activation peptide) were investigated. This process proceeds autocatalytically at acidic pH or with the assistance of the cysteine peptidase legumain. The proteolytic activity of LdCD was characterized using fluorogenic peptidic substrate and protein substrates, and kinetic parameters and pH optimum were determined. The inhibition specificity of LdCD was analyzed using a panel of peptidase inhibitors. LdCD was significantly inhibited by PDI (potato cathepsin D inhibitor), a protein inhibitor produced in potato leaves. This suggests that PDI is a natural defense protein, which is directed against LdCD in the midgut of Colorado potato beetle in order to block the digestion. The potential application of PDI in the construction of transgenic crops resistant against insects is discussed.

Chez les Eucaryotes, l’étape d’initiation est de loin la plus complexe et la plus lente du processus de traduction. Elle nécessite l’intervention de 12 facteurs protéiques, d’une coiffe m7GpppN située à l’extrémité 5’ des ARNm et d’une queue poly(A) en 3’. Les ARNm des histones « réplication-dépendantes » sont particuliers car dépourvus d’extrémité 3’ polyadénylée et dotés d’une extrémité 5’ non traduite extrêmement courte, de 9 nt seulement chez l’ARNm H4 de la souris. Pour traduire ces ARNm, un processus d’initiation non conventionnel a été décrit au laboratoire. L’objectif de ma thèse a été d’établir les bases structurales de ce mécanisme en combinant différentes approches expérimentales. Deux protocoles originaux de repliement ont été mis au point afin d’isoler l’ARNm H4 dans deux conformations distinctes et stables. Une caractérisation fonctionnelle et structurale de ces deux formes de l’ARNm a ensuite été réalisée. La stabilité et la structure de ces deux formes ont été étudiées par DLS, par SAXS et par équilibre de sédimentation. Puis, nous avons étudié la capacité de ces deux formes d’ARNm H4 à fixer le facteur d’initiation eIF4E et les ribosomes assemblés sur le codon d’initiation ainsi que leur aptitude à être traduits in vitro. Un modèle de repliement de la structure secondaire de l’ARNm H4 a été construit après sondage enzymatique et chimique des deux formes de l’ARNm. Ce modèle a servi de base pour le travail d’ingénierie de l’ARNm H4 qui a conduit à son découpage en sous-domaines. Des essais de cristallisation ont porté sur 18 de ces fragments ainsi que sur les deux formes de l’ARNm H4 complet. / In eukaryotes, the initiation step is far more complex and the slowest inside the translation process. It requires the intervention of 12 protein factors, an m7GpppN cap located to the 5 'end of the mRNA and a poly (A) tail at the 3'. Histone mRNAs "replication-dependent" are specific because they lack of polyadenylated tail at the 3’ end and have a 5' end untranslated extremely short (9 nt only in the mouse). To translate these mRNAs, a process of unconventional initiation has been described in the laboratory. The aim of my thesis was to establish the structural basis of the mechanism by combining different experimental approaches. Two original refolding protocols have been developed to isolate mRNA H4 in two distinct and stable conformations. A functional and structural characterization of these two shapes of H4 mRNA was then performed. The stability and the structure of these two shapes have been studied by DLS, SAXS and sedimentation equilibrium. Then, we studied the ability of these two conformations of H4 mRNA to bind the eIF4E initiation factor and ribosomes assembled on the start codon as well as their ability to be translated in vitro. Models of the secondary structure has been constructed after enzymatic and chemical probing of the two shapes of the mRNA. This model was the basis for the engineering of the H4 mRNA that led to its division into sub-domains. Crystallization trials focused on 18 of these fragments as well as on both H4 mRNA shapes.

Magnetic Resonance Imaging instruments rely on a contrast agent to provide high-resolution images of tissues in vivo. However, current clinical contrast agents are hindered by low relaxivity and fast correlation time, necessitating high injection dosages. These concerns, among others, have driven the development of a class of protein-based contrast agents (ProCAs), by design of lanthanide binding sites into a scaffold protein. ProCA1 has a higher reported relaxivity and dosage efficiency than current contrast agents. In this study, expression and Glutathione-S-Transferase purification procedures were optimized, and a refolding method for rapid production of ProCA1 has been developed to enable studies of conformation, metal binding, relaxivity, and in vivo applications. Several ProCA1 variants with 4-5 charged ligand residues were shown to have strong gadolinium binding affinity (Kd of 10-12 M) and metal selectivity. Several options to improve ProCA1 have been explored, including addition of a polyethylene chain or a bombesin tag.

Developmentally regulated G-proteins (DRGs) are a highly conserved family of GTP binding proteins found in archaea, plants, fungi and animals. Their function is poorly understood but they are implicated in cell division, proliferation, and growth, as well as several human medical conditions. The research reported here has utilised a variety of approaches including structural biology, biochemistry, expression profiling, and mutant analysis in order to investigate the cellular function of DRG proteins in plants. Recombinant, biologically active atDRG1 and atDRG2 protein from Arabidopsis thaliana was purified using in vitro refolding and was used in both structural studies and biochemical analysis. Crystallographic studies were carried out for both atDRG1 and atDRG2 across 3840 unique, independent crystallisation conditions for each protein. Heterogeneous nucleation was also used in a separate crystallography screen in order to induce nucleation and subsequent crystal growth however no diffraction quality protein crystal were produced in this study. The nucleotide binding and hydrolysis properties of recombinant atDRG1 and atDRG2 were measured in vitro, representing the first biochemical characterisation of DRG proteins. Both atDRG1 and atDRG2 were found to bind GDP and GTP in vitro without the assistance of exogenous exchange or activation factors. The Kcat for GTP hydrolysis by atDRG1 and atDRG2 was found to be 7.44 x 10-4 min-1 and 1.18 x 10-3 min-1 respectively which is consistent with proteins related to the DRG subfamily. An Arabidopsis thaliana atDRG2a knockout mutant was identified and characterised in this study as well representing the first DRG knockout mutant in a multicellular organism. We found that complete knockout of atDRG2a is not lethal in Arabidopsis and that the nearly identical atDRG2b protein is not upregulated in response to an absence of atDRG2a in the cell. The mutant did not display an obvious phenotype compared to wild-type. The expression profiles of the three Arabidopsis thaliana drg genes, drg1, drg2a, and drg2b, were characterised using drg promoter:GUS Arabidopsis transgenics and revealed several interesting features. Under normal conditions, drg1 and drg2a transcripts are present in all cells whilst drg2b transcripts are undetectable. When heat stress is applied, drg2b and drg1 are specifically up regulated and drg2a is not. During seed imbibition, drg2a and drg1 are specifically upregulated whilst drg2b is not. The expression pattern of the drg family closely mirrors that of chaperone/heat shock proteins and this would agree with previous research that suggests that DRG2a may perform a chaperone role. The ability of DRGs to bind nucleotides without assistance, their slow rate of GTP hydrolysis, heat stress activation, abundance in seeds, cytosolic localization, and domain conservation, all agree with the models proposed for spoOB associated G-protein (Obg) function, whereby Obgs stabilise or refold ribosomes or other proteins in response to stress. It is possible that DRGs perform a similar and complementary function to Obgs, specifically during heat stress, despite the low level of sequence conservation between Obgs and DRGs.

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.