Production of an anti-Aβ antibody fragment in Pichia pastoris and in vitro and in vivo validation of its therapeutic effect

ScFv-h3D6 has been shown as an efficient therapy in the 3xTg-AD mouse model of Alzheimer's Disease. Because one of the major bottlenecks for the therapeutic uses of proteins produced in Escherichia coli is their potential contamination with endotoxins, LPS were extensively removed by a rather low-efficient, expensive, and time-consuming purification step. In addition, disulfide scrambling is favored in the reducing bacterial cytoplasm albeit the use of reductase deficient strains. To overcome these hurdles, as well as to improve the yield, the yeast Pichia pastoris, an endotoxin-free host system for recombinant protein production, has been used to produce scFv-h3D6, both in flask and in a fed-batch bioreactor. Comparison of the thermal stability of the obtained protein with that from E. coli showed no differences. Opposite to the case of the protein obtained from E. coli, no disulfide scrambled conformations or LPS traces were detected in that produced in P. pastoris. Cytotoxicity assays in SH-SY5Y neuroblastoma cell-cultures demonstrated that proteins from both expression systems were similarly efficient in precluding Aβ-induced toxicity. Finally, the 3xTg-AD mouse model was used to test the therapeutic effect of both proteins. Quantification of Aβ levels from cortex and hippocampus protein extracts by ELISA, and Aβ-immunohistochemistry, showed that both proteins reduced Aβ burden. This work demonstrates that scFv-h3D6 obtained from P. pastoris shows the same benefits as those already known for that obtained from E. coli, with multiple advantages in terms of recombinant production and safety

Plasticity in the Oxidative Folding Pathway of the High Affinity Nerita Versicolor Carboxypeptidase Inhibitor (NvCI)

Nerita Versicolor carboxypeptidase inhibitor (NvCI) is the strongest inhibitor reported so far for the M14A subfamily of carboxypeptidases. It comprises 53 residues and a protein fold composed of a two-stranded antiparallel β sheet connected by three loops and stabilized by three disulfide bridges. Here we report the oxidative folding and reductive unfolding pathways of NvCI. Much debate has gone on whether protein conformational folding guides disulfide bond formation or instead they are disulfide bonds that favour the arrangement of local or global structural elements. We show here that for NvCI both possibilities apply. Under physiological conditions, this protein folds trough a funnelled pathway involving a network of kinetically connected native-like intermediates, all sharing the disulfide bond connecting the two β-strands. In contrast, under denaturing conditions, the folding of NvCI is under thermodynamic control and follows a "trial and error" mechanism, in which an initial quasi-stochastic population of intermediates rearrange their disulfide bonds to attain the stable native topology. Despite their striking mechanistic differences, the efficiency of both folding routes is similar. The present study illustrates thus a surprising plasticity in the folding of this extremely stable small disulfide-rich inhibitor and provides the basis for its redesign for biomedical applications

Proteïnes petites i riques en ponts disulfur com a models de producció recombinant i plegament

Consultable des del TDXTítol obtingut de la portada digitalitzadaEl PCI, la hirudina i el LCI formen part de la família de proteïnes petites i riques en ponts disulfur, que conté un gran nombre de biopèptids amb potencials aplicacions biotecnològiques, com són els factors de creixement, les toxines, els inhibidors de proteases o desintegrines. A causa de la presència dels ponts disulfur l'expressió recombinant d'aquestes proteïnes és complexa. En el primer treball de la tesi s'estudien tres sistemes de producció recombinant: un sistema eucariota (Pichia pastoris) i dos sistemes procariotes (Escherichia coli). S'optimitzen diversos paràmetres per tal de millorar el seu nivell de producció i es busquen sistemes que permetin simplificar el seu procés de purificació. S'aconsegueix desenvolupar un sistema en E.coli amb una producció final de proteïna que és 10 cops superior al sistema de partida. En el segon treball s'analitza el paper de les regions precursores del PCI en el plegament de la forma madura. S'analitza el procés de replegament oxidatiu in vitro de les diverses pro-formes del PCI per tal de caracteritzar la seva influència sobre la velocitat o la eficiència del plegament de la forma madura. També s'estudia la influència de la pro-regió N-terminal sobre el procés de plegament in vivo en E.coli del PCI madur i d'una sèrie de mutants que presenten un mal plegament o un baix nivell d'expressió recombinant. Finalment es duu a terme una caracterització estructural de la forma corresponent al PCI amb la pro-regió N-terminal a través d'estudis de bescanvi protó/deuteri, exoproteòlisi limitada, espectroscopia de dicroïsme circular i ressonància magnètica nuclear. En l'últim treball s'analitza el procés de plegament proteic de la hirudina i del LCI a través de l'estudi de les reaccions de bescanvi protó/deuteri seguides per espectrometria de masses MALDI-TOF. Es caracteritza l'adquisició d'estabilitat conformacional al llarg del procés de plegament de les dues proteïnes. S'aïllen una sèrie d'intermediaris de plegament i es determina la seva estabilitat; la qual cosa ens permet estudiar les diferències existents entre les vies de plegament de les dues proteïnes. Finalment s'estima la contribució de les interaccions covalents i no-covalents en l'estabilitat final de la proteïna nativa.PCI, hirudin and LCI belong to the familly of small disulfide-rich proteins, which also contains other biologically important biopeptides like growth factors, desintegrins, toxins or protease inhibitors. These proteins cannot be usually produced at high levels by recombinant DNA approaches due to the presence of the disulfide bridges. To deal with this, we selected three different systems for heterologous expression: one based in external secretion by the yeast Pichia pastoris and two others based in the periplasmic secretion in Escherichia coli, using powerful promoters in all cases. Several parameters were optimized to improve PCI production and to simplify the purification protocol. Finally, a new method was developed, which lead to a final PCI production 10-fold higher than the previously described system. In the second work of the thesis, the role of the PCI pro-regions on the mature peptide folding was analyzed. The in vitro oxidative refolding of mature PCI was compared with that of the pro-forms to characterize their influence on mature PCI folding rates and folding efficiency, using RP-HPLC to analyze the acid-trapped disulfide intermediates collected during the refolding process. The influence of the N-terminal pro-region on the expression or folding of several PCI variants reported to give a low expression yield or a low folding efficiency in vivo in E.coli was also studied. In addition, the presence of ordered structural elements in the N-terminal extension of PCI was investigated by deuteron to proton exchange, limited exoproteolysis, circular dicroism spectroscopy and nuclear magnetic ressonance. In the last work, the folding process of hirudin and LCI was analyzed by deuteron to proton exchange monitored by MALDI-TOF mass spectrometry. The comparison of the number of slow exchanging protons of different samples along the folding pathway provided an easy and fast way to follow the protein folding process. Additionally, the deuteron to proton exchange experiments on purified folding intermediates allowed us to characterize the differences between the two folding pathways and to measure the contribution of the disulfide bonds versus the non-covalent forces to the stability of the native protein

<i>Lactobacillus kefiri</i> shows inter-strain variations in the amino acid sequence of the S-layer proteins

The S-layer is a proteinaceous envelope constituted by subunits that self-assemble to form a two-dimensional lattice that covers the surface of different species of Bacteria and Archaea, and it could be involved in cell recognition of microbes among other several distinct functions. In this work, both proteomic and genomic approaches were used to gain knowledge about the sequences of the S-layer protein (SLPs) encoding genes expressed by six aggregative and sixteen non-aggregative strains of potentially probiotic Lactobacillus kefiri Peptide mass fingerprint (PMF) analysis confirmed the identity of SLPs extracted from L. kefiri, and based on the homology with phylogenetically related species, primers located outside and inside the SLP-genes were employed to amplify genomic DNA. The O-glycosylation site SASSAS was found in all L. kefiri SLPs. Ten strains were selected for sequencing of the complete genes. The total length of the mature proteins varies from 492 to 576 amino acids, and all SLPs have a calculated pI between 9.37 and 9.60. The N-terminal region is relatively conserved and shows a high percentage of positively charged amino acids. Major differences among strains are found in the C-terminal region. Different groups could be distinguished regarding the mature SLPs and the similarities observed in the PMF spectra. Interestingly, SLPs of the aggregative strains are 100% homologous, although these strains were isolated from different kefir grains. This knowledge provides relevant data for better understanding of the mechanisms involved in SLPs functionality and could contribute to the development of products of biotechnological interest from potentially probiotic bacteria.Facultad de Ciencias ExactasInstituto Multidisciplinario de Biología Celula

Lactobacillus kefiri shows inter-strain variations in the amino acid sequence of the S-layer proteins

The S-layer is a proteinaceous envelope constituted by subunits that self-assemble to form a two-dimensional lattice that covers the surface of different species of Bacteria and Archaea, and it could be involved in cell recognition of microbes among other several distinct functions. In this work, both proteomic and genomic approaches were used to gain knowledge about the sequences of the S-layer protein (SLPs) encoding genes expressed by six aggregative and sixteen non-aggregative strains of potentially probiotic Lactobacillus kefiri. Peptide mass fingerprint (PMF) analysis confirmed the identity of SLPs extracted from L. kefiri, and based on the homology with phylogenetically related species, primers located outside and inside the SLP-genes were employed to amplify genomic DNA. The O-glycosylation site SASSAS was found in all L. kefiri SLPs. Ten strains were selected for sequencing of the complete genes. The total length of the mature proteins varies from 492 to 576 amino acids, and all SLPs have a calculated pI between 9.37 and 9.60. The N-terminal region is relatively conserved and shows a high percentage of positively charged amino acids. Major differences among strains are found in the C-terminal region. Different groups could be distinguished regarding the mature SLPs and the similarities observed in the PMF spectra. Interestingly, SLPs of the aggregative strains are 100% homologous, although these strains were isolated from different kefir grains. This knowledge provides relevant data for better understanding of the mechanisms involved in SLPs functionality and could contribute to the development of products of biotechnological interest from potentially probiotic bacteria.Fil: Malamud, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Microbiología General; ArgentinaFil: Carasi, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Microbiología General; ArgentinaFil: Bronsoms, Sílvia. Universitat Autònoma de Barcelona; EspañaFil: Trejo, Sebastian Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina. Universitat Autònoma de Barcelona; EspañaFil: Serradell, María de los Ángeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Microbiología General; Argentina. Universidad Nacional Arturo Jauretche; Argentin

Study of a major intermediate in the oxidative folding of leech carboxypeptidase inhibitor: contribution of the fourth disulfide bond

The oxidative folding pathway of leech carboxypeptidase inhibitor (LCI; four disulfide bonds) proceeds through the formation of two major intermediates (III-A and III-B) that contain three native disulfide bonds and act as strong kinetic traps in the folding process. The III-B intermediate lacks the Cys19–Cys43 disulfide bond that links the β-sheet core with the α-helix in wild-type LCI. Here, an analog of this intermediate was constructed by replacing Cys19 and Cys43 with alanine residues. Its oxidative folding follows a rapid sequential flow through one, two, and three disulfide species to reach the native form; the low accumulation of two disulfide intermediates and three disulfide (scrambled) isomers accounts for a highly efficient reaction. The three-dimensional structure of this analog, alone and in complex with carboxypeptidase A (CPA), was determined by X-ray crystallography at 2.2 Å resolution. Its overall structure is very similar to that of wild-type LCI, although the residues in the region adjacent to the mutation sites show an increased flexibility, which is strongly reduced upon binding to CPA. The structure of the complex also demonstrates that the analog and the wild-type LCI bind to the enzyme in the same manner, as expected by their inhibitory capabilities, which were similar for all enzymes tested. Equilibrium unfolding experiments showed that this mutant is destabilized by ∼1.5 kcal mol−1 (40%) relative to the wild-type protein. Together, the data indicate that the fourth disulfide bond provides LCI with both high stability and structural specificity

Study of a major intermediate in the oxidative folding of leech carboxypeptidase inhibitor: contribution of the fourth disulfide bond

The oxidative folding pathway of leech carboxypeptidase inhibitor (LCI; four disulfide bonds) proceeds through the formation of two major intermediates (III-A and III-B) that contain three native disulfide bonds and act as strong kinetic traps in the folding process. The III-B intermediate lacks the Cys19–Cys43 disulfide bond that links the β-sheet core with the α-helix in wild-type LCI. Here, an analog of this intermediate was constructed by replacing Cys19 and Cys43 with alanine residues. Its oxidative folding follows a rapid sequential flow through one, two, and three disulfide species to reach the native form; the low accumulation of two disulfide intermediates and three disulfide (scrambled) isomers accounts for a highly efficient reaction. The three-dimensional structure of this analog, alone and in complex with carboxypeptidase A (CPA), was determined by X-ray crystallography at 2.2 Å resolution. Its overall structure is very similar to that of wild-type LCI, although the residues in the region adjacent to the mutation sites show an increased flexibility, which is strongly reduced upon binding to CPA. The structure of the complex also demonstrates that the analog and the wild-type LCI bind to the enzyme in the same manner, as expected by their inhibitory capabilities, which were similar for all enzymes tested. Equilibrium unfolding experiments showed that this mutant is destabilized by ∼1.5 kcal mol−1 (40%) relative to the wild-type protein. Together, the data indicate that the fourth disulfide bond provides LCI with both high stability and structural specificity

Profiling and identification of new proteins involved in brain ischemia using MALDI-imaging-mass-spectrometry

The identification of proteins involved in brain ischemia might allow the discovery of putative biomarkers or therapeutic targets for ischemic stroke. Our aim is to study the distribution of proteins within mouse brain after an ischemic insult using MALDI imaging-mass-spectrometry and to identify relevant proteins involved in brain damage. We occluded the middle cerebral artery of C57BL/6J mice. Brain slices were analyzed by MALDI-TOF and infarct (IC) and contralateral (CL) regions were compared using ClinProTools. The ion distribution maps of relevant m/z values were obtained by FlexImagin3.0. Protein identification was conducted through a bottom-up approach consisting on complementary sample fractionation methods. Some identifications were confirmed by immunohistochemistry and western blot. We identified 102 m/z values with different abundances between IC and CL (p 90% between IC and CL. Identification analyses confirmed altered expressions of ATP5i, COX6C and UMP-CMP kinase in IC compared to CL. Biological significance Using MALDI-IMS we identified for the first time new proteins that might be involved in brain ischemia representing potential diagnostic biomarkers or target molecules for neurological recovery.Fil: Llombart, Víctor. Universitat Autònoma de Barcelona; EspañaFil: Trejo, Sebastian Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina. Universitat Autònoma de Barcelona; EspañaFil: Bronsoms, Sílvia. Universitat Autònoma de Barcelona; EspañaFil: Morancho, Anna. Universitat Autònoma de Barcelona; EspañaFil: Feifei, Ma. Universitat Autònoma de Barcelona; EspañaFil: Faura, Júlia. Universitat Autònoma de Barcelona; EspañaFil: García Berrocoso, Teresa. Universitat Autònoma de Barcelona; EspañaFil: Simats, Alba. Universitat Autònoma de Barcelona; EspañaFil: Rosell, Anna. Universitat Autònoma de Barcelona; EspañaFil: Canals, Francesc. Vall d'Hebrón Institute of Oncology; EspañaFil: Hernández Guillamón, Mar. Universitat Autònoma de Barcelona; EspañaFil: Montaner, Joan. Universitat Autònoma de Barcelona; Españ

The adhesive (Figure A) and anti-adhesive (Figure B) abilities of fractions evaluated using the enterotoxigenic <i>E. coli</i> (ETEC) K88.

<p>A) Number of bacteria (log CFU per well) attached to wells coated with the different molecular-weight (MW) fractions obtained from wheat bran (WB) in the <i>in vitro</i> adhesion test (AT). The higher the log CFU counts than those of PBS, the higher the adhesive ability. The samples tested were the WB extract (14 mg/ml), the >300-kDa fraction (2.7 mg/ml) and the <300-kDa fraction (17 mg/ml). B) Number of bacteria (log CFU per well) that attached to IPEC-J2 cells after being co-incubated for 30 min with the different fractions obtained from WB. The lower the log CFU counts than those of PBS, the higher the blocking-adhesion ability. Different letters mean significant differences (P<0.05) between fractions. Data result from the experiments performed in triplicate in two independent assays. Error bars represent the standard error of the mean.</p

Recognition of ETEC K88 to the size-exclusion chromatography (SEC) fractions obtained.

<p>A) SEC of the >300-kDa fraction. Distribution of the eight fractions obtained. B) Number of bacteria (log CFU per well) attached to wells coated with the different fractions in the <i>in vitro</i> adhesion test (AT). The higher the log CFU counts than those of PBS, the higher the adhesive ability. C) Number of bacteria (log CFU per well) that attached to IPEC-J2 cells in the blocking test (BT). The lower the log CFU counts than those of PBS, the higher the blocking-adhesion ability. In Figures B and C, different letters mean significant differences (P<0.05) between fractions. Data result from two independent assays performed in triplicate. Error bars represent the standard error of the mean. D) Dot-blot analysis with purified fimbriae on immobilized WB extract, >300-kDa and <300-kDa fractions, CGMP, BSA, fetuin and the eight fractions (F1 to F8). (i) Binding of purified K88ac fimbriae of ETEC strain FV12408. (ii) Binding of purified K88ac fimbriae of ETEC strain 5/95. E) One-dimension SDS-PAGE of the eight fractions obtained by SEC.</p