Expression of schistosomal cathepsin L1 in Escherichia coli and evaluation of its protective capacity in an animal challenge

Schistosomes use proteinases to accomplish some tasks such as tissue penetration, tissue digestion for nutrition and evasion of host immune responses. The Cathepsin L is a cysteine proteinase of the papain superfamily detected in the gut lumen indicating that this enzyme contributes to the proteolysis of ingested hemoglobin. Due to these roles they play in the schistosome biology, proteolytic enzymes are considered potential targets to develop and direct anti-schistosomal therapies. In this work, the cDNA coding Cathepsin L1 of Schistosoma mansoni was cloned into the pAE vector that provides high-level expression of heterologous proteins in Escherichia coli. The recombinant protein was expressed as inclusion bodies, purified under denaturing conditions through nickel charged chromatography and used for experimental animal vaccination. ELISA was performed with the pooled sera. Although this protein showed to be immunogenic, mice immunized with three doses of recombinant protein plus aluminum hydroxide as adjuvant did not protect against S. mansoni infection.

Valor nutricional do triticale (X Triticosecale Wittimack) para uso como silagem na Zona da Mata de Minas Gerais / Nutritional value of triticale (X Triticosecale Wittimack) for ensiling in Zona da Mata - Minas Gerais State, Brazil

Avaliaram-se, em delineamento inteiramente ao acaso com quatro repetições, os efeitos da idade de corte (83, 90, 97, 104, 111 e 118 dias) do triticale (X Triticosecale Wittimack) sobre o valor nutricional da forragem e sobre os padrões de fermentação e de qualidade da sua silagem. Houve incremento de 0,26 e decréscimo de 0,12 unidades porcentuais, respectivamente, nos teores de matéria seca (MS) e de proteína bruta (PB) da silagem, para cada dia adicional na idade de corte, sendo os maiores teores de MS obtidos de plantas cortadas aos 104 e 118 dias. Foi observado decréscimo de 0,03 unidades no pH das silagens para cada dia adicional na idade de corte do triticale. Os menores valores de pH e a menor taxa de proteólise foram observados de 97 a 118 dias de crescimento das plantas. Os parâmetros de degradação ruminal in situ da MS da forragem verde variaram de 82,2 a 91,7 por cento (degradabilidade potencial), de 1,9 a 3,3 por cento/h (taxa de degradação) e de 40,5 a 52,9 por cento (degradabilidade efetiva, sob taxa de passagem ruminal de 5 por cento/h). Na faixa de idade de corte do triticale de 83 a 118 dias em cultivo de inverno com irrigação por aspersão, na Zona da Mata de Minas Gerais, as silagens obtidas de plantas cortadas aos 104 e 111 dias de crescimento apresentam melhor qualidade de fermentação. A forragem obtida aos 83 dias de crescimento das plantas apresentou bom valor nutricional para ser utilizada sob condição de pastejo ou fornecida verde picada no cocho.

The effects of cutting age of triticale (X Triticosecale Wittimack) on its nutritional value, fermentation patterns, and quality of its silage were evaluated in a completely randomized design with four replications. Cutting ages were 83, 90, 97, 104, 111, and 118 days after sowing. There was an increase of 0.26 and a decrease of 0.12 percent unit on dry matter and crude protein contents, respectively, for each additional day in the cutting age. The highest values of dry matter were observed in plants cut at 104 and 118 days. It was observed a 0.03 percent unit decrease in the silage pH for each additional day in the cutting age. The lowest pH values and the lowest ammoniacal nitrogen concentration were observed from 97 to 118 days. The in situ ruminal degradation parameters of fresh forage ranged from 82.2 to 91.7 percent (potential degradability), from 1.9 to 3.3 percent/h (degradation rate), and from 40.5 to 52.9 percent (effective degradability under 5 percent/h of passage rate). When triticale was growth under irrigation in Zona da Mata during the winter and cut for silage, the best fermentation was observed at the plant ages of 104 and 111 days. At the age of 83 days, the plants showed good nutritional value to be grazed or fed as fresh cut to the animals.

Stability improvement of the fatty acid binding protein Sm14 from S. mansoni by Cys replacement Structural and functional characterization of a vaccine candidate

The Schistosoma mansoni fatty acid binding protein (FABP), Sm14, is a vaccine candidate against, S. mansoni and F. hepatica. Previously, we demonstrated the importance of a correct fold to achieve protection in immunized animals after cercariae challenge [[10]. C.R.R. Ramos, R.C.R. Figueredo, T.A. Pertinhez, M.M. Vilar, A.L.T.O. Nascimento, M. Tendler, I. Raw, A. Spisni, P.L. Ho, Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein: structural, functional and immunoprotection analysis. J. Biol. Chem. 278 (2003) 12745-12751.]. Here we show that the reduction of vaccine efficacy over time is due to protein dimerization and subsequent aggregation. We produced the mutants Sm14-M20(C62S) and Sm14-M20(C62V) that, as expected, did not dimerize in SDS-PAGE. Molecular dynamics calculations and unfolding experiments highlighted a higher structural stability of these mutants with respect to the wild-type. In addition, we found that the mutated proteins, after thermal denaturation, refolded to their active native molecular architecture as proved by the recovery of the fatty acid binding ability. Sm14-M20(C62V) turned out to be the more stable form over time, providing the basis to determine the first 3D solution structure of a Sm14 protein in its apo-form. Overall, Sm14-M20(C62V) possesses an improved structural stability over time, an essential feature to preserve its immunization capability and, in experimentally immunized animals, it exhibits a protection effect against S. mansoni cercariae infections comparable to the one obtained with the wild-type protein. These facts indicate this protein as a good lead molecule for large-scale production and for developing an effective Sm14 based anti-helminthes vaccine.

A mutation in human VAP-B--MSP domain, present in ALS patients, affects the interaction with other cellular proteins.

Amyotrophic Lateral Sclerosis (ALS) is the most common adult-onset Motor Neuron Disease (MND), characterized by motor neurons death in the cortex, brainstem and spinal cord. Ten loci linked to Familial ALS have been mapped. ALS8 is caused by a substitution of a proline by a serine in the Vesicle-Associated Membrane Protein-Associated protein-B/C (VAP-B/C). VAP-B belongs to a highly conserved family of proteins implicated in Endoplasmic Reticulum-Golgi and intra-Golgi transport and microtubules stabilization. Previous studies demonstrated that the P56S mutation disrupts the subcellular localization of VAP-B and that this position would be essential for Unfolded Protein Response (UPR) induced by VAP-B. In the present work we expressed and purified recombinant wild-type and P56S mutant VAP-B-MSP domain for the analysis of its interactions with other cellular proteins. Our findings suggest that the P56S mutation may lead to a less stable interaction of this endoplasmic reticulum protein with at least two other proteins: tubulin and GAPDH. These two proteins have been previously related to other forms of neurodegenerative diseases and are potential key points to understand ALS8 pathogenesis and other forms of MND. Understanding the role of these protein interactions may help the treatment of this devastating disease in the future.

A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide

We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.

A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide.

We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.

Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein Molecular, functional, and immunoprotection analysis

The Schistosoma mansoni Sm14 antigen belongs to the fatty acid-binding protein family and is considered a vaccine candidate against at least two parasite worms, Fasciola hepatica and S. mansoni. Here the genomic sequence and the polymorphism of Sm14 have been characterized for the first time. We found that the conserved methionine at position 20 is polymorphic, being exchangeable with threonine (M20T). To evaluate the function of the amino acid residue at this position, we have also constructed the mutant Sm14-A20 besides the two native isoforms (Sm14-M20 and Sm14-T20). The three purified recombinant His6-tagged Sm14 proteins (rSm14-M20, rSm14-T20, and rSm14-A20) present a predominant â-barrel structure as shown by CD spectroscopy. Thermal and urea unfolding studies evidenced a higher structural stability of rSm14-M20 over the other forms (rSm14M20>rSm14-T20>rSm14-A20). All of the Sm14 proteins were able to bind 11-(dansylamino)undecanoic acid (DAUDA) without substantial difference in the binding affinity. However, rSm14-M20 exhibited a higher affinity for natural fatty acids than the rSm14-T20 and rSm14-A20 proteins as judged by competitive experiments against DAUDA (rSm14-M20>rSm14-T20> rSm14-A20). The rSm14-M20 or rSm14-T20 isoforms but not the rSm14-A20 mutant was able to induce significant protection against S. mansoni cercariae challenge in immunized mice. The level of protection efficacy correlates with the extent of structure stability of the recombinant Sm14 isoforms and mutant.

r-Sm14 - pRSETA efficacy in experimental animals.

Previous studies carried out with Sm14 in experimental vaccination against Schistosoma mansoni or Fasciola hepatica infections were performed with recombinant Sm14 (rSm14) produced in Escherichia coli by the pGEMEX system (Promega). The rSm14 was expressed as a 40 kDa fusion protein with the major bacteriophage T7 capsid protein. Vaccination experiments with this rSm14 in animal models resulted in consistent high protective activity against S. mansoni cercariae challenge and enabled rSm14 to be included among the vaccine antigens endorsed by the World Health Organization for phase I/II clinical trials. Since the preparation of pGEMEX based rSm14 is time consuming and results in low yield for large scale production, we have tested other E. coli expression systems which would be more suitable for scale up and downstream processing. We expressed two different 6XHis-tagged Sm14 fusion proteins in a T7 promoter based plasmids. The 6XHis-tag fusions allowed rapid purification of the recombinant proteins through a Ni+2-charged resin. The resulted recombinant 18 and 16 kDa proteins were recognized by anti-Sm14 antibodies and also by antiserum against adult S. mansoni soluble secreted/excreted proteins in Western-Blot. Both proteins were also protective against S. mansoni cercariae infection to the same extent as the rSm14 expressed by the pGEMEX system.