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.

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.