Guidelines to reach high-quality purified recombinant proteins

The final goal in recombinant protein production is to obtain high-quality pure protein samples. Indeed, the successful downstream application of a recombinant protein depends on its quality. Besides production, which is conditioned by the host, the quality of a recombinant protein product relies mainly on the purification procedure. Thus, the purification strategy must be carefully designed from the molecular level. On the other hand, the quality control of a protein sample must be performed to ensure its purity, homogeneity, and structural conformity, in order to validate the recombinant production and purification process. Therefore, this review aims at providing succinct information on the rational purification design of recombinant proteins produced in Escherichia coli, specifically the tagging purification, as well as on accessible tools for evaluating and optimizing protein quality. The classical techniques for structural protein characterization - denaturing protein gel electrophoresis (SDS-PAGE), size exclusion chromatography (SEC), dynamic light scattering (DLS), and circular dichroism (CD) - are revisited with focus on the protein, and their main advantages and disadvantages. Furthermore, methods for determining protein concentration and protein storage are also presented. The guidelines compiled herein will aid preparing pure, soluble and homogeneous functional recombinant proteins from the very beginning of the molecular cloning design.This study was funded by the Fundação para a Ciência e a Tecnologia (FCT), Portugal, under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01- 0145-FEDER-006684) and the Post-Doctoral grant SFRH/BPD/ 110640/2015, and by the BioTecNorte operation (NORTE-01-0145- FEDER-000004) supported by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Interferência ambiental do sombreamento da araucária (Araucaria angustifolia L.) na lavoura cafeeira.

O cultivo de café é de fundamental importância para a economia do Brasil, em especial do Estado de Minas Gerais. Entretanto, considerando a crescente demanda por produtos agrícolas, surge a necessidade de se buscarem técnicas de manejo que conciliem produção e conservação ambiental. Neste contexto, este trabalho teve como objetivo avaliar os efeitos de três intensidades de sombreamento do café por araucária (Araucaria angustifolia L.) sobre a qualidade ambiental e sobre a produtividade da lavoura. A área foi dividida em três glebas distintas, considerando-se o grau de sombreamento que já existia naturalmente no local: Café com sombreamento intenso (CSI), Café com sombreamento médio (CSM) e Café a sol pleno (CSP). Foram avaliadas características físicas e químicas do solo, macrofauna e microbiota, fitomassa, folhas e produção de grãos, além de aspectos ambientais como atenuação da radiação e área sombreada. Os dados foram analisados estatisticamente por meio de teste de médias (Duncan a 5%), análise de correlação, boxplot, análise de agrupamentos e análise de componentes principais. Verificou-se que o sombreamento proporcionado pelas araucárias favorece o equilíbrio ambiental da lavoura e em condições de sombra de média intensidade a produção de café/planta se compara àquela obtida a pleno sol

Produção de β-galactosidase por levedura recombinante : desenvolvimento de um sistema de produção estável

A tecnologia do DNA recombinante é uma ferramenta muito utilizada na produção de roteínas em sistemas heterólogos, proteínas essas de elevado valor acrescentado que são, por natureza, produzidas em baixas quantidades. Com este trabalho pretendeu-se construir estirpes floculantes de Saccharomyces cerevisiae que apresentassem uma produção estável de β-galactosidase de Aspergillus niger por integração do gene que codifica para a expressão desta enzima (lacA) nas sequências δ do genoma desta levedura. Foram utilizados dois sistemas de integração distintos. Um deles utiliza como marca de selecção dominante a resistência ao antibiótico G418 e permite a integração de cópias tandem enquanto que o outro sistema utiliza a marca URA3 e a integração é efectuada em diferentes sítios, com uma única cópia em cada sítio. O gene lacA, ladeado pelo promotor e terminador ADH1, foi clonado em ambos os sistemas que foram seguidamente utilizados para transformar as estirpes S. cerevisiae NCYC869-wt (MatαFlo1) e S. cerevisiae NCYC869-A3 (MatαFlo1ura3), respectivamente. Para o primeiro sistema foram testadas concentrações de antibiótico de 0,2 a 1,5 g/l enquanto que para o segundo sistema foram efectuadas 3 rondas de transformação usando sempre a mesma marca de selecção uma vez que o gene URA3 vai sendo eliminado por recombinação das sequências repetidas que o flanqueiam (hisG). Os transformantes foram seleccionados para estudos posteriores pela actividade de β-galactosidase devido à presença de X-gal nas placas selectivas, ou seja, foram seleccionados pela intensidade de cor azul. Ambos os sistemas de transformação permitiram obter transformantes de S. cerevisiae utilizadores de lactose. Independentemente da marca de selecção utilizada, foram observados diferentes níveis de floculação e diferentes níveis de produção de β-galactosidase. Globalmente, os níveis de produção de β-galactosidase foram mais elevados para o primeiro sistema. Os transformantes que susceptibilizaram maior interesse foram caracterizados geneticamente por hibridação Southern. Para o primeiro sistema, foram observados, no máximo, dois sítios de integração usados em simultâneo com cópias tandem e 8 cópias integradas no total, enquanto que para o segundo, após as 3 rondas de transformação e perda do gene URA3, observou-se apenas dois sítios de integração usados em simultâneo correspondentes a 2 cópias integradas. O melhor transformante seleccionado, pertencente ao primeiro sistema (1,5 g/l de G418), apresenta actividade de β-galactosidase em glucose comparável ao sistema de referência, baseado em plasmídeo epissomal (S. cerevisiae NCYC869-A3/pVK1.1), e apresenta estabilidade das inserções após 8 culturas sequenciais com pelo menos 10 gerações cada. Apesar de crescer em lactose como única fonte de carbono e energia, a produção de β-galactosidase, o nível de floculação e a rapidez no consumo de lactose são inferiores à estirpe de referência.Recombinant DNA technology is a widely used tool in the production of proteins in heterologous systems, namely for high value proteins that are, naturally, produced in low quantities. This work aims to construct flocculent Saccharomyces cerevisiae strains with Aspergillus niger β-galactosidase stable production by using the repeated chromosomal δ sequences of the yeast as target sites for the lacA gene (coding for A. niger β-galactosidase) integration. Two different integration systems were used. One uses as dominant selection marker the G418 antibiotic resistance and allows for tandem integrations, while the other uses the URA3 selection marker and integration occurs at different sites with one single copy of the gene at each site. The lacA gene, flanked by ADH1 promotor and terminator, was cloned in both systems that were used in the transformation of S. cerevisiae NCYC869-wt (MatαFlo1) and S. cerevisiae NCYC869-A3 (MatαFlo1ura3) strains, respectively. In the first system different G418 concentrations were used raging from 0.2 to 1.5 g/l while in the other system 3 rounds of transformation using the same selection marker were made, once the URA3 gene is “pooped” out by recombination between flanking direct hisG repeats. In addition, transformants were selected for further studies based on the blue tonality of the colony. Due to the presence of X-gal in selective medium plates, a deeper blue colour of the colony indicated increased β-galactosidase activity. Both integration systems resulted in recombinant S. cerevisiae strains that grow on lactose. Independently of the selection marker used, different flocculation and β-galactosidase expression levels were observed. Overall, transformants obtained from the first system presented higher β-galactosidase extracellular production. The most interesting transformants were characterized genetically by Southern analyses. For the first system, one or two integration sites were observed, with tandem copies and 8 gene copies integrated in maximum, while for the second system, after 3 rounds of transformation and URA3 gene loss, 2 different sites were used for integration, corresponding to 2 gene copies. The best transformant obtained, belonging to the first system (selected with G418 1.5 g/l), has β-galactosidase activity in glucose comparable to the reference epissomal based plasmid strain (S. cerevisiae NCYC869-A3/pVK1.1) and reveals great integration stability after 8 sequential batch cultures with, at least, 10 generations each. Despite of growing on lactose as the only carbon and energy source, β-galactosidase expression level, flocculation level and lactose consumption time were lower than those obtained with the reference strain

Traveling through heritage : Brazilian popular dance to the creative process in contemporary dance

Orientador: Inaicyra Falcão dos SantosAcompanha 01 DVD "Gengibre", 01 caderno "O sol cai", 01 caderno "Rosarina", 01 CD-ROM "Rosarina", 01 envelope de Bibliografias, 01 envelope de agradecimentos, 01 envelope "Ponto de partida" acondicionados em pastaDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de ArtesResumo: Este trabalho tem como foco a pesquisa em danças do Brasil; articulando as matrizes afro-brasileiras do Congado de São José do Triunfo Zona da Mata mineira, MG, para a construção dos processos criativos nas linguagens de performance e em dança contemporânea. Através de imagens, textos e recursos multimídia, a pesquisa visa articular, os contextos do Real, Ritual e Virtual aliando-os aos recursos do processo de criação em dança contemporânea aos elementos técnicos de montagem cênica. Reflete como tais processos criativos envolvendo tradição e tradução, projetam as artes para novos territórios, possíveis platôs para uma sociedade transformadaAbstract: The purpose of this paper is to research Brazilian dances by tracing the African Brazilian origins of the Congado from São José do Triunfo, in the southeast of the State of Minas Gerais, for the construction of creative processes in the performance languages and contemporary dance. Using images, texts, and multimedia resources, the study aims to trace the Real, Ritual, and Virtual contexts by joining the resources of the contemporary dance creative process to the technical elements of staging. It reflects on how these creative processes, involving tradition and translation, push the arts into new territories, possible grounds for a transformed societyMestradoMestre em Arte

TOURAINE, Alain. Um novo paradigma: para compreender o mundo de hoje. Petrópolis: Vozes, 2006. 261 p.

Resenha deTOURAINE, Alain. Um novo paradigma: para compreender o mundo de hoje. Petrópolis: Vozes, 2006. 261 p.

Análise da indicação da cesariana na pespectiva das puérperas e dos critérios clínicos prescritos para sua realização

The cesarean birth was conceived to reduce risks and complications to the pair mother/baby during pregnancy and birth label on situations in which vaginal birth is unfavorable. The goal of this work was to evaluate the indication of cesarean in the perspective of the puerperae and the clinical criteria to its execution. A quantitative study, of transversal cut realized in the Public Maternity Hospital Dona Regina Siqueira Campos, Palmas-TO. The population was composed of 239 puerperae. The data gathering was executed through surveys and documental analysis, through queries on medical records. The puerperae were 26,5±6,53 years old, 98(43%) had concluded high school education and 112(46,9%), were in stable union with a partner. Regarding the obstetric data, 88,5% had 3 previous gestations and 45 of them abortion, they presented 38,5± 3,86 weeks of gestational age, 238 (99,6%) did prenatal, being 210(88,2%) of them accompanied in public health system, they had 9,44±5,24 medical appointments and 33(35,5%) had arterial hypertension and a gestational intercurrence. Concerning the guidance received on the prenatal on giving birth, 55% states not having received orientation, among the puerperae that received guidance, 31(29%), states having received information on the benefits of normal birth, 18(17%), received information on the difference of normal and cesarean birth, 13 (12%) puerperae were informed about the birth signs, other 13(13%) , still on prenatal, were informed that their births would be cesarean and to 07(7%) received information on what activities should be performed in order to facilitate birth. About the cesarean indication register in medical record, it was observed that 3 indications reported were not on agreement with scientific evidence about cesarean indication, being them: women’s lack of cooperation, urinary tract infection and tubectomy. Regarding information received on the motivation that took them to submit themselves to a cesarean, 201 of the interviewed, states having received explanations and that these happened mostly (90%) before birth, the most related were categorized on the followings indications: progression dystonia (34%), arterial hypertension (21%), cephalon pelvic disproportion (21%), fetal suffering (18%), poor fetal positioning (12%) and previous cesarean (7%). About the cesarean indications reported by the puerperae being consistent with the ones registered on medical records, 60 (25%) of the interviews done were not possible to compare the indication register of the medical record to the reports of the puerperae, among the bought interviews, 139 (58%) the information obtained was in accordance to the clinical criteria report, and on 40 (17%) were distinct. Therefore, as cesarean indication in the puerperae perspective is evaluated and the clinical criteria to its execution, it was verified that a great part of the interviews the obtained information was in accordance to the clinical criteria, however in some of them they were distinct, sometimes not being adequate not even to the obstetric record of the patient.O parto cesáreo foi concebido no intuito de reduzir riscos e complicações ao binômio mãe/bebe durante a gravidez e o trabalho de parto em situações que o parto varginal for desfavorável. O objetivo do trabalho foi avaliar a indicação da cesariana na perspectiva das puérperas e os critérios clínicos para a realização da mesma. Estudo quantitativo, de corte transversal realizado no Hospital e Maternidade Publica Dona Regina Siqueira Campos, Palmas-TO. A população foi composta por 239 puérperas. A coleta de dados foi realizada por meio de entrevista e de analise documental, através de consultas ao prontuário. As puérperas tinham 26,5±6,53 anos de idade, 98 (43%) delas o ensino médio completo e 112 (46,9%), união estável. Em relação aos dados obstétricos, 88,5% tiverem ate 3 gestações anteriores e 45 delas aborto, apresentavam 38,5± 3,86 semanas de idade gestacional, 238 (99,6%) realizaram pré-natal, sendo 210 (88,2%) delas acompanhadas em rede pública, tinham 9,44±5,24 número de consultas e 33 (35,5%) tiveram como intercorrência na gestação Hipertensão Arterial. Sobre as orientações recebidas no pré-natal sobre o parto, 55% relataram não ter recebido nenhuma orientação, dentre as puérperas que receberam orientações, 31(29%) delas, relataram ter obtidos informações sobre os benefícios do parto normal, 18 (17%) delas, receberam informações sobre a diferença do parto normal para o parto cesáreo, 13 (12%) puérperas sobre os sinais de parto, outras 13 (13%), ainda no pré-natal, recebeu a definição de que seu parto seria cesáreo e para 07 (7%) delas foram explicado quais atividades deveriam ser realizadas para facilitar o parto. Quanto ao registro da indicação da cesariana em prontuário clínico, observou-se que 3 indicações relatadas não estavam de acordo com as evidencias cientificas sobre a indicação da cesariana, sendo elas: não cooperação da mulher, infecção do trato urinário e laqueadura. Em relação as informações recebidas dos motivos que as levaram a se submeter a uma cesariana, 201 das entrevistadas, afirmaram ter recebido explicações e que essas aconteceram em sua maioria (90%) antes do parto, as mais relatadas foram categorizadas nas seguintes indicações: distócia de progressão (34%), hipertensão arterial (21%), desproporção cefalo-pélvica (21%), sofrimento fetal(18%), má posição fetal (12%) e cesárea previa(7%). Sobre a indicação da cesariana relatada pelas puérperas ser condizente com as registradas nos prontuários clínicos, 60 (25%) das entrevistas realizadas não foram possíveis comparar o registro da indicação do prontuário clinico aos relados pelas puerperais, dentre as entrevistas compradas, 139 (58%) delas, a informação obtida estava de acordo com o critério clinico relato, e em 40 (17%) delas, estavam distintas. Portanto ao avaliar a indicação da cesariana na perspectiva das puérperas e os critérios clínicos para a realização da mesma, verificou-se que grande parte das entrevistas a informação obtida estava de acordo com o critério clinico relato, mas em algumas delas estavam distintas, acontecendo as vezes de não estarem adequados nem ao menos ao histórico obstétrico da paciente

Expression and production of recombinant frutalin in different expression systems and evaluation of its biomedical applications

Este resumo faz parte de: Book of abstracts of the Meeting of the Institute for Biotechnology and Bioengineering, 2, Braga, Portugal, 2010. A versão completa do livro de atas está disponível em: http://hdl.handle.net/1822/10968Frutalin is the alpha-D-galactose-binding lectin expressed in breadfruit seeds (Artocarpus incisa). This lectin may be used in cancer diagnostics/therapeutics due to its potential ability to recognise specific carbohydrates expressed in cancer cells membranes and/or cells surface receptors. However, frutalin extraction from plant seeds is a time-consuming process and typically results in a heterogeneous mixture of different natural isoforms. To overcome these limitations, frutalin was cloned and expressed in Pichia pastoris and Escherichia coli. Recombinant frutalin was detected in cultures of these microorganisms by SDS-PAGE and Western blot analysis. The higher recombinant frutalin yield was obtained in the P. pastoris expression system (up to 20 mg/L) [1]. Molecular and biological differences were found between each recombinant frutalin and native frutalin. Potential biomedical applications for native frutalin and recombinant frutalin produced in P. pastoris were studied. Recombinant frutalin demonstrated higher capacity than native frutalin to differentiate malign from benign human prostate diseases by immunohistochemistry (with a significant positive statistical correlation, P<0.00001), in spite of its lower carbohydrate-binding affinity [2]. In addition, native and recombinant frutalin showed an identical magnitude of cytotoxicity on HeLa cervical cancer cells growth (IC50=100 microgram/mL, 24 h), by inducing cell apoptosis and inhibiting cell proliferation and migration. Interaction studies conducted by confocal microscopy showed that native and recombinant frutalin were internalised and targeted to HeLa cell’s nucleus within 1 h of incubation. Therefore, frutalin with promising application in cancer diagnosis and therapy might be obtained from the recombinant P. pastoris expression system in alternative to its natural source. References [1] Oliveira C, Felix W, Moreira RA, Teixeira JA, Domingues L, “Expression of frutalin, an alpha-D-galactose-binding jacalin related lectin, in the yeast Pichia pastoris”, Prot. Exp. Pur. (2008) 60:188-193. [2] Oliveira C, Teixeira JA, Schmitt F, Domingues L, “A comparative study of recombinant and native frutalin binding to human prostate tissues”, BMC Biotechnol. (2009) 9:78

Study of the effects of High Hydrostatic Pressure (HHP) and Pulsed Light (PL) on BSA structure and hydrolysis

Non-thermal technologies, such as High Hydrostatic Pressure (HHP) and Pulsed light (PL), affect protein inducing modifications in its conformational structure. For this reason the hydrolysis reaction of the protein can be modulated if it is conducted in combination with these technologies which are able to change the availability of peptide bonds exposed to the enzymatic action. The aim is to study the effects of HHP and PL on the structure and the extent of hydrolysis reaction of a target protein: the Bovine Serum Albumin (BSA). BSA protein (5 mg/mL) in sodium phosphate buffer (50 mM, pH = 8) were treated with PL and HHP at different processing conditions, namely pressure level and treatment time in the case of HHP and treatment time and energy input in the case of PL. Structural modification of the protein solutions were analyzed by determining the sulphidrilic groups and the changes of the secondary structure. The effect of the two treatments on the hydrolysis degree (HD) at 37 °C was also evaluated by OPA method. Chymotrypsin and trypsin (E/S ratio = 1/10) were used to hydrolyze the BSA protein solutions. The hydrolysis was carried out in HHP assisted or PL assisted conditions or the protein solutions were treated with HHP or PL processes and immediately after hydrolyzed with the enzymes. Results obtained so far demonstrated that the two technologies tested are able to induce protein modifications and the occurrence and importance of this phenomenon depends on processing parameters causing protein unfolding, namely pressure level and number of pulses. When the maximum protein unfolding is obtained, higher HD values are detected. The highest HD value is obtained in HHP assisted hydrolysis with longer treatment time, and when, before undergoing hydrolysis, the PL treatment is applied to the solution placed at the higher distance from the lamp

Recombinant microbial systems for improved β-galactosidase production and biotechnological applications

β-Galactosidases (EC 3.2.1.23) constitute a large family of proteins that are known to catalyze both hydrolytic and transgalactosylation reactions. The hydrolytic activity has been applied in the food industry for decades for reducing the lactose content in milk, while the transgalactosylation activity has been used to synthesize galacto-oligosaccharides and galactose containing chemicals in recent years. The main focus of this review is on the expression and production of Aspergillus niger, Kluyveromyces lactis and bacterial β-galactosidases in different microbial hosts. Furthermore, emphasis is given on the reported applications of the recombinant enzymes. Current developments on novel β-galactosidases, derived from newly identified microbial sources or by protein engineering means, together with the use of efficient recombinant microbial production systems are converting this enzyme into a relevant synthetic tool. Thermostable β-galactosidases (cold-adapted or thermophilic) in addition to the growing market for functional foods will likely redouble its industrial interest.C. Oliveira and P. M. R. Guimaraes acknowledge support from Fundacao para a Ciencia e a Tecnologia (FCT), Portugal (grants SFRH/BDP/63831/2009 and SFRH/BDP/44328/2008, respectively)