A esquistossomose é a mais importante das doenças helmínticas humanas em termos de morbidez e mortalidade. A proteína Sm14 de Schistosoma mansoni, que pertence à família de proteínas ligadoras de ácidos graxos (fatty acid-binding proteins, FABPs) (Moser et al., 1991), mostrou um bom nível de proteção (65%) contra a esquistossomose em animais experimentais (Tendler et al., 1996). No presente trabalho foram desenvolvidos sistemas de expressão que possibilitará a produção da proteína Sm14 em larga escala em E.coli. Com o intuito de conhecer a estrutura do gene da proteína Sm14, foi clonado um fragmento de DNA genômico de S. mansoni que contém a seqüência codificante da proteína Sm14. Como os outros membros da família gênica das FABP, o gene para a proteína Sm14 contém quatro "exons" separados por três "introns" de 674, 585 e 42 bp. Esta é a primeira descrição da estrutura gênica de um membro das FABP correspondente a um helminto. A Sm14 é uma proteína que pode ser potencialmente usada como vacina. Estudamos a existência de polimorfismo em duas linhagens de S. mansoni endêmicas do Brasil: LE e BH. Para a análise de polimorfismo, a ORF correspondente à proteína Sm14 foi amplificada por RT-PCR do RNA total de vermes adultos de S. mansoni. Os produtos de amplificação independentes foram clonados no vetor pGEM-T e seqüenciados. As análises de seqüências mostraram duas isoformas principais para a proteína Sm14: Sm14-M20...
A neurocisticercose (NC) é uma doença provocada por larvas de Taenia solium (Tso) no sistema nervoso central. Seu diagnóstico fundamenta-se em critérios clínicos, epidemiológicos e laboratoriais. A utilização de antígenos parasitários no imunodiagnóstico apresenta desvantagens como: necessidade de animais, ausência de homogeneidade entre lotes, baixo rendimento, e contaminação com proteínas suínas. Assim, os antígenos recombinantes podem otimizar o imunodiagnóstico da NC, pois são reagentes simples e reprodutíveis, sem requerer animais. Este estudo teve como objetivo a obtenção, caracterização e análise da reatividade de proteína recombinante baseada em antígenos de líquido vesicular de Taenia crassiceps (Tcra). Assim, o cDNA foi obtido por amplificação a partir de RNAm de cisticercos de Tcra. A proteína recombinante Tc14 foi produzida em Escherichia coli (DE3) BL21 utilizando-se o vetor de expressão pET-22b e purificada por cromatografia de afinidade. A caracterização antigênica deu-se por Imunoblot (IB) utilizando anticorpos monoclonais (AcMo). Houve reatividade com todos os AcMo utilizados (AcMo anti-antígeno de excreção/secreção de Tcra, AcMo anti-líquido vesicular de Tcra, AcMo antilíquido vesicular de Tso e AcMo anti-antígeno total de Tso)...
Visando novos estudos com a proteína amilóide sérica A (SAA), propusemos a produção de seu recombinante humano em Escherichia coli, mais especificamente, a isoforma encontrada na fase aguda (A-SAA1) e da constitutivamente expressa (C-SAA4). Realizamos a expressão, identificação e purificação das proteínas recombinantes. Concomitantemente, também avaliamos o efeito da hipóxia na expressão e produção da proteína SAA nativa em linhagens de pré-adipócitos murinos 3T3-L1, não diferenciados e diferenciados e adipócitos humanos. Aparentemente quanto maior o grau de diferenciação celular, maior a expressão e produção da proteína. Para os adipócitos humanos, o perfil de expressão de mRNA da SAA mostra que SAA1>SAA2>SAA4 nas relações 500:150:1. Na hipóxia, há um aumento na expressão de SAA, entretanto não associamos esta expressão a um aumento da concentração da proteína. A importância do reconhecimento de que SAA pode ser umas das proteínas induzidas pela hipóxia em adipócitos é discutida em relação ao seu papel pró-inflamatório.; In order to provide more complex studies with the protein serum amyloid A (SAA), this study proposed the production of its human recombinant in bacteria (Escherichia coli)...
A papilomatose bovina é uma doença infectocontagiosa de ocorrência mundial, que assola o rebanho brasileiro, sem qualquer atitude efetiva de controle, e que tem como enfermidades associadas a tumores de bexiga hematúria enzoótica e tumores de trato digestório superior caraguatá, responsáveis por sensíveis perdas para a pecuária. Várias tentativas vacinais têm sido empreendidas com finalidades profiláticas ou terapêuticas, porém sem resultados eficazes. Esta situação se deve a aspectos relacionados à estrutura viral que dificultam uma manipulação eficiente para produção de produtos vacinais. Para que tais dados possam ser obtidos, faz-se necessário uma melhor compreensão da ação das proteínas recombinantes. A clonagem em vetores bacterianos para a expressão e purificação dessas proteínas serve a diferentes propósitos. Entre eles, a produção de insumos imunológicos, como testes diagnósticos ou mesmo vacinas. O presente projeto visou à expressão e purificação da proteína de capsídeo recombinante L2 de BPV-2. A proteína foi expressa em bactéria e tentou-se a purificação por coluna de afinidade; entretanto, problemas na purificação não possibilitaram a conclusão deste objetivo. Todas as abordagens e protocolos utilizados nesse trabalho são discutidos para contribuição ao conhecimento do processo.; The bovine papillomatosis is an infectious disease of worldwide occurrence...
Proteins production requires a successful correlation between expression, solubility and
purification steps. As an expression system, Escherichia coli combines its low cost and ease of use
with rapid expression, being widely used for heterologous protein production. This host cell has
however several drawbacks, namely at the expression of insoluble proteins aggregated into
inclusion bodies. Many efforts have been made to overcome such problems, including the
optimization of expression conditions and the use of solubility fusion tags. The use of fusion tags
for protein production remains challenging since none of the available fusion systems work
universally with every partner protein. A novel fusion system had been recently discovered and
submitted to a patenting process by Hitag Biotechnology, Lda. This fusion system consists of low
molecular weight peptides/proteins from recombinant antigens, which have demonstrated to
increase soluble protein expression levels in E. coli.
This work aims at the evaluation of the effects of two novel fusion tags on soluble protein
expression in E. coli. Specific primers were designed to amplify and sub?clone gene sequences
that encodes for frutalin, Cryptosporidium parvum 12kDa protein and Giardia lamblia cyst wall
protein. These target proteins present therapeutic and diagnostic interests and had shown to be
difficult?to?express in E. coli. Proteins were first fused to novel tags and than expressed in E. coli.
Proteins purification was carried out by affinity chromatography...
Cell-to-cell communication is required for many biological processes in development and adult life. One of the most common systems utilized by a wide range of eukaryotes is the Notch signalling pathway. Four Notch receptors and five ligands have been identified in mammals that interact via their extracellular domains leading to transcription activation. Studies have shown that the Notch ligands expression is undetectable in normal breast tissues, but moderate to high expression has been detected in breast cancer. Thus, any of the Notch1 ligands can be studied as possible therapeutic targets for breast cancer.
To study Notch pathway proteins there is the need to obtain stable protein solutions. E. coli is the host of excellence for recombinant proteins for the ease of use, fast growth and high cell densities. However, the expression of mammalian proteins in such systems may overwhelm the bacterial cellular machinery, which does not possess the ability for post-translational modifications, or dedicated compartments for protein synthesis. Mammalian cells are therefore preferred, despite their technical and financial increased demands.
We aim to determine the best expression and purification conditions for the different ligand protein constructs...
Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein-1 min-1). This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa)...
With the advent of recombinant DNA technology, recombinant protein expression has become an important tool in the study of the structure, function and identification of new proteins, especially those with therapeutic functions. Escherichia coli has been the predominant prokaryote used in genetic engineering studies due to the abundance of information about its metabolism. Despite significant advances in molecular biology and immunology of infections, there are as yet no prophylactic drugs capable of preventing visceral leishmaniasis. It is therefore important to identify specific antigens in order to develop vaccines and diagnostic kits against this disease. The objective of this study was to evaluate the influence of culture medium on the production of eIF antigen from Leishmania chagasi in recombinant Escherichia coli. An induction procedure using IPTG was carried out in a series of trials, to observe the influence of culture medium (2xTY, TB) under expression of the recombinant eIF protein. Results showed that recombinant protein expression was associated to growth and that the highest eIF antigen expression was obtained in the 2xTY medium.
ColE1-like plasmids constitute the most popular vectors for recombinant protein expression. ColE1 plasmid replication is tightly controlled by an antisense RNA mechanism that is highly dynamic, tuning plasmid metabolic burden to the physiological state of the host. Plasmid homeostasis is upset upon induction of recombinant protein expression because of non-physiological levels of expression and because of the frequently biased amino acid composition of recombinant proteins. Disregulation of plasmid replication is the main cause of collapse of plasmid-based expression systems because of a simultaneous increase in the metabolic burden (due to increased average copy number) and in the probability of generation of plasmid-free cells (due to increased copy number variation). Interference between regulatory elements of co-resident plasmids causes comparable effects on plasmid stability (plasmid incompatibility). Modulating plasmid copy number for recombinant gene expression aims at achieving a high gene dosage while preserving the stability of the expression system. Here I present strategies targeting plasmid replication for optimizing recombinant gene expression. Specifically, I review approaches aimed at modulating the antisense regulatory system (as well as their implications for plasmid incompatibility) and innovative strategies involving modulation of host factors...
Factors affecting protein expression have been intensely studied to the benefit of recombinant protein production. Through mutational analysis at the +2 amino acid position of recombinant Ig?, we examined the effect of all 20 amino acids on protein expression. The results showed that amino acids at the +2 position were affected 10-fold in the recombinant protein expression. Specifically, Ala, Cys, Pro, Ser, Thr, and Lys at the +2 position resulted in significantly higher expression of recombinant Ig? than other amino acids, while Met, His and Glu resulted in greatly reduced protein expression. This expression difference depended on the amino acid instead of their codon usages. Consistent with the mutational results, a statistically significant enrichment in Ala and Ser at the +2 position was observed among highly expressed E. coli genes. This work suggests a general approach to enhance protein expression by incorporating an Ala or Ser after the initiation codon.
We describe here a unique ethanol-inducible process for expression of recombinant proteins in transgenic plants. The process is based on inducible release of viral RNA replicons from stably integrated DNA proreplicons. A simple treatment with ethanol releases the replicon leading to RNA amplification and high-level protein production. To achieve tight control of replicon activation and spread in the uninduced state, the viral vector has been deconstructed, and its two components, the replicon and the cell-to-cell movement protein, have each been placed separately under the control of an inducible promoter. Transgenic Nicotiana benthamiana plants incorporating this double-inducible system demonstrate negligible background expression, high (over 0.5 × 104-fold) induction multiples, and high absolute levels of protein expression upon induction (up to 4.3 mg/g fresh biomass). The process can be easily scaled up, supports expression of practically important recombinant proteins, and thus can be directly used for industrial manufacturing.
Despite major progresses in genetic studies of hyperthermophilic archaea, recombinant protein production in these organisms always suffers from low yields and a robust expression system is still in great demand. Here we report a versatile vector that confers high levels of protein expression in Sulfolobus islandicus, a hyperthermophilic crenarchaeon. Two expression vectors, pSeSD and pEXA, harboring 11 unique restriction sites were constructed. They contain coding sequences of two hexahistidine (6×His) peptide tags and those coding for two protease sites, the latter of which make it possible to remove the peptide tags from expressed recombinant proteins. While pEXA employed an araS promoter for protein expression, pSeSD utilized ParaS-SD, an araS derivative promoter carrying an engineered ribosome-binding site (RBS; a Shine-Dalgarno [SD] sequence). We found that ParaS-SD directed high levels of target gene expression. More strikingly, N-terminal amino acid sequencing of recombinant proteins unraveled that the protein synthesized from pEXA-N-lacS lacked the designed 6×His tag and that translation initiation did not start at the ATG codon of the fusion gene. Instead, it started at multiple sites downstream of the 6×His codons. Intriguingly...
We report here the successful scale up of transient recombinant protein expression to litre scale using Semliki Forest Virus System. The expression of bacterial ?-galactosidase was initially compared in BHK and CHO cells and the conditions for optimal infection of BHK cells were identified. 10% FCS in a medium at pH 6.9 and infection in small volumes were found to be optimal. A high MOI results in an increased recombinant protein yield. Stirring does not affect the infection process. Finally we applied these optimal conditions to the production of a microsomal enzyme, human cyclooxygenase-2 in suspension spinners. Five independant productions at the 1 litre scale yielded reproducible substantial amounts of recombinant protein (16 mg microsomal protein 109 cells?1) with an average specific activity of 3942 ± 765 pg PGE2 ?g?1 microsomal protein 5 min?1.
The firefly luciferase gene was introduced into the Semliki Forest virus (SFV) vector and high titer recombinant SFV particles generated. The broad host range of SFV allowed efficient infection and high level expression of four mammalian cell lines growing in serum-free suspension cultures. The incubation temperature had dramatic effects on the level and duration of recombinant protein expression. For example, the luciferase activity was significantly higher in the rodent BHK and CHO cell lines incubated at 33 °C compared to 37 °C when harvested 19 h post-infection. At 33 °C the specific expression levels increased 10–20 fold during prolongation of the post-infection time up to 50 h. In contrast, a significant decrease in luciferase activity was observed from 26 h post-infection for cell cultures incubated at 37 °C. Only a slight temperature effect on luciferase expression was seen in the human cell line HEK293 and no effect was observed for the subclone293(EBNA).
As more genes are being identified through genomic techniques,the need to rapidly express recombinant proteins for functionalstudies has become increasingly acute. Transient expression ofrecombinant protein using COS-1, CV-1 and 293 cells is widelyused to address this need. To improve the robustness of hostcells for transient expression, the effect of over-expression ofProtein Kinase B? has been explored. In this report wedemonstrate that over-expression of Protein Kinase B? canimprove transient recombinant protein expression 40% to >200%depending on the protein being expressed and the cell line used.
The recombinant expression of proteins has been the method of choice to meet the demands from proteomics and structural genomics studies. Despite its successful production of many heterologous proteins, Escherichia coli failed to produce many other proteins in their native forms. This may be related to the fact that the stresses resulting from the overproduction interfere with cellular processes. To better understand the physiological change during the overproduction phase, we profiled the metabolites along the time course of the recombinant protein expression. We identified 32 metabolites collected from different time points in the protein production phase. The stress induced by protein production can be characterized by (A) the increased usage of aspartic acid, choline, glycerol, and N-acetyllysine; and (B) the accumulation of adenosine, alanine, oxidized glutathione, glycine, N-acetylputrescine, and uracil. We envision that this work can be used to create a strategy for the production of usable proteins in large quantities.
Purpose of work: A comparative analysis of new and established insect cell lines, in regard to process relevant parameters, provide data that can be exploited for designing more robust and effective protein production processes. The baculovirus-insect cell expression system has been efficiently used for the production of heterologous proteins. Three different insect cell lines Tnao38, High Five and Sf9 were compared in terms of virus susceptibility, baculovirus production and product yield of an intra-cellularly (YFP) and extra-cellularly (influenza A virus hemagglutinin)-expressed recombinant protein. The Tnao38 and High Five cell lines exhibited higher (tenfold) susceptibility to baculovirus infection than Sf9 cells, whereas Sf9 cells showed a higher (100-fold) capacity for production of infectious virus particles. Analysis of recombinant protein expression revealed considerably higher product yields in Tnao38 and High Five cells as compared to Sf9 cells, for both model proteins. Overall, the two Trichoplusia ni-derived cell lines, High Five and Tnao38, were significantly more efficient in terms of secreting proteins such as the glycoprotein hemagglutinin of influenza A virus.
A metabolic engineering perspective which views recombinant protein expression as a multistep pathway allows us to move beyond vector design and identify the downstream rate limiting steps in expression. In E.coli these are typically at the translational level and the supply of precursors in the form of energy, amino acids and nucleotides. Further recombinant protein production triggers a global cellular stress response which feedback inhibits both growth and product formation. Countering this requires a system level analysis followed by a rational host cell engineering to sustain expression for longer time periods. Another strategy to increase protein yields could be to divert the metabolic flux away from biomass formation and towards recombinant protein production. This would require a growth stoppage mechanism which does not affect the metabolic activity of the cell or the transcriptional or translational efficiencies. Finally cells have to be designed for efficient export to prevent buildup of proteins inside the cytoplasm and also simplify downstream processing. The rational and the high throughput strategies that can be used for the construction of such improved host cell platforms for recombinant protein expression is the focus of this review.
The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.