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Refolded dengue virus type 2 NS1 protein expressed in Escherichia coli preserves structural and immunological properties of the native protein

AMORIM, Jaime Henrique; PORCHIA, Bruna F. M. M.; BALAN, Andrea; CAVALCANTE, Rafael C. M.; COSTA, Simone Morais da; ALVES, Ada Maria de Barcelos; FERREIRA, Luis Carlos de Souza
Fonte: ELSEVIER SCIENCE BV Publicador: ELSEVIER SCIENCE BV
Tipo: Artigo de Revista Científica
Português
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46%
The dengue virus NS1 protein has been shown to be a protective antigen under different experimental conditions but the recombinant protein produced in bacterial expression systems is usually not soluble and loses structural and immunological features of the native viral protein In the present study, experimental conditions leading to purification and refolding of the recombinant dengue virus type 2 (DENV-2) NS1 protein expressed in Escherichia coil are described The refolded recombinant protein was recovered as heat-stable soluble dimers with preserved structural features, as demonstrated by spectroscopic methods In addition, antibodies against epitopes of the NS1 protein expressed in eukaryotic cells recognized the refolded protein expressed in E coli but not the denatured form or the same protein submitted to a different refolding condition Collectively, the results demonstrate that the recombinant NS1 protein preserved important conformation and antigenic determinants of the native virus protein and represents a valuable reagent either for the development of vaccines or for diagnostic methods. (C) 2010 Elsevier B V All rights reserved; FAPESP; Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); FAPERJ; Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ); CNPq; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Produção e purificação de um fragmento recombinante da proteína A de superfície do clado 3 (PspA3) de Streptococcus pneumoniae  em Escherichia coli.; Production and purification of a recombinant fragment of pneumococcal surface protein A clade 3 (PspA3) from Streptococcus pneumoniae in Escherichia coli.

Carvalho, Rimenys Junior
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: dissertação de mestrado Formato: application/pdf
Publicado em 28/08/2009 Português
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A proteína A de superfície de pneumococo (PspA) é indispensável para a virulência da bactéria e foi escolhida para a elaboração de uma nova vacina conjugada contra S. pneumoniae. Para tanto foi desenvolvido um processo industrial de produção e purificação do fragmento recombinante da PspA clado 3 em E. coli. Cultivos descontínuos alimentados foram estabelecidos com glicose ou glicerol em reator de 5L, obtendo-se 62g/L de células secas e 3g/L de PspA3. As células foram lisadas por homogeneizador contínuo de alta pressão com eficiência de 96,7%. A centrifugação foi definida como etapa de clarificação. A sequência cromatográfica troca aniônica seguida de afinidade por Ni+2 rendeu os melhores resultados de pureza (81%) e recuperação (70%). A cromatografia de troca catiônica foi selecionada como terceira etapa do processo, definindo assim um processo de produção e purificação escalonável que possibilitou a obtenção de PspA3 com alto grau de pureza (90%).; The pneumococcal surface protein A (PspA) is indispensable for virulence of S. pneumoniae and it was the first choice as carrier for a new conjugated vaccine against S.pneumoniae. Hence, the purpose of this work was to develop an industrial production and purification process of a recombinant fragment PspA clade 3 (rfPspA3) in E. coli. Fed-batch cultivations in 5 L bioreactors with defined medium were carried out using glucose or glycerol as carbon sources. It was obtained 62 g/L of dry cell weight and 3 g/L of rfPspA3. Cells were disrupted with 96.7% of efficiency by high pressure continuous homogenizer. Centrifugation was defined for the clarification step. The sequence with Q- followed by IMAC-Sepharose yielded the best purity and recovery of rfPspA3 (81 and 70%...

Cloning, overexpression, and purification of functional human purine nucleoside phosphorylase

Silva, Rafael G.; Carvalho, Luiz Pedro S.; Oliveira, Jaim S.; Pinto, Clotilde A.; Mendes, Maria A.; Palma, Mario Sergio; Basso, Luiz A.; Santos, Diógenes S.
Tipo: Artigo de Revista Científica Formato: 158-164
Português
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Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for human PNP causes T-cell deficiency as the major physiological defect. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant tissue rejection, psoriasis, rheumatoid arthritis, lupus, and T-cell lymphomas. Human PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation. In addition, bacterial PNP has been used as reactant in a fast and sensitive spectrophotometric method that allows both quantitation of inorganic phosphate (Pi) and continuous assay of reactions that generate P i such as those catalyzed by ATPases and GTPases. Human PNP may therefore be an important biotechnological tool for P i detection. However, low expression of human PNP in bacterial hosts, protein purification protocols involving many steps, and low protein yields represent technical obstacles to be overcome if human PNP is to be used in either high-throughput drug screening or as a reagent in an affordable P i detection method. Here, we describe PCR amplification of human PNP from a liver cDNA library...

Hyaluronidase from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae): Cloning, structural modeling, purification, and immunological analysis

Justo Jacomini, Débora Laís; Campos Pereira, Franco Dani; Aparecido dos Santos Pinto, José Roberto; dos Santos, Lucilene Delazari; da Silva Neto, Antonio Joaquim; Giratto, Danielli Thieza; Palma, Mario Sergio; de Lima Zollner, Ricardo; Brochetto Braga,
Tipo: Artigo de Revista Científica Formato: 70-80
Português
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In this study, we describe the cDNA cloning, sequencing, and 3-D structure of the allergen hyaluronidase from Polybia paulista venom (Pp-Hyal). Using a proteomic approach, the native form of Pp-Hyal was purified to homogeneity and used to produce a Pp-specific polyclonal antibody. The results revealed that Pp-Hyal can be classified as a glycosyl hydrolase and that the full-length Pp-Hyal cDNA (1315 bp; GI: 302201582) is similar (80-90%) to hyaluronidase from the venoms of endemic Northern wasp species. The isolated mature protein is comprised of 338 amino acids, with a theoretical pI of 8.77 and a molecular mass of 39,648.8 Da versus a pI of 8.13 and 43,277.0 Da indicated by MS. The Pp-Hyal 3D-structural model revealed a central core (?/?)7 barrel, two sulfide bonds (Cys 19-308 and Cys 185-197), and three putative glycosylation sites (Asn79, Asn187, and Asn325), two of which are also found in the rVes v 2 protein. Based on the model, residues Ser299, Asp107, and Glu109 interact with the substrate and potential epitopes (five conformational and seven linear) located at surface-exposed regions of the structure. Purified native Pp-Hyal showed high similarity (97%) with hyaluronidase from Polistes annularis venom (Q9U6V9). Immunoblotting analysis confirmed the specificity of the Pp-Hyal-specific antibody as it recognized the Pp-Hyal protein in both the purified fraction and P. paulista crude venom. No reaction was observed with the venoms of Apis mellifera...

The Fh8 tag : a fusion partner for simple and cost-effective protein purification in Escherichia coli

Costa, Sofia Judite; Coelho, Eduardo; Franco, Lara Oliveira; Almeida, André; Castro, António G.; Domingues, Lucília
Fonte: Elsevier; Academic Press Publicador: Elsevier; Academic Press
Tipo: Artigo de Revista Científica
Publicado em //2013 Português
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Downstream processing is still a major bottleneck in recombinant protein production representing most of its costs. Hence, there is a continuing demand of novel and cost-effective purification processes aiming at the recovery of pure and active target protein. In this work, a novel purification methodology is presented, using the Fh8 solubility enhancer tag as fusion handle. The binding properties of Fh8 tag to a hydrophobic matrix were first studied via hydrophobic interaction chromatography (HIC). The Fh8 tag was then evaluated as a purification handle by its fusion to green fluorescent protein and superoxide dismutase. The purification efficiency of the Fh8-HIC strategy was compared to the immobilized metal ion affinity chromatography (IMAC) using the His6 tag. Results showed that the Fh8-HIC binding mechanism is calcium-dependent in a low salt medium, making the purification process highly selective. Both target proteins were biologically active, even when fused to Fh8, and were successfully purified by HIC, achieving efficiencies identical to those of IMAC. Thus, the Fh8 acts as an effective affinity tag that, together with its previously reported solubility enhancer capability, allows for the design of inexpensive and successful recombinant protein production processes in Escherichia coli.

Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli : the novel Fh8 system

Costa, Sofia; Almeida, André; Castro, António; Domingues, Lucília
Tipo: Artigo de Revista Científica
Publicado em //2014 Português
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Proteins are now widely produced in diverse microbial cell factories. The Escherichia coli is still the dominant host for recombinant protein production but, as a bacterial cell, it also has its issues: the aggregation of foreign proteins into insoluble inclusion bodies is perhaps the main limiting factor of the E. coli expression system. Conversely, E. coli benefits of cost, ease of use and scale make it essential to design new approaches directed for improved recombinant protein production in this host cell. With the aid of genetic and protein engineering novel tailored-made strategies can be designed to suit user or process requirements. Gene fusion technology has been widely used for the improvement of soluble protein production and/or purification in E. coli, and for increasing peptide’s immunogenicity as well. New fusion partners are constantly emerging and complementing the traditional solutions, as for instance, the Fh8 fusion tag that has been recently studied and ranked among the best solubility enhancer partners. In this review, we provide an overview of current strategies to improve recombinant protein production in E. coli, including the key factors for successful protein production, highlighting soluble protein production...

Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

Avilán,L.; Yarzábal,A.; Jürgensen,C.; Bastidas,M.; Cruz,J.; Puig,J.
Fonte: Associação Brasileira de Divulgação Científica Publicador: Associação Brasileira de Divulgação Científica
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/1997 Português
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We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing

Expression and purification of the non-tagged LipL32 of pathogenic Leptospira

Hauk,P.; Carvalho,E.; Ho,P.L.
Fonte: Associação Brasileira de Divulgação Científica Publicador: Associação Brasileira de Divulgação Científica
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/04/2011 Português
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Leptospirosis is a reemerging infectious disease and the most disseminated zoonosis worldwide. A leptospiral surface protein, LipL32, only occurs in pathogenic Leptospira, and is the most abundant protein on the bacterial surface, being described as an important factor in host immunogenic response and also in bacterial infection. We describe here an alternative and simple purification protocol for non-tagged recombinant LipL32. The recombinant LipL32(21-272) was expressed in Escherichia coli without His-tag or any other tag used to facilitate recombinant protein purification. The recombinant protein was expressed in the soluble form, and the purification was based on ion exchange (anionic and cationic) and hydrophobic interactions. The final purification yielded 3 mg soluble LipL32(21-272) per liter of the induced culture. Antiserum produced against the recombinant protein was effective to detect native LipL32 from cell extracts of several Leptospira serovars. The purified recombinant LipL32(21-272) produced by this protocol can be used for structural, biochemical and functional studies and avoids the risk of possible interactions and interferences of the tags commonly used as well as the time consuming and almost always inefficient methods to cleave these tags when a tag-free LipL32 is needed. Non-tagged LipL32 may represent an alternative antigen for biochemical studies...

Expression and purification of the non-tagged LipL32 of pathogenic Leptospira

Hauk,P.; Carvalho,E.; Ho,P.L.
Fonte: Associação Brasileira de Divulgação Científica Publicador: Associação Brasileira de Divulgação Científica
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/04/2011 Português
Relevância na Pesquisa
56.15%
Leptospirosis is a reemerging infectious disease and the most disseminated zoonosis worldwide. A leptospiral surface protein, LipL32, only occurs in pathogenic Leptospira, and is the most abundant protein on the bacterial surface, being described as an important factor in host immunogenic response and also in bacterial infection. We describe here an alternative and simple purification protocol for non-tagged recombinant LipL32. The recombinant LipL32(21-272) was expressed in Escherichia coli without His-tag or any other tag used to facilitate recombinant protein purification. The recombinant protein was expressed in the soluble form, and the purification was based on ion exchange (anionic and cationic) and hydrophobic interactions. The final purification yielded 3 mg soluble LipL32(21-272) per liter of the induced culture. Antiserum produced against the recombinant protein was effective to detect native LipL32 from cell extracts of several Leptospira serovars. The purified recombinant LipL32(21-272) produced by this protocol can be used for structural, biochemical and functional studies and avoids the risk of possible interactions and interferences of the tags commonly used as well as the time consuming and almost always inefficient methods to cleave these tags when a tag-free LipL32 is needed. Non-tagged LipL32 may represent an alternative antigen for biochemical studies...

Two-phase aqueous micellar systems: an alternative method for protein purification

Rangel-Yagui,C. O.; Pessoa-Jr,A.; Blankschtein,D.
Fonte: Brazilian Society of Chemical Engineering Publicador: Brazilian Society of Chemical Engineering
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/2004 Português
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Two-phase aqueous micellar systems can be exploited in separation science for the extraction/purification of desired biomolecules. This article reviews recent experimental and theoretical work by Blankschtein and co-workers on the use of two-phase aqueous micellar systems for the separation of hydrophilic proteins. The experimental partitioning behavior of the enzyme glucose-6-phosphate dehydrogenase (G6PD) in two-phase aqueous micellar systems is also reviewed and new results are presented. Specifically, we discuss very recent work on the purification of G6PD using: i) a two-phase aqueous micellar system composed of the nonionic surfactant n-decyl tetra(ethylene oxide) (C10E4), and (ii) a two-phase aqueous mixed micellar system composed of C10E4 and the cationic surfactant decyltrimethylammonium bromide (C10TAB). Our results indicate that the two-phase aqueous mixed (C10E4/C10TAB) micellar system can improve significantly the partitioning behavior of G6PD relative to that observed in the two-phase aqueous C10E4 micellar system.

P174-T Affinity Protein Purification by Automation Using a Magtration Robotic System

Patel, S.; Duan, X.; TerBush, D.; Yang, Q.; Obata, K.
Fonte: The Association of Biomolecular Resource Facilities Publicador: The Association of Biomolecular Resource Facilities
Tipo: text
Publicado em /02/2007 Português
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Affinity purification is a powerful tool for protein enrichment in proteomics studies. We here present a fully automated system for purification of His-tag proteins and IgG using Ni2+/Co2+ and Protein A magnetic beads, respectively. Reagents for His-tag protein or IgG purification are pre-dispensed in a sealed cartridge for automated runs on a Magtration 12GC robot. The automated purification is based on Magtration technology to perform magnetic bead separation similar to a filtration process in a pipette tip. An optimized protocol has been developed for the automated protein purification. High protein purity and yields were obtained using this automated system. His-tag protein human galectin-1 was purified to approx. 1.6 mg with 12 samples processed in parallel within 30 min on the 12GC robot. This system was also used to screen the expression of His-tag water-soluble proteins and inclusion bodies in bacterial cells, even at a very low expression level. Using Protein A magnetic beads and corresponding pre-filled reagent cartridges, various amounts of human serum (15–80 ?L) and the magnetic beads (100–200 mg) were tested on the robotic system. With 30 ?L serum and 150 mg magnetic beads, we purified IgG with a high yield of 230 ?g. A total of approx. 2.8 mg IgG can be obtained within 60 min with 12 samples run in parallel on the robot. The magnetic beads after the affinity purification can be regenerated by automation for repeated use. Magtration robotic system can be extended for purification of GST-tag fusion proteins and Immunoprecipitation by automation. We have provided an automated protein purification system with a Magtration robot and pre-filled reagent cartridges for rapid and multiparallel processing of different proteins.

Dual Mode Fluorophore-Doped Nickel Nitrilotriacetic Acid-Modified Silica Nanoparticles Combine Histidine-Tagged Protein Purification with Site-Specific Fluorophore Labeling

Kim, Sung Hoon; Jeyakumar, M.; Katzenellenbogen, John A.
Tipo: text
Português
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We present the first example of a fluorophore-doped nickel chelate surface- modified silica nanoparticle that functions in a dual mode, combining histidine-tagged protein purification with site-specific fluorophore labeling. Tetramethylrhodamine (TMR)-doped silica nanoparticles, estimated to contain 700–900 TMRs per ca. 23-nm particle, were surface modified with nitrilotriacetic acid (NTA), producing TMR-SiO2-NTA-Ni+2. Silica-embedded TMR retains very high quantum yield, is resistant to quenching by buffer components and is modestly quenched and only to a certain depth (ca. 2 nm) by surface-attached Ni+2. When exposed to a bacterial lysate containing estrogen receptor ? ligand binding domain (ER?) as a minor component, these beads showed very high specificity binding, enabling protein purification in one step. The capacity and specificity of these beads for binding a his-tagged protein were characterized by electrophoresis, radiometric counting, and MALDI-TOF MS. ER?, bound to TMR-SiO2-NTA-Ni++ beads in a site-specific manner, exhibited good activity for ligand binding and for ligand-induced binding to coactivators in solution FRET experiments and protein microarray fluorometric and FRET assays. This dual-mode type TMR-SiO2-NTA-Ni++ system represents a powerful combination of one-step histidine-tagged protein purification and site-specific labeling with multiple fluorophore species.

Selection of aptamers for a protein target in cell lysate and their application to protein purification

Javaherian, Sahar; Musheev, Michael U.; Kanoatov, Mirzo; Berezovski, Maxim V.; Krylov, Sergey N.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: text
Português
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Functional genomics requires structural and functional studies of a large number of proteins. While the production of proteins through over-expression in cultured cells is a relatively routine procedure, the subsequent protein purification from the cell lysate often represents a significant challenge. The most direct way of protein purification from a cell lysate is affinity purification using an affinity probe to the target protein. It is extremely difficult to develop antibodies, classical affinity probes, for a protein in the cell lysate; their development requires a pure protein. Thus, isolating the protein from the cell lysate requires antibodies, while developing antibodies requires a pure protein. Here we resolve this loop problem. We introduce AptaPIC, Aptamer-facilitated Protein Isolation from Cells, a technology that integrates (i) the development of aptamers for a protein in cell lysate and (ii) the utilization of the developed aptamers for protein isolation from the cell lysate. Using MutS protein as a target, we demonstrate that this technology is applicable to the target protein being at an expression level as low as 0.8% of the total protein in the lysate. AptaPIC has the potential to considerably speed up the purification of proteins and...

Automation of protein purification for structural genomics

Kim, Youngchang; Dementieva, Irina; Zhou, Min; Wu, Ruiying; Lezondra, Lour; Quartey, Pearl; Joachimiak, Grazyna; Korolev, Olga; Li, Hui; Joachimiak, Andrzej
Tipo: text
Publicado em //2004 Português
Relevância na Pesquisa
46.13%
A critical issue in structural genomics, and in structural biology in general, is the availability of high-quality samples. The additional challenge in structural genomics is the need to produce high numbers of proteins with low sequence similarities and poorly characterized or unknown properties. ‘Structural-biology-grade’ proteins must be generated in a quantity and quality suitable for structure determination experiments using X-ray crystallography or nuclear magnetic resonance (NMR). The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. The purification procedure must yield a homogeneous protein and must be highly reproducible in order to supply milligram quantities of protein and/or its derivative containing marker atom(s). At the Midwest Center for Structural Genomics we have developed protocols for high-throughput protein purification. These protocols have been implemented on AKTA EXPLORER 3D and AKTA FPLC 3D workstations capable of performing multidimensional chromatography. The automated chromatography has been successfully applied to many soluble proteins of microbial origin. Various MCSG purification strategies, their implementation, and their success rates are discussed in this paper.

HIGH-THROUGHPUT PROTEIN PURIFICATION FOR X-RAY CRYSTALLOGRAPHY AND NMR

KIM, YOUNGCHANG; BIGELOW, LANCE; BOROVILOS, MARIA; DEMENTIEVA, IRINA; DUGGAN, ERIKA; HATZOS, CATHERINE; JOACHIMIAK, GRAZYNA; LI, HUI; MULLIGAN, RORY; QUARTEY, PEARL; SATHER, ALICIA; STOLS, LUCY; VOLKART, LOUR; ZHOU, MIN
Tipo: text
Português
Relevância na Pesquisa
46.19%
In structural biology, the most critical issue is the availability of high-quality samples. “Structural-biology-grade” proteins must be generated in a quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance. The additional challenge for structural genomics is the need for high numbers of proteins at low cost where protein targets quite often have low sequence similarities, unknown properties and are poorly characterized. The purification procedures must reproducibly yield homogeneous proteins or their derivatives containing marker atom(s) in milligram quantities. The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. Where the ultimate goal of structural biology is the same—to understand the structural basis of proteins in cellular processes, the structural genomics approach is different in that the functional aspects of individual protein or family are not ignored, however, emphasis here is on the number of unique structures, covering most of the protein folding space and developing new technologies with high efficiency. At the Mid-west Center Structural Genomics (MCSG), we have developed semiautomated protocols for high-throughput parallel protein purification. In brief...

Development of the Fc-III Tagged Protein Expression System for Protein Purification and Detection

Feng, Shan; Tian, Enbing; Zhang, Lei; Wang, Qingtao; Deng, Haiteng
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: text
Publicado em 29/08/2012 Português
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46.1%
In the present work, we developed the Fc-III tagged protein expression system for protein purification and detection. The Fc-III sequence encodes for a 13 residue peptide and this peptide is cyclized by disulfide bond formation when the fusion protein is expressed. The Fc-III-fusion proteins selectively bind to immunoglobulin Fc domains (IgG-Fc) expressed from E. coli. We showed the efficient purification of Fc-III tagged proteins by immobilized non-native IgG-Fc and the detection of the cellular locations of fusion proteins by fluorescent-conjugated IgG-Fc. Our results prove that Fc-III tagged protein expression system is a simple and efficient tool for protein purification and detection and is a useful addition to the biochemistry and proteomics toolbox.

High-throughput Protein Purification and Quality Assessment for Crystallization

Kim, Youngchang; Babnigg, Gyorgy; Jedrzejczak, Robert; Eschenfeldt, William H.; Li, Hui; Maltseva, Natalia; Hatzos-Skintges, Catherine; Gu, Minyi; Makowska-Grzyska, Magdalena; Wu, Ruiying; An, Hao; Chhor, Gekleng; Joachimiak, Andrzej
Tipo: text
Português
Relevância na Pesquisa
46.18%
The ultimate goal of structural biology is to understand the structural basis of proteins in cellular processes. In structural biology, the most critical issue is the availability of high-quality samples. “Structural biology-grade” proteins must be generated in the quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. The purification procedures must reproducibly yield homogeneous proteins or their derivatives containing marker atom(s) in milligram quantities. The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. With structural genomics emphasizing a genome-based approach in understanding protein structure and function, a number of unique structures covering most of the protein folding space have been determined and new technologies with high efficiency have been developed. At the Midwest Center for Structural Genomics (MCSG), we have developed semi-automated protocols for high-throughput parallel protein expression and purification. A protein, expressed as a fusion with a cleavable affinity tag, is purified in two consecutive immobilized metal affinity chromatography (IMAC) steps: (i) the first step is an IMAC coupled with buffer-exchange...

Ligation-independent cloning and self-cleaving intein as a tool for high-throughput protein purification

Warren, Tiana D.; Coolbaugh, Michael J.; Wood, David W.
Tipo: text
Português
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46.13%
The rapid production of purified recombinant proteins has become increasingly important for countless applications. Many purification methods involve expression of target proteins in fusion to purification tags, which often must be removed from the target proteins after purification. Recently, engineered inteins have been used to create convenient self-cleaving tags for tag removal. Although intein methods can greatly simplify protein purification, commercially available expression vectors still rely on conventional restriction/ligation cloning methods for target gene insertion. We have streamlined this process by introducing Ligation-Independent Cloning (LIC) capability to our intein expression plasmids, which provides a simple method for constructing self-cleaving tag-target gene fusions. In this work, we demonstrate efficient gene insertion via this system, as well as target protein expression and purification consistent with previously reported results. Through this newly developed system, arbitrary protein genes can be rapidly incorporated into self-cleaving tag expression vectors, and their products purified using convenient platform methods.

A Phosphorylation Tag for Uranyl Mediated Protein Purification and Photo Assisted Tag Removal

Zhang, Qiang; Jørgensen, Thomas J. D.; Nielsen, Peter E.; Møllegaard, Niels Erik
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: text
Publicado em 05/03/2014 Português
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Most protein purification procedures include an affinity tag fused to either the N or C-terminal end of the protein of interest as well as a procedure for tag removal. Tag removal is not straightforward and especially tag removal from the C-terminal end is a challenge due to the characteristics of enzymes available for this purpose. In the present study, we demonstrate the utility of the divalent uranyl ion in a new procedure for protein purification and tag removal. By employment of a GFP (green florescence protein) recombinant protein we show that uranyl binding to a phosphorylated C-terminal tag enables target protein purification from an E. coli extract by immobilized uranyl affinity chromatography. Subsequently, the tag can be efficiently removed by UV-irradiation assisted uranyl photocleavage. We therefore suggest that the divalent uranyl ion (UO22+) may provide a dual function in protein purification and subsequent C-terminal tag removal procedures.

Simplified Protein Purification Using an Autoprocessing, Inducible Enzyme Tag

Shen, Aimee
Tipo: text
Publicado em //2014 Português
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46.08%
The development of affinity tags has greatly simplified protein purification procedures. A variety of affinity tags are now available to improve expression, solubility, and/or tag removal. In this chapter, we describe a method for purifying recombinant proteins expressed in Escherichia coli that uses a highly specific, inducible, C-terminal autoprocessing protease tag. This method streamlines affinity purification, cleavage, and tag separation into a one-step purification procedure, avoiding the need to remove fusion tags from target proteins with exogenous proteases. In addition to accelerating protein purification, we show that this method can enhance the expression, stability, and solubility of select proteins.