Efficient E. coli Expression Strategies for Production of Soluble Human Crystallin ALDH3A1

Voulgaridou, Georgia-Persephoni; Mantso, Theodora; Chlichlia, Katerina; Panayiotidis, Mihalis Ι.; Pappa, Aglaia

doi:10.1371/journal.pone.0056582

Cited by 37 publications

(20 citation statements)

References 43 publications

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“…The diminished protein degradation seems to have been critical for recovery. Modifications of culture conditions and induction time may favor parameters such as reduced molecular hydrophobic interactions, inhibition inclusion body formation, and lower protein synthesis rate, which in turn favor the correct folding and reduce the activity of proteases (58). Thus, the protocol presented in this work for purification of DENV NS5 results in the highest recovery values so far reported.…”

Section: Discussionmentioning

confidence: 95%

Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen

Alves

Pereira

Fabris

et al. 2016

Clin Vaccine Immunol

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Dengue fever is caused by any of the four known dengue virus serotypes (DENV1 to DENV4) that affect millions of people worldwide, causing a significant number of deaths. There are vaccines based on chimeric viruses, but they still are not in clinical use. Anti-DENV vaccine strategies based on nonstructural proteins are promising alternatives to those based on whole virus or structural proteins. The DENV nonstructural protein 5 (NS5) is the main target of anti-DENV T cell-based immune responses in humans. In this study, we purified a soluble recombinant form of DENV2 NS5 expressed in Escherichia coli at large amounts and high purity after optimization of expression conditions and purification steps. The purified DENV2 NS5 was recognized by serum from DENV1-, DENV2-, DENV3-, or DENV4-infected patients in an epitope-conformation-dependent manner. In addition, immunization of BALB/c mice with NS5 induced high levels of NS5-specific antibodies and expansion of gamma interferon-and tumor necrosis factor alpha-producing T cells. Moreover, mice immunized with purified NS5 were partially protected from lethal challenges with the DENV2 NGC strain and with a clinical isolate (JHA1). These results indicate that the recombinant NS5 protein preserves immunological determinants of the native protein and is a promising vaccine antigen capable of inducing protective immune responses. Dengue fever is an acute disease caused by dengue virus (DENV), an arbovirus belonging to the Flaviviridae family, transmitted by Aedes mosquitoes (1, 2). In urban settings, DENV is found as four immunologically distinct types (serotypes), DENV1 to DENV4, which have approximately 30% divergence in their genomic nucleotide sequences (3-7). The disease may present with different degrees of manifestations that are classified by the WHO as dengue fever, dengue with warning signs, and severe dengue (8). A recent study showed that, annually, an average of 96 million DENV-infected people develop symptoms with severity sufficient to change their routines (1); according to previous studies, about 500,000 cases develop into severe forms of disease, which may include hemorrhage and shock syndrome (9). The mortality rate in this group reaches 10% in hospitalized patients, increasing to 30% in nontreated DENV-infected patients (9-11). Despite the high epidemiological importance of dengue fever, no effective anti-DENV drug or vaccine formulation is in clinical use for treatment or prevention of the disease.Several strategies have been explored, aiming toward the development of an effective dengue vaccine (12-21). Nonetheless, a tetravalent vaccine formulation requires a neutralizing response to all four serotypes (22)(23)(24)(25). A poor vaccine-induced response to structural proteins of any of the four serotypes may pose a risk of the onset of antibody-dependent enhancement (ADE) of virus replication upon infection by a subneutralized serotype (22-25). Moreover, protection based on the induction of neutralizing antibodies targeting structural proteins may be neg...

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Section: Discussionmentioning

confidence: 95%

Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen

Alves

Pereira

Fabris

et al. 2016

Clin Vaccine Immunol

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“…This methodology previously has been demonstrated to be a highly effective way to increase the soluble expression of various recombinant proteins in E. coli [25, 26], and furthermore using a chaperone transformation strategy [25, 40]. The resulting expression studies showed that two groups of molecular chaperones from E.coli (GroES/GroEL and DnaK/DnaJ/GrpE) were the most effective with greater than 20% of the monomeric cyclin A being expressed in the soluble fraction.…”

Section: Discussionmentioning

confidence: 99%

“…A chaperone expression strategy previously described [25], was exploited to achieve elevated amounts of heterologous soluble over-expression of a 6-His tagged- human protein with the two groups of molecular chaperones from E. coli (GroES/ GroEL and DnaK/DnaJ/GrpE). This strategy resulted in higher yield of the soluble, active and natively folded form of ALDH3A1 [26]. In addition, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS, from Actinosynnema pretiosum ssp.…”

Section: Introductionmentioning

confidence: 99%

Efficient soluble expression of active recombinant human cyclin A2 mediated by E. coli molecular chaperones

Grigoroudis

McInnes

Premnath

et al. 2015

Protein Expression and Purification

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Bacterial expression of human proteins continues to present a critical challenge in protein crystallography and drug design. While human cyclin A constructs have been extensively characterized in complex with cyclin dependent kinase 2 (CDK2), efforts to express the monomeric human cyclin A2 in Escherichia coli in a stable form, without the kinase subunit, have been laden with technical difficulties, including solubility, yield and purity. Here, optimized conditions are described with the aim of generating for first time, sufficient quantities of human recombinant cyclin A2 in a soluble and active form for crystallization and ligand characterization purposes. The studies involve implementation of a His-tagged heterologous expression system under conditions of auto-induction and mediated by molecular chaperone-expressing plasmids. A high yield of human cyclin A2 was obtained in natively folded and soluble form, through co-expression with groups of molecular chaperones from E. coli in various combinations. A one-step affinity chromatography method was utilized to purify the fusion protein products to homogeneity, and the biological activity confirmed through ligand-binding affinity to inhibitory peptides, representing alternatives for the key determinants of the CDK2 substrate recruitment site on the cyclin regulatory subunit. As a whole, obtaining the active cyclin A without the CDK partner (referred as monomeric in this work) in a straightforward and facile manner will obviate protein –production issues with the CDK2/cyclin A complex and enable drug discovery efforts for non-ATP competitive CDK inhibition through the cyclin groove.

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“…The most extensively used chaperone systems that have facilitated protein production in E.coli are DnaK-DnaJ-GrpE and GroEL-GroES[19,20]. In insect cells, host protein biosynthesis shuts down as a result of infection by the recombinant baculovirus, which can adversely affect levels of molecular chaperones important for the folding of secreted proteins and membrane proteins in the endoplasmic reticulum (ER), particularly in relation to the high levels of protein synthesis resulting from high mRNA levels produced from the polyhedrin promoter.…”

Section: Folding and Secretory Pathway Engineeringmentioning

confidence: 99%

Engineering cells to improve protein expression

Xiao

Shiloach

Betenbaugh

2014

Current Opinion in Structural Biology

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Cellular engineering of bacteria, fungi, insect cells and mammalian cells is a promising methodology to improve recombinant protein production for structural, biochemical, and commercial applications. Increased understanding of the host organism biology has suggested engineering strategies targeting bottlenecks in transcription, translation, protein processing and secretory pathways, as well as cell growth and survival. A combination of metabolic engineering and synthetic biology has been used to improve the properties of cells for protein production, which has resulted in enhanced yields of multiple protein classes.

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Efficient E. coli Expression Strategies for Production of Soluble Human Crystallin ALDH3A1

Cited by 37 publications

References 43 publications

Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen

Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen

Efficient soluble expression of active recombinant human cyclin A2 mediated by E. coli molecular chaperones

Engineering cells to improve protein expression

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