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      Folding and Oligomerization of the gp2b/gp3/gp4 Spike Proteins of Equine Arteritis Virus in Vitro

      research-article
      , *
      Viruses
      MDPI
      equine arteritis virus, glycoproteins, gp2b, gp3, gp4, protein folding, oligomerization

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          Abstract

          Equine arteritis virus (EAV) is a small, positive-stranded RNA virus. The glycoproteins gp2b, gp3 and gp4 form a heterotrimer in the viral envelope, which is required for cell entry of EAV. We describe expression of the ectodomains of the proteins in E. coli and their refolding from inclusion bodies. After extraction of inclusion bodies and dialysis, Gst-, but not His-tagged proteins, refold into a soluble conformation. However, when dialyzed together with Gst-gp3 or with Gst-gp4, His-gp2b and His-gp4 remain soluble and oligomers are obtained by affinity-chromatography. Thus, folding and oligomerization of gp2b, gp3 and gp4 in vitro are interdependent processes.

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          Most cited references20

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          Recombinant protein folding and misfolding in Escherichia coli.

          The past 20 years have seen enormous progress in the understanding of the mechanisms used by the enteric bacterium Escherichia coli to promote protein folding, support protein translocation and handle protein misfolding. Insights from these studies have been exploited to tackle the problems of inclusion body formation, proteolytic degradation and disulfide bond generation that have long impeded the production of complex heterologous proteins in a properly folded and biologically active form. The application of this information to industrial processes, together with emerging strategies for creating designer folding modulators and performing glycosylation all but guarantee that E. coli will remain an important host for the production of both commodity and high value added proteins.
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            The molecular biology of arteriviruses.

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              Protein quality in bacterial inclusion bodies.

              A common limitation of recombinant protein production in bacteria is the formation of insoluble protein aggregates known as inclusion bodies. The propensity of a given protein to aggregate is unpredictable, and the goal of a properly folded, soluble species has been pursued using four main approaches: modification of the protein sequence; increasing the availability of folding assistant proteins; increasing the performance of the translation machinery; and minimizing physicochemical conditions favoring conformational stress and aggregation. From a molecular point of view, inclusion bodies are considered to be formed by unspecific hydrophobic interactions between disorderly deposited polypeptides, and are observed as "molecular dust-balls" in productive cells. However, recent data suggest that these protein aggregates might be a reservoir of alternative conformational states, their formation being no less specific than the acquisition of the native-state structure.
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                Author and article information

                Journal
                Viruses
                Viruses
                viruses
                Viruses
                MDPI
                1999-4915
                22 March 2012
                March 2012
                : 4
                : 3
                : 414-423
                Affiliations
                Department of Immunology and Molecular Biology, Veterinary Faculty, Free University, Philippstraße 13, Berlin 10115, Germany; Email: ajk21@ 123456gmx.de
                Author notes
                [* ] Author to whom correspondence should be addressed; Email: mveit@ 123456zedat.fu-berlin.de ; Tel.: +49-30-2093-6272; Fax: +49-30-2093-6171.
                Article
                viruses-04-00414
                10.3390/v4030414
                3347035
                22590679
                fde20f4d-e296-4e4e-bef8-c2d8bb89f199
                © 2012 by the authors; licensee MDPI, Basel, Switzerland.

                This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/3.0/).

                History
                : 05 January 2012
                : 13 March 2012
                : 18 March 2012
                Categories
                Communication

                Microbiology & Virology
                equine arteritis virus,protein folding, oligomerization,gp2b,gp4,glycoproteins,gp3

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