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      High-throughput production of human proteins for crystallization: The SGC experience

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          Abstract

          Producing purified human proteins with high yield and purity remains a considerable challenge. We describe the methods utilized in the Structural Genomics Consortium (SGC) in Oxford, resulting in successful purification of 48% of human proteins attempted; of those, the structures of ∼40% were solved by X-ray crystallography. The main driver has been the parallel processing of multiple (typically 9–20) truncated constructs of each target; modest diversity in vectors and host systems; and standardized purification procedures. We provide method details as well as data on the properties of the constructs leading to crystallized proteins and the impact of methodological variants. These can be used to formulate guidelines for initial approaches to expression of new eukaryotic proteins.

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          SMART, a simple modular architecture research tool: identification of signaling domains.

          J. Schultz, F Milpetz, P. Bork … (1998)
          Accurate multiple alignments of 86 domains that occur in signaling proteins have been constructed and used to provide a Web-based tool (SMART: simple modular architecture research tool) that allows rapid identification and annotation of signaling domain sequences. The majority of signaling proteins are multidomain in character with a considerable variety of domain combinations known. Comparison with established databases showed that 25% of our domain set could not be deduced from SwissProt and 41% could not be annotated by Pfam. SMART is able to determine the modular architectures of single sequences or genomes; application to the entire yeast genome revealed that at least 6.7% of its genes contain one or more signaling domains, approximately 350 greater than previously annotated. The process of constructing SMART predicted (i) novel domain homologues in unexpected locations such as band 4.1-homologous domains in focal adhesion kinases; (ii) previously unknown domain families, including a citron-homology domain; (iii) putative functions of domain families after identification of additional family members, for example, a ubiquitin-binding role for ubiquitin-associated domains (UBA); (iv) cellular roles for proteins, such predicted DEATH domains in netrin receptors further implicating these molecules in axonal guidance; (v) signaling domains in known disease genes such as SPRY domains in both marenostrin/pyrin and Midline 1; (vi) domains in unexpected phylogenetic contexts such as diacylglycerol kinase homologues in yeast and bacteria; and (vii) likely protein misclassifications exemplified by a predicted pleckstrin homology domain in a Candida albicans protein, previously described as an integrin.
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            The PSIPRED protein structure prediction server.

            Howland Jones, W McGuffin, K. Bryson … (2000)
            The PSIPRED protein structure prediction server allows users to submit a protein sequence, perform a prediction of their choice and receive the results of the prediction both textually via e-mail and graphically via the web. The user may select one of three prediction methods to apply to their sequence: PSIPRED, a highly accurate secondary structure prediction method; MEMSAT 2, a new version of a widely used transmembrane topology prediction method; or GenTHREADER, a sequence profile based fold recognition method. Freely available to non-commercial users at http://globin.bio.warwick.ac.uk/psipred/
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              Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses.

              P H Yancey (2005)
              Organic osmolytes are small solutes used by cells of numerous water-stressed organisms and tissues to maintain cell volume. Similar compounds are accumulated by some organisms in anhydrobiotic, thermal and possibly pressure stresses. These solutes are amino acids and derivatives, polyols and sugars, methylamines, methylsulfonium compounds and urea. Except for urea, they are often called ;compatible solutes', a term indicating lack of perturbing effects on cellular macromolecules and implying interchangeability. However, these features may not always exist, for three reasons. First, some of these solutes may have unique protective metabolic roles, such as acting as antioxidants (e.g. polyols, taurine, hypotaurine), providing redox balance (e.g. glycerol) and detoxifying sulfide (hypotaurine in animals at hydrothermal vents and seeps). Second, some of these solutes stabilize macromolecules and counteract perturbants in non-interchangeable ways. Methylamines [e.g. trimethylamine N-oxide (TMAO)] can enhance protein folding and ligand binding and counteract perturbations by urea (e.g. in elasmobranchs and mammalian kidney), inorganic ions, and hydrostatic pressure in deep-sea animals. Trehalose and proline in overwintering insects stabilize membranes at subzero temperatures. Trehalose in insects and yeast, and anionic polyols in microorganisms around hydrothermal vents, can protect proteins from denaturation by high temperatures. Third, stabilizing solutes appear to be used in nature only to counteract perturbants of macromolecules, perhaps because stabilization is detrimental in the absence of perturbation. Some of these solutes have applications in biotechnology, agriculture and medicine, including in vitro rescue of the misfolded protein of cystic fibrosis. However, caution is warranted if high levels cause overstabilization of proteins.
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                Author and article information

                Journal
                Journal ID (nlm-ta): J Struct Biol
                Journal ID (iso-abbrev): J. Struct. Biol
                Title: Journal of Structural Biology
                Publisher: Academic Press
                ISSN (Print): 1047-8477
                ISSN (Electronic): 1095-8657
                Publication date PMC-release: October 2010
                Publication date (Print): October 2010
                Volume: 172
                Issue: 1
                Pages: 3-13
                Affiliations
                [a ]Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK
                [b ]Nuffield Department of Clinical Medicine, Old Road Campus, Headington, Oxford OX3 7BN, UK
                Author notes
                [* ]Corresponding author. Fax: +44 1865 617575. opher.gileadi@ 123456sgc.ox.ac.uk
                [1]

                For clone requests please contact: contact@sgc.ox.ac.uk.

                Article
                Publisher ID: YJSBI5815
                DOI: 10.1016/j.jsb.2010.06.008
                PMC ID: 2938586
                PubMed ID: 20541610
                SO-VID: 0c149688-c810-428c-ae29-4050ea9a3c27
                Copyright © © 2010 Elsevier Inc.
                License:

                This document may be redistributed and reused, subject to certain conditions.

                History
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                Subject: Article

                ScienceOpen disciplines: Biophysics
                Keywords: structural genomics,ligation-independent cloning,protein expression,open source,protein purification,structure prediction
                Data availability:
                ScienceOpen disciplines: Biophysics
                Keywords: structural genomics, ligation-independent cloning, protein expression, open source, protein purification, structure prediction

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