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Publications (3)6.62 Total impact

  • Johanna Sagemark · Per Kraulis · Johan Weigelt ·
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    ABSTRACT: Structural and biochemical analysis of proteins requires access to purified protein material. Modern molecular biology technologies facilitate straightforward molecular cloning and expression analysis of multiple protein constructs in parallel, and such approaches have proven very efficient to identify samples suitable for further analysis. A variety of information can be used to support rational design of protein constructs. This includes, e.g. prediction of secondary structure elements, protein domain predictions, and structure prediction methods such as threading. To fully access the available information, collation of data extracted from several different sources is required. This can be cumbersome and sometimes also confusing due to for example different implementation of amino acid residue numbering schemes. The SGC Domain Boundary Analyser tool provides a graphical interface that simplifies and accelerates rational design of protein expression constructs.
    Protein Expression and Purification 03/2010; 72(2):175-8. DOI:10.1016/j.pep.2010.03.020 · 1.70 Impact Factor
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    ABSTRACT: Paraplegin is an m-AAA protease of the mitochondrial inner membrane that is linked to hereditary spastic paraplegias. The gene encodes an FtsH-homology protease domain in tandem with an AAA+ homology ATPase domain. The protein is believed to form a hexamer that uses ATPase-driven conformational changes in its AAA-domain to deliver substrate peptides to its protease domain. We present the crystal structure of the AAA-domain of human paraplegin bound to ADP at 2.2 Å. This enables assignment of the roles of specific side chains within the catalytic cycle, and provides the structural basis for understanding the mechanism of disease mutations. Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.
    PLoS ONE 10/2009; 4(10):e6975. DOI:10.1371/journal.pone.0006975 · 3.23 Impact Factor
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    ABSTRACT: Bacterial over-expression of proteins is a powerful tool to obtain soluble protein amenable to biochemical, biophysical and/or structural characterization. However, it is well established that many recombinant proteins cannot be produced in a soluble form. Several theoretical and empirical methods to improve soluble production have been suggested, although there is to date no universally accepted protocol. This report describes, and quantitatively analyses, a systematic multi-construct approach to obtain soluble protein. Although commonly used in several laboratories, quantitative analyses of the merits of the strategy applied to a larger number of target proteins are missing from the literature. In this study, typically 10 different protein constructs were tested for each targeted domain of nearly 400 human proteins. Overall, soluble expression was obtained for nearly 50% of the human target proteins upon over-expression in Escherichia coli. The chance of obtaining soluble expression was almost doubled using the multi-construct method as compared to more traditional approaches. Soluble protein constructs were subsequently subjected to crystallization trials and the multi-construct approach yielded a more than fourfold increase, from 15 proteins to 65, for the likelihood of obtaining well-diffracting crystals. The results also demonstrate the value of testing multiple constructs in crystallization trials. Finally, a retrospective analysis of gel filtration profiles indicates that these could be used with caution to prioritize protein targets for crystallization trials.
    Protein Expression and Purification 05/2008; 58(2):210-21. DOI:10.1016/j.pep.2007.11.008 · 1.70 Impact Factor