Research Report 2003 - Max-Planck-Institut für molekulare Genetik

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Department of Vertebrate Genomics 12 Protein Structure Factory Head: Dr. Konrad Büssow Phone: +49 (0)30-32639 2802 Fax: +49 (0)30-32639 2833 Email: buessow@molgen.mpg.de PSF / Protein Expression: Christoph Scheich (engineer) Dr. Volker Sievert (scientist) Janett Tischer (technician) Claudia Quedenau (PhD student) PSF / Protein Crystallisation: Joachim Lebik (engineer) NGFN core area platform 4 / protein expression Dr. Hendrik Weiner (scientist) Thomas Faupel (PhD student) Brigitte Hieke (technician) Scientific overview The Protein Structure Factory is a structural genomics project for the systematic structural analysis of human proteins. The goal of the Protein Structure Factory is to systematically select suitable human proteins by sequence analysis, express the proteins in recombinant form and proceed with structural analysis with those proteins that can be produced easily. The PSF is part of the International Structure Genomics Organization (ISGO) and has organised the International Congress of Structural Genomics 2002. Funded in 1999, the Protein Structure Factory is one of the earliest endeavours towards fast and cost-effective high-throughput protein production and structure determination. The Department of Vertebrate Genomics has initiated the Protein Structure Factory as a BMBFfounded “Leitprojekt” as partner of a multidisciplinary consortium of structural biology and other research groups and companies (http://www.proteinstrukturfabrik.de). Our scientific partners in the Protein Structure Factory and the expertise provided are: • Prof. Dr. Udo Heinemann, MDC Berlin – X-ray diffraction at BESSY synchroton • Prof. Dr. Wolfgang Saenger, FU Berlin – protein crystallisation • Prof. Dr. Hartmut Oschkinat, FMP Berlin – NMR of protein domains • PD Dr. Christine Lang, TU-Berlin – protein expression in yeast • Prof. Dr. K.-P. Hofmann, Charité Berlin – biophysical characterisation of proteins • Alpha Bioverfahrenstechnik GmbH, Kleinmachnow – protein expression and purification • Deutsches Resourcenzentrum GmbH, Berlin – cDNA clone resources • Prof. Dr. Peer Bork, EMBL Heidelberg – bioinformatics, selection of target proteins Current status of research Many structural genomics projects have been initiated since the start of the Protein Structure Factory in 1999. It is clear today that the major bottleneck in the systematic structural analysis of proteins is their production and crystallisation. Especially the recombinant production of eukaryotic proteins is difficult and has a high failure rate. One way to overcome this limitation is to generate and analyse a large number of targets and to proceed with the ones that can be produced readily. Alternative expression systems are another option - yeast is used in addition to E. coli at
the Protein Structure Factory. Finally, in vitro folding of proteins can transfer insolubly expressed proteins into the folded form (see below). High-throughput protein expression Our part in the project is the generation of E. coli expression clones for human proteins and the characterization of the clones. We are responsible to supply the Protein Structure Factory with a sufficient number of clones that express human proteins in good yield and soluble form. We have established expression systems that allow efficient protein purification by affinity chromatography via affinity tags and removal of affinity tags after protein purification (Heinemann et al., 2003). All steps required for subcloning of cDNAs into our optimised expression vectors and subsequent characterisation of expression clones by protein expression and affinity purification were established and automated in 96-well format on a pipetting robot (Scheich et al, 2003, Holz et al., 2003). In co-operation with the group of Peer Bork, we selected 860 human proteins that have a high probability to be successfully expressed in E. coli, e.g. excluding membrane proteins. Another selection criterion was the availability of full-length cDNA clones, since these clones enabled us to use high-throughput cloning procedures. So far, our group supplied constructs for soluble expression of 243 human proteins. Using these clones, 52 distinct proteins were expressed and purified in sufficient amount and quality for crystallization trials in the year 2002. In 2003, this number was already reached after eight months. Using these crystals, two structures were solved by our partners in the Protein Structure Factory and six more proteins with well-diffracting crystals were obtained in this time period (Manjasetty et al., in press). The current status of generated clones, proteins and structures is available at: http://www.proteinstrukturfabrik.de/public/PSF_Status_1.shtml. A database and additional software were developed for management of data in the group and to provide co-workers in the Protein Structure Factory with information on sequences and clones of target proteins. Part of this software development was published (Büssow et al., 2002). A large proportion of human proteins do not fold and form insoluble aggregates when expressed in E. coli cells. In vitro folding can transfer such aggregates into correctly folded proteins. We have developed a automated method for protein crystallisation, that allows to perform fast screenings for suitable folding buffer conditions using a biophysical readout (Scheich et al., in preparation). Automated protein crystallisation The department of Vertebrate Genomics has automated protein crystallisation in the Protein Structure Factory in a co-operative effort with the group of Prof. Saenger, Free University of Berlin (Mueller et al., 2001). Automation is a requirement for the crystallisation of larger number of target proteins, since manual crystallisation is very time-consuming. We have established automated set-up and analysis of crystallisation screens. A micro-dispensing technology for crystallisation in sub-microlitre volumes in microtitre plates has been established. Crystallisation is currently monitored by a system that takes pictures of crystallisation drops in a 96-well plate automatically. An additional system is under development that will combine a microtitre-plate storage device with a detection system and that will be able to automatically record images of crystallisation experiments at certain time intervals. Protein-protein interaction studies As part of the “Nationales Genomforschungsnetz” NGFN, a project has been established at the Protein Structure Factory for expression of human proteins involved in neurodegenerative diseases for the in vitro characterization of protein-protein interaction. We are developing a technology for identification of protein interaction partners using highdensity protein arrays on filter membranes (Weiner et al., in press). This project is a collaboration with Prof. Erich Wanker of the MDC Berlin-Buch, in which we share a common set of 100 proteins involved in neurodegenerative disease to identify novel protein-protein interactions. We have identified sets of interaction partners for different target proteins. Interactions are currently being verified by in vitro pull-down assays and other methods. (NGFN Platform 4, http:/ /www.ngfn.de/protein) MPI for Molecular Genetics Research Report 2003 13
Page 1 and 2: Max-Planck-Institut für molekulare
Page 3 and 4: Table of Contents The Max Planck In
Page 5 and 6: MPI for Molecular Genetics Research
Page 7 and 8: The Max Planck Institute for Molecu
Page 9 and 10: Department of Vertebrate Genomics I
Page 11 and 12: To generate high resolution express
Page 13: Competitive position Since a long t
Page 17 and 18: Mass Spectrometry Group Head: Dr. J
Page 19 and 20: different tissues of A. thaliana, w
Page 21 and 22: Bioinformatics Group Heads: Ralf He
Page 23 and 24: a) Evolutionary studies In a recent
Page 25 and 26: Fuchs T, Malecova B, Linhart C, Sha
Page 27 and 28: Mouse, Medaka & MHC Group Head: Dr.
Page 29 and 30: General information Publications 19
Page 31 and 32: Jochen Wittbrodt, EMBL, Heidelberg
Page 35 and 36: THETA - Two Hybrid Transfected-cell
Page 37 and 38: Academical co-operations Andreas Ra
Page 39 and 40: The investigation of signaling path
Page 41 and 42: In vitro Ligand Screening Group Hea
Page 45 and 46: Neurodegenerative Disorders Group H
Page 47 and 48: Automation Group Graduate students:
Page 49 and 50: In addition, novel methods for the
Page 51 and 52: Sittler A, Walter S, Wedemeyer N, H
Page 53 and 54: Prof. Dr. Joachim Klose, Institut f
Page 55 and 56: eral years. This ambiguity is partl
Page 57 and 58: Clark MD, Panopoulou GD, Cahill DJ,
Page 59 and 60: integrations were passaged to the g
Page 63 and 64: Protein Group Scientists: Sabine Ba
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Kersten B, Bürkle L, Kuhn EJ, Giav
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Cardiovascular Genetics Group Head:
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protein interaction partners and at
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General information Publications 19
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Chromosome 21 Group Head: Marie-Lau
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Department of Human Molecular Genet
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Network (NGFN). Recently, DNA from
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Finally, several of these groups ar
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Neurochemistry Group & Mouse Lab Gr
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studies. Furthermore, these mice wi
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Noonan syndrome and related disorde
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Chromosome Rearrangements & Disease
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General information Selected Public
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DNA Microarrays Scientists: Dr. Fik
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General information Publications He
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that in addition to reduced prolife
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Christoph Redies, Prof. Dr., Instit
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Since my move to the MPIMG in 11/20
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Biochemistry of Inherited Brain Dis
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Schweiger S, Chaoui R, Tennstedt C,
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Until 2002, 13 genes have been impl
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General information Selected public
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Department of Computational Molecul
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EU: The European Molecular Biology
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EST tissue distribution Besides the
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Protein Families & Evolution Group
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We make use of the well-known HMMer
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Theses Benjamin Georgi: A graph-bas
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wide analysis of tyrosine as well a
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Further projects include multiple t
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Co-operations Identification and fu
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upstream of the translational start
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Department of Developmental Genetic
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2000; see figure 1). So far, we hav
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partners are. We also want to find
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Emeritus Group General Molecular Ge
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Sethmann S, Ceglowski P, Willert J,
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Research Group Development & Diseas
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MPI for Molecular Genetics Research
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Goals An important future goal is t
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Independent Junior Research Groups
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over, daf-9 is tightly regulated by
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European C.elegans Meeting, Blanken
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and at the ribosomal DNA locus. One
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lencer, has so far remained unchara
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Molecular control of skeletal devel
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BMPs, into a common control network
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this end I plan to analyze mouse mu
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Ribosome Group The Ribosome group c
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General information Selected Public
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group during the same period. In ad
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11) Agmon I, Auerbach T, Baram D, B
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Reconstitution & function The ribos
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Wendrich TM, Blaha G, Wilson DN, Ma
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Miscellaneous Research Groups There
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vealed that the extraordinary stabi
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34. Thorsted PB, Macartney DP, Akht
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esis and DNA packaging of this phag
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Other launched projects concern con
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BMBF 01GR0105, Plattform 1.1: NGFN
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DNA synthesis, lipid biosynthesis,
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Administration & Research Support H
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Technical Management and Workshops
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tion based on the simplified GOOD a
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Library Librarians: Dipl.-Fachinf.
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How to get to the Institute Max Pla
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Research Report 2003 - Max-Planck-Institut für molekulare Genetik

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