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  HUMAN GLYCOPROTEINS AND DERIVED VARIANTS FROM RECOMBINANT MAMMALIAN CELLLINES

  Dittmar, K. E. J.; Conradt, H. S.; Hauser, H.; Hofer, B.; Lindenmaier, W.; GBF, Gesellschaft fiir Biotechnologische Forschung, Mascheroder Weg 1, D-3300 Braunschweig (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)

  The expression of foreign genes using recombinant DNA technology in various host systems has permitted the production of human proteins of therapeutic interest in high amounts. Manyclinically important human proteins are posttranslationally modified. However, the inability of microbes to perform mammalian-type of posttranslational modifications of proteins is a major shortcoming. Alternative expression systems are insect and mammaliancells. Principle mammalian types of protein modifications are N- and O-glycosylation. Insect cells, fungi and yeasts are unable to perform the same terminal glycosylation reactions on glycoproteins as mammalian cells. Recombinant DNA technology used for the production of pharmaceutically useful polypeptides has mainly been focused on microbial expression systems (bacteria like E. coli, yeast and fungi). The advantage of microbial expression systems is the high amount of expressed protein that can be obtained. The present communication considers aspects of glycoprotein research relevant to the field of biotechnology and protein design. Results are presented that have been obtained by our group during the last four years concerning the expression of the glycoproteins human Interleukin 2 (Il-2) and Interferon-8 (IFN-8) in different mammalian cell lines, the determination of their carbohydrate residues, the effect of site-directed mutagenesis on their carbohydrate attachment sites and the insertion of peptide domains which function as acceptors for carbohydrates.
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  COMPUTERAIDED PROTEIN DESIGN: METHODS AND APPLICATIONS

  Schomburg, Dietmar; GBF(Gesellschaft fiir Biotechnologische Forschung) Mascheroder Weg 1, D-3300 Braunschweig, WEST GERMANY (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)

  Since the first reports on the use ofsite directed mutagenesis in 1982! protein engineering or - when rationally aimed - protein design has been Tecognized as a promising and fascinating field of research in many countries. In Japan (PERI) and the U.S.A. (CARB)researchinstitutes have been founded with the focus on protein design. More and more researchinstitutes in the United States, Canada, Japan and Europe have been Starting broad research projects on protein design (UK: SERC, W. Germany: GBF, EMBL). Possible prospects for applications of designed proteins with modified activities or other new properties are very high,in the areas of pharmacology, enzyme applications in food industry’, waste treatment and chemical synthesis, vaccine design, biosensors etc.*". This conception wasvery clearly lined out in an excellent article by Kevin Ulmerin 19835, Encouraging results have so far been obtained only for a small number ofcases including insulin, proteases and peptidic protease inhibitors, and some others®’. On the other hand many unpredicted andsurprising results of site directed mutagenesis experiments are reported onscientific meetings and in the literature*’, That demonstrates that our methods andtools in thatarea arestill rather crude and urgently require improvement!", Simplecalculations show that the random approach to protein-engineeringis a very slow one. There are 10°* ways to arrange aminoacids in a medium sized protein chain of 250 amino acids. Ca. 10” molecules would form the whole estimated mass ofour universe. But even whenthe information about the seven most important aminoacids is available and only five changes should be tested for each of the positions, about 80,000 different protein-mutants have to be prepared andtested. This implies that a knowledge of the 3D-protein-structure and a good understandingofthe functionactivity relationship is absolutely essential in order to do rational protein-design. Research projects in protein-design require a close cooperation between groupsspecialising in protein-isolation and purification, in fermentation techniques, in genetic-engineering, in DNAsynthesis and protein-crystallography (protein-NMR techniques are being established). This interdisciplinary connection between protein chemists, molecular biologists and stereo-chemists is essential for the protein-design cycle (Fig. 1) consisting of design, cloning, expression andtesting new proteins starting from known ones.
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  NEW DEVELOPMENTSIN PROTEIN CRYSTALLOGRAPHY

  Hol, W. G. J.; Laboratory of Chemical Physics University of Groningen Nijenborgh 16 9747 AG Groningen The Netherlands (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1988)

  Protein crystallography is currently undergoing a rapid change in many different ways. One development is the explosion of interest by molecular biologists and immunologists since knowledge of protein sequences, obtained via DNA sequencing, is expanding rapidly, but does often not increase immediately insight into the functioning of the protein. Another change is the recombinant DNA technique which make it possible to obtain large amounts of proteins which were previously only available in minute quantities. A third change is the wide-spread awareness that detailed knowledge of wellselected protein structures is a promising starting point for designing new pharmaceuticals and vaccines, for obtaining new proteins via protein engineering techniques and for inspiring synthetic chemists in their biomimetic endeavours. At the same time many technical aspects of protein crystallography are undergoing a rapid development. Someof them will be describedin this paper. Crystal structures of proteins can be obtained currently in two quite different ways: (i) the "multiple isomorphous replacement" (MIR) method [1-3] for de novo structure determinations, often using additional anomalous scattering information (MIRAS) [4,5]; and, (ii) the "molecular replacement" (MR) method [6-8] for solving new structures related to a known structure. We will discuss the steps involved in obtaining high resolution X-ray structures as outlined in Figure 1. A detailed account of these steps can be found in two volumes of Methods of Enzymology [9].
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  DESIGN AND STRUCTURES OF DISULFIDE CONTAINING SUBTILISIN VARIANTS

  Katz, Bradley A.; Department of Pharmaceutical Chemistry University of California, San Francisco San Francisco, California 94143 Department of Biomolecular Chemistry Genentech Inc. 460 Point San Bruno Blvd South San Francisco, California 94080 and Research Department Genencor, Inc. 180 Kimball Way South San Francisco, California 94080 present address: Triton Biosciences Inc. 1501 Harbor Bay Parkway Alameda, California 94501 (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)

  The crystal structures of 4 variants of subtilisin, each one containing an engineered disulfide crosslink have been determined. The geometries of the engineered disulfide groups are atypical. For the Cys24-Cys87 and Cys22-Cys87 disulfides there is a relationship between their measured redox potentials and their calculated dihedral energies. Disulfide introduction produced cavities in the protein structures. The cavity produced by removal of Met119 in A29C/M119C (Ala29 to Cys, Met119 to Cys) was partially filled by a disordering of nearby Asn117. The cavities were often filled with ordered water molecules that replaced interactions of the removed groups. Molecular modelling provided insight into the location where a disulfide could be incorporated, and into its resulting geometry. The structures of A29C/M119C and of V26C/A232C showed that introduction of disulfides into buried hydrophobic regions resulted in long range concerted rearrangements.
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  CHARACTERISATION OF ENGINEERED PROTEINS: SOME CRITICAL REFLECTIONS

  Dittmar, Kurt E.; Woolley, Paul; Gesellschaft fiir biotechnologische Forschung, Mascheroder Weg 1, D-3300 Braunschweig #Kemisk Institut, Aarhus Universitet, DK-8000 Arhus C (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1989)

  This essay is an attempt to point up the gap between, on the one hand, the methods currently available to the biologist in the laboratory and, on the other, the kind of data that he or she would need in order to characterise genetically engineered proteins of topical biological interest in such a way as to make use of the techniques of protein engineering. Sgren Kirkegaard was Denmark's greatest philosopher, and he was well aware of the fact. One day he reflected: “To be Denmark's greatest philosopher, ah, that is indeed a fine satire.” By this he presumably meant that he was the only one. These words have encouraged us to philosophize a little about the protein engineering cycle, of which our version is shownin Figure 1. We have dissected the cycle according to two principles, information-theoretical (vertical axis) and epistemological (horizontal axis). The cycle starts from a gene and proceeds via expression to the corresponding protein, which we associate with a set of properties by testing or suitable characterisation. The understanding of these leads by way of theory, experience or intuition to a new gene, and thereafter the cycle continues, a process of which we have seen many impressive examples.

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