Dicty News Electronic Edition Volume 17, number 7 September 29, 2001 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at DictyBase--http://dictybase.org. ============== Abstracts ============== EXPRESSION SYSTEMS FOR PRODUCTION OF HETEROLOGOUS PROTEINS Meena Rai and Harish Padh1 1B. V. Patel Pharmaceutical Education & Research Development Centre Thaltej-Gandhinagar Highway, Thaltej, Ahmedabad -380054 Tel: 079- 7439375; Telfax: 079- 7450449 E-mail: perd@wilnetonline.net Current Science Vol. 80(9) 1121 - 1128, 2001 SUMMARY In this review we outline steps involved in the production of heterologous proteins and then evaluate in detail available expression systems and factors affecting heterologous protein expression. Prokaryotic and eukaryotic are the two general categories of expression systems. Prokaryotic systems are generally easier to handle and are satisfactory for most purposes. However, there are serious limitations in using prokaryotic cells for the production of eukaryotic proteins. For example, many of the eukaryotic proteins undergo a variety of post- translational modifications like proper folding, glycosylation, phosphorylation, formation of disulfide bridges etc, There is no universal expression system for heterologous proteins. All expression systems have some advantages as well as some disadvantages that should be considered in selecting which one to use. Choosing the best one requires evaluating the options- from yield to glycosylation, to proper folding, to economics of scale up. The development of reliable transformation systems for Dictyostelium discoideum has provided the possibility of expressing heterologous genes in this microbe23.24. Dictyostelium is a simple eukaryotic micro-organism with a haploid genome of 5 x 107 bp and a life cycle that alternates between single celled and multicellular stages. They grow to a high cell density without the serum factors or special aeration needed by animal cell cultures. There is no cell wall and the high copy number plasmid vectors allow the expression of protein in cell-associated, membrane attached, or secreted form under the control of regulatable promoters25. The cells of Dictyostelium can do both 0- as well as N- glycosylation26,27. The major advantages of this system includes a very simple and cheap growth medium and the potential for large scale production of proteins. Eventual objective of producing a desired protein in an economical heterologous host is influenced by a variety of factors. The new found art of maximising production of heterologous proteins for commercial application is still an art. We have begun to understand factors influencing the eventual production. These factors, described in detail in this article are varied and at times poorly understood. Largely the approach remains empirical. However our collective experience will permit us to rationalise our approach in designing heterologous production of commercially important proteins in a variety of expression system. Subsequent to production, stabilisation and formulation of proteins will pose significant hurdles in utilising the natural biological catalysts and other proteins for therapeutic and industrial purposes. ----------------------------------------------------------------------------- Cyclophilins of a novel subfamily interact with SNW/SKIP coregulator in D.discoideum and S.pombe Michal Skruzny, Monika Ambrozkova, Iva Fukova, Katerina Martinkova, Anna Blahuskova, Ludmila Hamplova, Frantisek Puta, and Petr Folk Department of Physiology and Developmental Biology, Charles University, Vinicna7, Praha2, 128 00, Czech Republic accepted: Biochimica et Biophysica Acta We screened the D.discoideum two-hybrid cDNA library with the SNW/SKIP transcription coregulator SnwA and identified anovel cyclophilin CypE. Independently, the S.pombe cDNA library was screened with the SnwA ortholog Snw1 and the ortholog of CypE (named Cyp2) was found. Both cyclophilins bind the respective SNW protein in their autologous systems. The interaction was localized to the Nterminal part of SnwA as well as of Snw1. CypE was confirmed in vitro to be a cyclosporinA sensitive peptidyl- prolyl cis-trans isomerase. Remarkably, both SNW proteins bind the cyclophilins in acyclosporinA independent manner, possibly serving as adaptors for these novel isomerases. These results are the first characterization of the members of a novel cyclophilin subfamily, which includes the human CGI124/PPIL1 protein. ----------------------------------------------------------------------------- Cross-induction of cell types in Dictyostelium : evidence that DIF-1 is made by prespore cells Robert R. Kay and Christopher R. L. Thompson MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England. Development, in press. Abstract To investigate how cell type proportions are regulated during Dictyostelium development, we have attempted to find out which cell type produces DIF-1, a diffusible signal molecule inducing the differentiation of prestalk-O cells. DIF-1 is a chlorinated alkyl phenone that is synthesized from a C12 polyketide precursor by chlorination and methylation, with the final step catalyzed by the dmtA methyltransferase. All our evidence points to the prespore cells as the major source of DIF-1: (1) dmtA mRNA and enzyme activity are greatly enriched in prespore compared to prestalk cells. The chlorinating activity is also somewhat prespore-enriched. (2) Expression of dmtA is induced by cyclic-AMP and this induction is inhibited by DIF-1. This regulatory behaviour is characteristic of prespore products. (3) Short-term labelling experiments, using the polyketide precursor, show that purified prespore cells produce DIF-1 at more than 20-times the rate of prestalk cells. (4) Although DIF-1 has little effect on its own synthesis in short-term labelling experiments, in long-term experiments, using 36Cl- as label, it is strongly inhibitory (IC50 about 5 nM), presumably because it represses expression of dmtA; this is again consistent with DIF-1 production by prespore cells. Inhibition takes about 1 hr to become effective. We propose that prespore cells cross-induce the differentiation of prestalk-O cells by making DIF-1, and that this is one of the regulatory loops that sets the proportion of prespore-to-prestalk cells in the aggregate. ----------------------------------------------------------------------------- [End Dicty News, volume 17, number 7]