Copy Link
Add to Bookmark
Report
dictyNews Volume 17 Number 07
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]
