biotechnology symposium 2005 abstracts - Universität Leipzig

4.6 Recombinant expression and structure
determination of phosphodiesterases
Roy Eylenstein Eylenstein, Nancy Kühn, Petra Schäfer, E. Bartholomeus
Küttner, Susanne Moschütz, Norbert Sträter
PDEs catalyzing the hydrolysis of the 3’-5’ phosphodiester bond of cyclic nucleotides
(cAMP, cGMP) comprise a family of enzymes that modulate the immune response,
infl ammation, cell growth and many other functions. 11 families of PDEs have been
described including multiple isoforms with varying selectivities for cAMP and/or cGMP.
The isoforms are differentially expressed and regulated in different cell types, and they
appear to modulate the location, substrate selectivity, kinetics, and response to activators
or inhibitors of the enzyme. As essential regulators of cyclic nucleotide signalling, PDEs
are recognized as important drug targets for the treatment of diseases such as heart failure,
asthma and erectile dysfunction.
The aim of the current project is to develop expression constructs for the production
of suffi cient material for crystallization of full length PDEs and catalytic domains. The
X-ray structures of these PDEs will allow for the rational design of specifi c inhibitors as
therapeutics. 3D structures of the catalytic domains of PDE1, -3, -4, -5 and -9 are currently
available.
For the expression of the catalytic domains we designed different truncations of PDEs
to fi nd good constructs for subsequent protein purifi cation and crystallization. Expression
in E. coli as well as in P. pastoris often yields insoluble products. To avoid the refolding
processes of the proteins we also try to express the catalytic domains in an E. coli based
cell-free in vitro system. This system allows for the simple modifi cation of many expression
parameters and hence it is maybe possible to directly produce soluble and active protein.
Roy Eylenstein
Universität Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Professorship for Structural Analysis of Biopolymers
and
Faculty of Chemistry and Mineralogy
E-Mail: roy.eylenstein@bbz.uni-leipzig.de
www.uni-leipzig.de/~strater/
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