Research Report 2010 2011 - Helmholtz-Zentrum für ...

SCIENTIFIC REPORTS | Technological Platforms 127
02 Recombinant Protein Expression
HEAD | Dr. Joop van den Heuvel | Research Group Recombinant Protein Expression | jvh@helmholtz-hzi.de
SCIENTIFIC COLLABORATOR | Dr. Konrad Büssow | Dr. Volker Jäger | Prof. Dr. Ursula Rinas
The recombinant protein expression platform (RPEX) is
essential for the production of ultra pure protein for high
resolution structural analysis using X-ray crystallography
and NMR spectroscopy. Four major expression systems have
been established in the RPEX facility: E. coli, P. pastoris,
insect cell and mammalian cell culture. This allows the
production of “simple proteins” as well as proteins with
complicated modifications.
For production of the challenging mammalian protein
targets an animal cell culture facility has been established.
The size of the cell culture reactors ranges from 1.6 to 6
litres in order to meet the increasing requirements for
routine production of proteins on a 10-50 mg scale. For the
purification of poorly expressed recombinant multi-protein
complexes a 30-litres-bioreactor has been set up that allows
the pilot-scale production of protein from several hundred
litres of medium.
The system includes all the necessary equipment for continuous
media exchange via tangential flow microfiltration
or internal membrane perfusion and subsequent protein
purification steps. As a supplement to recombinant protein
production, large amounts of HeLa cell biomass can be
produced as a source for purification of naturally occurring
human protein complexes.
Research A research group within the PSPF is focussing
on the development of new and fast strategies for creating
stable expression cell lines. This group has already established
a new and unique methodology for rapidly generating
highly overproducing stable mammalian cell lines (RMCE)
(Wilke et al., 2010). The expression level varies between
1-5 mg/L and can be easily, and reproducibly, scaled up to
10-100 L. In cooperation with the project group of K. Büssow
the newly developed CHO-Lec-RMCE cell lines will be
improved in robustness and validated for their efficiency
of generating a substantial set of new expression cell lines.
The performance of these production cell lines in cell culture
bioreactors will be characterized. Development of new
cell lines for the co-expression of multi-protein complexes
on the basis of the CHO-Lec-RMCE cell lines will be one of
the major challenges in future.
PSPF-Training course in basic techniques in insect cell
cultivation and baculoviral expression of recombinant protein.
Photo: HZI, Bierstedt
Support The Protein Sample Production Facility (PSPF)
is a part of the HZI protein expression platform and was
established in 2007 in cooperation with the Max Delbrück
Centre for Molecular Medicine (MDC). The main goal of this
Helmholtz cooperation is the support of structural biologists
in Germany via circumvention of the major bottleneck of
protein production. The PSPF service is offered either on a
subsidised fee-for-service billing scheme or as a scientific
cooperation (www.pspf.de). Currently, 50% of the capacity of
the unit is used for internal HZI projects.
During 2010, a total of 20 PSPF projects have been processed.
Eight of these projects have been successfully finished, four
have been terminated and eight are still in progress. In 2010,
three scientists were trained and supported to establish the
expression of their target gene in the baculovirus expression
system in our lab. Several in-house HZI cooperation projects
for the analysis of multi-protein complexes have been undertaken
and include: TLRs (Schubert (University of the Western
Cape)/Heinz), HGF-cMet receptor interaction (Krausze/
Heinz/Gherardi), HCV Protease NS3/4 complexed with novel
inhibitory peptides (Collins/Schmelz) and the ABCB6 membrane
protein (Menzel/Heinz). The production of these proteins
is based either on the baculovirus expression system or
the Acembl/Multibac expression system, animal stable glycosylation
deficient CHO cell line cultivation or expression in
Pichia pastoris. Many projects are on-going as the production
of sufficient amounts of soluble and stable protein is still
limiting and requires extensive redesign and recloning of the
expression systems. For several of the projects the process of
crystallisation is still continuing and requires a continuous
supply of fresh and pure material.