C6 Nanoanalytics for protein production processes
The production of new recombinant
proteins (e.g., glycosyl transferases and antibodies) in biotechnological
processes with genetically modified microorganisms, such as Aspergillus
niger and Bacillus megaterium, is central topic of the SFB 578.
In order to optimize such production processes, the influence of
the physical and biochemical growth parameters on morphology and
protein production rate is described and modelled. Here the project
C6 (Schilling/Ludwig) will contribute by means of investigations
of the initial stage of the growth of the cultures using high-resolution
microscopy and by means of a new method for the measurement of the
protein concentration using magnetically labelled antibodies.
Our scanning force microscope is used for the in-vivo investigation
of the growth of individual microorganisms (spores / bacteria) with
a resolution of about 10 nm. Preliminary studies on samples of various
growth stage with and without specially patterned substrates provided
by the SFB were successfully carried out with the microscope. In
addition, our scanning force microscope allows us to record distance-force
curves which provide information on intermolecular interactions.
The investigated microorganisms A.
niger and B. megaterium have the ability to release the produced
proteins into the culture medium. The concentration of the proteins,
e.g., during the secretion, shall be studied using a new labelling
method. For this, antibodies on the surface of superparamagnetic
nanoparticles as markers are used to measure growth and production
kinetics. Superconducting quantum interference devices (SQUIDs)
and fluxgates, both being fabricated at our institute for years,
are used as magnetic field sensors. The magnetic field measuring
technique has been the main working field of the applicants for
more than 10 years. Nanoparticles marked with antibodies in various
modifications are commercially available. The labelling of the magnetic
nanoparticles in order to achieve the specificity for the target
proteins of the SFB is performed in collaboration with project A6 (Dübel) and using commercially available antibodies. Compared
to fluorescent labels the particular magnetic properties of the
markers allow one to use them in opaque media and without washout
steps. In addition, they can be manipulated in numerous ways by
force measurements and the magnetic separation.
Aimed at the development of such an analysis method, we developed
in the first year of the project a differential fluxgate setup for
magnetorelaxometry measurements. In magnetorelaxometry (MRX) the
superparamagnetic nanoparticles are aligned by an external magnetic
field – in our case provided by a Helmholtz coil system -,
and after turning off the magnetic field the relaxation behaviour
of the nanoparticles is analyzed. This arrangement allows MRX measurements
without any magnetic shielding. Systematic investigations of dilution
series of magnetite nanoparticles have shown that 100 nmol Fe can
be detected for a sample volume of 150 ?l. Currently we work on
the functionalization of the nanoparticles with regard to the realization
of biologically relevant experiments.