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C6 Nanoanalytics for protein production processes

(Schilling/Ludwig)

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.

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