Team:Freiburg/Methods/Cellfree
Methodology Cell-free Expression
Why do we use cell-free expression?
Cell-based protein expression is a well established method to obtain a large amount of a target protein. It enables to accumulate and purify the protein in quantities sufficient for various in vitro applications. Nonetheless, it a tedious task to generate all the genetically modified organisms if a variety of proteins needs to be expressed. Additionally, protein purification is not a generalized procedure, but has to be optimized for every protein itself. Cell-free expression represents a possibility to overcome many challenges of conventional protein expression in general and offers many advantages concerning our project in particular. Generally speaking, it saves a lot of time and money to avoid the generation of genetically modified organisms for every protein. Suitable DNA sequences are constructed once and can be stored until needed. In terms of purification, cell-free expression avoids the need for cell lysis preserving the protein from this harsh procedure. In cell-based expression, too strong induction often results in aggregating and therefore non-functional protein. This probability is minimized using cell-free expression because the expressed protein is dispersed in a far larger volume than the intracellular space. For copying a DNA template into a protein microarray in a microfluidic set-up, cell-free expression is the method of choice, because such a system is capable of expressing many different sequences at once. Additionally, the microfluidic system provides the opportunity to supplement the expression constantly with consumed (?) components, like dNTPs or amino acids, enabling high yields of protein expression.
Our system
We developed our own way of expressing proteins in a prokaryotic cell-free expression system based on Escherichia coli lysate. Starting with the lysate, the amino acids and the T7 polymerase, we prepared most of the components ourselves. To verify our system we used various commercial kits.
Proof of protein
We validated our expression via SDS PAGE and Western Blot. Moreover we checked with GFP and luciferase for fluorescence and luminescence, respectively.
Step by step validation
Since we aim to use cell-free expression in the DiaCHIP we decided to validate the process in single steps. Starting with expression in a tube we spotted already expressed proteins on iRIF slides to check wether or not we expressed detectable antigens that interacted with our antibodies at hand. As a next step we spotted our expression system on the slide without starting the reaction beforehand. By letting it incubate on a specific surface we could check for further possibilities. Lastly, we performed a cell-free expression from immobilized DNA in the finished setup of glass and PDMS slide.
Spotting of expressed GFP on slide
To establish, whether our cell-free expression system reaches protein concentrations high enough to detect in the iRIf, we spotted readily cell-free expressed GFP on iRIF slides. This not only gave us information about the amount of protein, but also on its folding. By seeing an interaction with a corresponding antibody in the iRIf measurement, we could draw conclusions on the epitope.
On-slide expression of GFP
Firstly we could detect wether the interaction between tagged proteins and the surface was strong enough to bind the proteins as soon as they were formed. This would show when measuring the slide in the iRIf afterwards. Secondly we wanted to assess if the surface was specific enough to not bind components of the mix itself.
In-chamber expression of GFP
This step would show us if we would already get our setup to work as a whole. Moreover, we could estimate, whether the diffusion patterns in a flow chamber differed too much from that in a suitable reaction tube.
Cell-free expression of antigens
Our biggest goal was to express antigens with the required tags in a concentration and folding state that suffices for an iRIf measurement. Here we focussed on a c. tetani epitope flagged by a double His6 and a Spy-Tag. The antigen was expressed in a tube and spotted on a Ni-NTA surface after expression.
Testing different conditions
To ameliorate the quantity of the cell-free expressed proteins, we varied MgOAc and DNA template concentrations. Here we tested for optimal amounts by using luciferase DNA for direct detection.
