We developed our own low-budget protocol for expressing proteins in a prokaryotic cell-free expression system based on an Escherichia coli lysate. Most of the established cell-free expression systems are based on rabbit reticulocytes, wheat germ cells, insect cells or E. coli cells. Due to the prokaryotic origin of most of the proteins expressed during our project, we decided to develop our own low-budget prokaryotic cell-free expression system, the DiaMIX, based on an E. coli cell culture.
To produce the DiaMIX we used the E. coli strain BL21, as it is carrying a gene coding for the T7 RNA polymerase, which facilitates an improved expression of proteins during the cell-free expression. The cells were induced with IPTG prior to further treatment to initialize T7 polymerase expression, therefore allowing a high rate of protein synthesis.
Since all the coding sequences used in our project were manufactured synthetically, the E. coli BL21 cells were additionally transformed with a plasmid (pRARE2) containing the coding sequence of tRNAs for the translation of rare codons (AGA, AGG; AUA, CUA, GGA, CCC and CGG).
According to an optimized protocol adapted from the European Molecular Biology Lab (EMBL) Heidelberg, the lysate was prepared from E. coli BL21 cells, frozen in liquid nitrogen and afterwards stored at -80°C. This lysate contains the whole transcription and translation apparatus of the cell, allowing a successful expression of proteins from DNA templates.
However, the E. coli lysate is just one of the main components of the DiaMIX. To finally be able to express proteins, the system needs additional buffers, amino acids, nucleotides and energy sources. We obtained an L-amino acid kit from Sigma-Aldrich and prepared an accurate mix ourselves. The energy generating system based on creatine phosphate, phosphokinases and buffers as well as the mix containing nucleotides and amino acids (see protocol LINK) were prepared and added to the lysate directly before expression.
After adding all three components the complete cell-free expression mix was subsequently used to express proteins. Therefore, the reaction was started by adding DNA coding for the respective proteins.
To verify the functionality of our system we compared it to a commercially available kit.

To validate the expression of correctly folded proteins applying the DiaMIX we used various methods like fluorescence measurements (link protocol), luciferase assays (link protocol) and Western Blots (link protocol). One of our first approaches was the expression of GFP (LINK), which can be detected via its fluorescence. For expression of GFP we used a plasmid template (LINK) that was added to the self-prepared expression mix. Expression was then visualized via a microplate reader measurement that monitored the increase in emitted light at a wavelength of 520nm during progression of the expression. To verify the obtained data from this measurement a Western Blot was performed additionally. We used GFP antibodies to show the expression of GFP in our mix.
If you are interested in the results of this experiment, have a look here.

As a second test for our system we performed a luciferase assay. In contrast to the expression of GFP which can be followed by a gradually rising of GFP over time, the luciferase assay is based on the emittance of light when luciferin is conversed into oxyluciferin. This reaction is catalyzed by the enzyme luciferase and doesn’t allow a tracking of expression over time. Moreover it is not dependent on correct folding and offers detection within seconds. We used the coding sequence of the firefly luciferase (pBESTluc^TM) to express the protein with our cell-free expression system. The substrate luciferin was added to the mix after expression was finished. Then, the luminescence was measured at once using a microplate reader.
For more information about the outcome of this analysis, check out our results. This analysis helped us to evaluate the concentration of expressed protein in the cell-free expression system.

The enzyme luciferase catalyzes the reaction of D-luciferin to D-luciferyl using ATP as an energy source. D-luciferyl is instantly converted into oxyluciferin, whereby energy is emitted as light. The intensity of bioluminescence is thus proportional to the amount of expressed luciferase.

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, whether 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.

One of the core components of the DiaCHIP is the copying mechanism that allows production of a protein microarray from a DNA template. The following section summarizes our achievements concerning this procedure. Altogether we established a cell-free expression system that can be used to translate proteins from DNA immobilized on a PDMS slide. The expressed protein is then immobilized on the opposite glass slide via a specific tag system resulting in a distinct pattern. A microfluidic system is used to flush the slide with an antibody solution. Specific antibody-antigen interactions were successfully detected by imaging reflectometric interference (iRIf), a label-free detection method. For diagnostic applications, the immobilized proteins are antigenic peptides, specific for a certain pathogen.

The first step needed for copying the DNA array is to genetically fuse antigen coding sequences to a 10xHis tag that is used for surface immobilization later on. The whole expression cassette including promoter and terminator regions is amplified by PCR using an amino-labeled reverse primer. Via this amino group, the DNA is immobilized on an activated PDMS surface. The forward primer used for this PCR is labeled with Cy3. As it is shown in figure 1, spotting the DNA on the activated surface resulted in a distinct pattern visualized by Cy3 fluorescence.

Figure 2: Spotting of expressed GFP Cellfree expressed GFP-Lysate was spotted on the activated glass slide after expression was performed for 2 hours at 37°C. For detailed reaction check our labjournal.

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 already 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.

Figure 2: On-slide expression of GFP Cell-free expression mix was spotted on the activated glass slide and expression was performed for 2 hours at 37°C. For detailed reaction check our labjournal.

On-slide Expression of GFP

Figure 2: In-chamber expression of GFP Cell-free expression mix was pipetted into the flowchamber with immobilied DNA on the silicone slide and an activated glass slide. The expression was performed for 2 hours at 37°C. For detailed reaction check our labjournal.

In-chamber Expression of GFP

This step would show us if we would 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

Testing Different Conditions