Methodology Cell-free Expression

Why Do We Use Cell-free Expression?

Cell-based protein expression is a well established method to obtain large amounts of a target protein. It enables accumulation and purification of the protein in quantities sufficient for various in vitro applications. Nonetheless, it is a tedious task to generate all the genetically modified organisms if a variety of proteins needs to be expressed. Additionally, purification of a distinct protein can not be performed according to a generalized protocol, but has to be optimized for every protein separately.
Cell-free expression represents a possibility to overcome many challenges of conventional protein expression and offers many advantages concerning our project in particular.¹⁾
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, for example by having it synthesized by a company, and can be stored until needed. In terms of purification, cell-free expression avoids the need for cell lysis and therefore circumvents this harsh procedure, thus preserving the protein. In cell-based expression, too strong induction often results in aggregated and therefore non-functional protein. This risk is minimized by using cell-free expression, since the expressed protein is dispersed in a far larger volume than the intracellular space.²⁾
For translating DNA templates into protein microarrays in a microfluidic set-up, cell-free expression is the method of choice. This system is capable of expressing many different sequences at once. Additionally, the microfluidic setup provides the opportunity to constantly supplement the expression. Replacing depleted components, like dNTPs, amino acids or energy sources like creatine phosphate, enables higher yields of protein.

Basics of Cell-free expression

Two basic components are needed to conduct in vitro protein expression:

the genetic template (mRNA or DNA) encoding the target protein
a reaction solution containing the necessary transcriptional and translational molecular machinery

Cell extracts supply all or most of the molecules of the reaction solution, including:

RNA polymerases for mRNA transcription
ribosomes for polypeptide translation
tRNA and amino acids
enzymatic cofactors and an energy source
cellular components essential for proper protein folding

Normally, cell lysate already contains most of the components needed for cell-free expression. The machinery that usually conducts Translation and transcription of various proteins in the organism can be "reprogrammed" to produce only the protein of choice. Our building blocks, the aminoacids and NTPs are already present but are also added in an increased amount to raise the efficiency of our system. As the normal energy regeneration system is missing in a cell lysate an artifical one is added. Here for example creatine phosphokinase is used. The other compontents present in an cell-free expression mix buffer the sensitive system and imitate a cytosolic environment.

Besides a bacterial based system, there are several other options with specific advantages and disadvantages. An E. coli system convinces with easyness, cost-efficiency and high yield. However, for the production of complex eukaryotic proteins, it might not be the best option. In contrast, eucaryotic cell-free lysates provide post-translational modifications such as core glycosylation or phosphorylation, and natural membrane components (e.g. microsomes) for membrane protein insertion. Here, the systems are based on wheat germ embryos, rabbit reticulozytes, insect cells and various other animal and human cell lines.

There are two variants to perform a cell-free reaction. In batch reactions, transcription and translation are carried out in a reaction vessel containing all necessary components. Due to different reasons like fast depletion of energy supply, degradation of components like nucleotides and decreasing concentrations of free Mg²⁺-ions, the reaction in the batch system usually reaches a plateau after about 1-2 hours.
In a dialysis mode, the cell-free transcription and translation reaction is carried out in a small reaction chamber that is seperated by a dialysis membrane from a 10-20x larger reservoir containing low molecular weight reagents. The reservoir supplies the reaction chamber with ions, energy substrates, nucleotides and amino acids. In turn, the low molecular weight by-products are efficiently diluted via the membrane from the reaction chamber into the reservoir. This setup will drive the reaction longer (usually 20-24 hrs) and results in higher levels of produced protein.

A further aspect that has to be discussed is the design of the DNA template.
In general plasmids, linear templates from for example PCR production or RNA can be used. The structural design of the DNA or RNA is in every case of great importance for yield and quality of produced protein. In bacterial 5′-UTRs such optimization can be achieved by placing the Shine-Dalgarno (SD) sequence at an optimal distance, typically eight nucleotides, from the AUG start codon. The SD sequence interacts with the 3′-end of 16S ribosomal RNA (rRNA) that is part of the small ribosomal (30S) subunit, to allow efficient initiation. Furthermore, the integration of an A/U-rich enhancer sequence further upstream allows the SD sequence to interact more effectively with the rRNA. Also a worthwhile trial in this context is the use of differnt tags to enhance expression as reported by Haberstock et al. (2012)³⁾. In eukaryotes, the Internal Ribosome Entry Site (IRES), is a highly structured element found within viral mRNA that is able to induce eukaryotic initiation. This solves the problem of ineffective capping, which presents a major restriction of high production. For example, the IRES from CrPV showed the ability to increase expression substantially across several species.

Our DiaMIX

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).

Link to pRARE2 (2015017LK)

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.

Proof of Protein

To validate the expression of correctly folded proteins applying the DiaMIX we used various methods like fluorescence measurements, luciferase assays and Western Blots. 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 520 nm 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.

Figure 1: Luciferase reaction. 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.

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, 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.

Immobilizing DNA on PDMS

Insert picture (20150917lk)

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 discover, whether our cell-free expression system reaches protein concentration high enough to be detected with iRIf, we spotted cell-free expressed GFP on iRIf slides. We then measured the binding of anti-GFP to the cell-free expressed GFP, to gather information about the amount of expressed protein and also about its folding status.

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

To estimate the specificity of our surface we evaluated the proportion of cell-free expressed GFP that bound to the surface. To investigate if lots of non-target proteins bound unspecific to the surface we measured the slide with iRIf using anti-GFP antibodies. Unspecific binding of proteins present in the DiaMIX would lead to decrease in signal, whereas a highly specific surface will only bind GFP and thus show a strong signal for the GFP spot.

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 if our whole system, from the cell-free expression until detection of the expressed proteins with antibodies is really working. Expression of the antigens inside the flowchamber with our DiaMIX and subsequent diffusion of the His-tagged proteins through the chamber will result in a distinct spot on the glass slide. When cell-free expression is finished the bound antigens would form an antigen array that ccan be used for detection of antigen-antibody binding events.

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 focused on a C. tetani epitope flagged by a double His6 and a Spy-Tag. For cloning of this construct we used our own cloning site and a vector that we optimized for cell-free expression.
The antigen was expressed in a tube reaction and spotted onto a Ni-NTA surface afterwards. Sadly, we could not show binding of the respective antibodies/serum with the iRIf measurement device. This could be due to folding problems of the antigen during cell-free expression. Moreover, the concentration of expressed C. tetani antigen might not have been high enough to be detected by iRif measurements.

Testing Different Conditions

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To identify the optimal DNA concentration that results in a maximum protein expression, we testet different conditions. For evaluation we used DNA concentrations in the range of 1 to 5 ng per 50 µl reaction. To verify our results every reaction was performed in triplicates with every reaction being treated the same way. We could show that the protein amount was highest when a DNA concentration of 2 µg was used (fig. X)
For this experiment, we used DNA coding for the enzyme luciferase. The protein concentration could therefore be measured at once using the luciferase assay.

Another component whose concentration is crucial for optimal cell-free expression, is magnesium. To evaluate the effects of different magnesium concentrations we started multiple cell-free expression mixes using different magnesium starting concentrations (12 to 16 mM). Magnesium was added to the mix in the form of magnesium acetate and cell-free expression took place for 2 hours. We again used triplicates for each reaction and treated all samples in the same manner (link protocol).
After expression, a luciferase assay could visualize the protein concentration in the different reaction mixes. We could show that a magnesium acetate concentration of 12 mM resulted in the highest protein concentration (fig.X).

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