Team:Waseda/Racemase
Racemase ~War in the mirror~
Introduction
In the zombie vs. Samurai storyline, we envisioned a scenario in which one side deprives their opponent of the food they need. A battle between two enantiomers, that is to say, one side has D amino acid and the other has L amino acid derived food as their food source, and both sides converts their opponent’s amino acid and deprives the of their food
Although there is not yet a translation and transcription system that efficiently encorporates D amino acids, it is known that the natural enyme chemically synthesized with D amino acids would be active.A protein made up of only d-amino acids act on substrates with opposite chirality, as demonstrated using the HIV protease.[Milton et.al 1992]
Besides, practical usage of D-amino acids is advancing in the field of Biotechnology today. Peptides composed of D-amino acids are not susceptible to degradation by protease in L-body organisms, thus are highly stable. [Tugyi et.al 2005][ Garton et.al 2018] Consequently, researchers are seeking a way to make the target of D-amino acid Peptide Aptamers(A type of antibody) L-body organisms’ protein. [Oberthür et.al 2015][ Majier et.al 2016][ Schumacher et.al 1996][ Funke et.al 2009]Currently, target proteins are synthesized with D-bodies, and L-body peptide aptamers that bonds to the protein are artificially evolutionized. When protein is expressed with the obtained amino acid sequence with D-amino acids, the binding site would consist of L-bodies. [Oberthür et.al 2015][ Majier et.al 2016][ Schumacher et.al 1996][ Funke et.al 2009]
Such storyline can be applied to our real experimental system by adopting racemase, an enzyme which catalyzes interconversion of D amino acid and L amino acid. Racemase catalyzes interconversion between L-amino acid and D-amino acid. There are many kinds of racemases and each corresponds to respective amino acid. This year, we focused especially on alanine racemase. Alanine racemase (AR) is a fold type III racemase enzyme catalyze the conversion of L-alanine to D-alanine. [Walish 1989]This enzyme provides D-alanine consuming L-alanine and using pyridoxal 5- phosphate (PLP) as a cofactor. AR is unique to prokaryotes and plays an important role in a biosynthesis. [Walish 1989] [Azam et.al 2016] Since D-alanine is used as a necessary component of the peptidoglycan layer of bacterial cell walls, lack of alanine racemase can result in the inhibition of growth of prokaryotes. [Azam et.al 2016] As it is generally absent in higher eukaryotes such as human but is ubiquitous throughout bacteria. Therefore, alanine racemase is recognized as an attractive target for antibacterial drug development. [Azam et.al 2016]
Experiment
In this experiment, alanine racemase (AR) gene carried on our new part (BBa_K3580200) on a T5 promoter-controlled pCA24N vector was transformed into the BL21(DE3) star strain. After expression induction by IPTG, the BL21 cells were collected by centrifugation, then sonicated and His-Tag purification was performed(Fig.)(Fig.). The meanings of each abbreviation are given in Table 1.
To assess the activity of purified racemase, fluorescence values of GFPS1 synthesized by a combination of GFPS1 plasmid, L-alanine, D-alanine, and purified AR were measured in the Pure System, a reconstituted CFPS that contains no enzymes other than the translation system(Fig. 3-2-4)[Shimizu et.al 2001].
The natural translation system is difficult to take up D-amino acids. [Kuncha 2019]19 amino acids other than alanine are L-bodies, so if only alanine is D-body, even if the sequence is correct, it has no activity as an enzyme(Fig. 3-2-5).
Since GFP is composed of L-amino acids, when GFPS1 takes up L-alanine, the system would give off fluorescent effect, and when D-alanine and AR are added, the racemized L-alanine can be used for translation and the fluorescence value was expected to be restored(Fig. 3-2-6). The evaluation of the effect of amino acids on the synthesis of a reporter protein is the most appropriate methodology for this scenario as a "food source".
We decided to assay AR by using PURE frex donated by GeneFrontier, Inc. As PURE frex does not contain any extra metabolic enzymes other than those involved in transcription and translation, we would be able to perform the assays without having to control the metabolic system, which have plagued the assays with extract based CFPS. First, we identified problems with commercial D-alanine that may be contaminated with L-alanine (Fig. 3-2-7). The fluorescence values of the expressed GFP were measured by real-time PCR and the measured fluorescence values were quantitatively calibrated by FITC.
Fig. 3-2-7 shows a comparison of the fluorescence values at a plateau. Although slightly synthesized GFP was identified by contaminating L-alanine with commercially available D-alanine, was found to be acceptable as a negative control, so the assay with AR was confirmed by the combination of each alanine and racemase (Fig. 3-2-8) (Fig. 3-2-9).
Fig. 3-2-8 shows the temporal variation of GFP fluorescence value.
Fig. 3-2-9 shows a comparison of the fluorescence values of each sample at 120 min, when GFP expression seems to have plateaued.
The results demonstrated that GFP could not be synthesized by D-alanine alone, but L-alanine produced by racemase-mediated racemization of D-alanine and L-alanine could be used for translation, creating a situation in which fluorescence was restored by the synthesized GFP. In other words, we succeeded in creating a situation in which the opponent’s food was converted into food for themselves in the Zombie vs. Samurai scenario. Additionally, when the amount of D alanine reacting with alanine racemase was reduced, the fluorescence value of the synthesized GFP was lower but significantly higher than that of the negative control, confirming the deprivation even in a situation where food was originally low (Fig. 3-2-9 ).
This result indicates that the system is highly sensitive to the substrate, as the corresponding expression of the reporter was lowered in a low substrate situation.
We also used an extract-based CFPS system for assays of purified racemase, prior to performing experiments in the PURE system. As with the PURE system method, fluorescence of GFPS1 synthesized by a combination of GFPS1 plasmid, L-alanine, D-alanine, and purified AR were measured in CFPS of dialyzed extract of E. coli. The results of this experiment are shown in Fig3-2-11.
As the results show, we were not able to consistently achieve the results we expected. Despite various subsequent trials and errors, we were unable to successfully achieve the theoretical results. We hypothesized that the main reason for this is that L-alanine is replenished by various enzymatic metabolic pathways in the extract solution and cannot be fully controlled, hence, we attempted to assay AR after adding the enzyme inhibitor, beta-Chloro-L-alanine[Whalen et,al 1985] [Arfin et.al 1971], in E.coli crude extract. Since beta-Chloro-L-alanine is known to irreversibly inhibit PLP dependent enzyme including AR[Manning et.al 1974] [Mobashery et.al 1987], the beta-Chloro-L-alanine reacted extract was dialyzed to reduce the amount of beta-Chloro-L-alanine in the sample. Thus, AR was added to the dialyzed reaction solution which theoretically does not contain β-Chloro-L-alanine(Fig.3-2-12).
Fig3-2-12 shows that the inhibition of metabolic synthesis of L-alanine by β-chloro-L-alanine is weak and difficult to control. After that, we tried to react the extract with β-chloro-L-alanine at higher concentrations, but its ability as an extract for CFPS was greatly reduced.
As a result, despite various subsequent trials and errors, we were unable to successfully achieve the theoretical results. Conversely, it is clear that the robustness found in living life in general is also found in the extract-based CFPS such as the one used in this experiment.
Summary
In our project, alanine racemase was expressed in E. coli and purified to create a situation with and without L-alanine as a substrate required for the cell-free protein synthesis of the reporter protein. Different situations resulted in different expression of GFP in the cell-free system, which indirectly assayed racemization by racemase. Our experiments were performed in the PURE system and achieved theoretical results in each condition, consistent with the 'war in the mirror' scenario. Furthermore, the system was confirmed to be sensitive to substrate amount.
Alanine racemase is generally absent in higher eukaryotes such as human but is ubiquitous throughout bacteria. Some bacteria are known to be resistant in a variety of settings (optimal pH[Watanabe et.al 2002], etc) and should be considered when performing drug discovery assays.
Material and methods
Expression and purification of alanine racemase
The composition of buffers used was as follows:
Buffer S, 50mM Tris-Cl pH 7.6, 300mM NaCl, 0.05% TritonX-100, 1mM DTT;
Buffer W, 50mM Tris-Cl pH 7.6, 300mM NaCl, 1mM imidazole, 1mM DTT;
Buffer E, 50mM Tris-Cl pH 7.6, 300mM NaCl, 100mM imidazole, 1mM DTT;
Buffer D, 50mM Tris-Cl pH 8.0, 100mM NaCl, 100µM EDTA, 1mM DTT;
For the purification of alanine racemase, our new part (BBa_K3580201) on the E. coli BL21(DE3)star strain on a T5 promoter-controlled, chloramphenicl-resistant pCA24N vector was transformed by the heat shock method on LB agar media. The transformed BL21(DE3)star was grown overnight on 3 ml of LB media with 25ng/µl chloramphenicol(LB-Cm). The culture medium was diluted 100-fold in 200 ml of fresh LB-Cm and incubated at 37°C, 140 rpm until OD 600nm =0.7-0.8. IPTG was then added to the culture medium to a final concentration of 1mM and incubated at 30°C, 140 rpm for 19 hours. After incubation, the cells were collected by centrifugation and frozen at -80°C. The bacteria suspended in Buffer S was sonicated and the lysate was centrifuged for 10 min at 12,000 rpm and the supernatant was collected. The supernatant was rotated with equilibrated Ni beads (50% slurry, His 60 Ni super flow resin, takara) for 1 hour at 4°C. Rotated material was passed through a 20 ml Open column (Econopak, biorad) and washed twice with Buffer W. The alanine racemase His-Tag was purified by elution of 1 ml each with Buffer E. SDS-PAGE confirmed a concentrated fraction and the sample was dialyzed with Buffer D. The dialyzed samples were stored at -80°C. The concentration of the purified racemase was quantified by the Bradford method using a dilution series of BSA(Bio-Rad).
Racemase assay in a PURE system
Indirect assays for AR were performed using the PURE frex donated by Gene Frontier Inc. The combinations are shown on Table.3-2-2. The racemization reaction was performed in solution with final concentrations of 5 mM D-alanine, 10 µM PLP, 50 mM potassium phosphate, and 57.5 µg/ml AR mixed together and incubated at 37°C for 1 h. The amount of racemase used was determined taking into account the amount of D-alanine substrate and Vmax[Strych et.al 2007][Ju et.al 2010].The sample was Filtered using centrifugal filter device at 14,000g for 30min (Amicon Ultra-0.5 Centrifugal Filter Devices 3K) and the filtered sample was collected. The filter device was washed prior to centrifugation of the sample using the buffer used in the AR reaction. the sample was centrifuged to collect the filtered product. Filtering allowed us to collect substances other than AR from the incubated product.CFPS reactions were performed on a 10µl scale and racemization reactions were performed on a 100µl scale.
Racemase assay in an extract-based CFPS
The purified AR was assayed indirectly by an extract-based cell-free protein synthesis system (CFPS). Fluorescence values of GFPS1 synthesized in CFPS of dialyzed E. coli crude extract by a combination of GFPS1 plasmid, L-alanine, D-alanine and purified AR were measured. The compositions are as follows(Table3-2-3 ).The composition of mixA and mixB are as follows(Table.3-2-4)(Table.3-2-5).
Cell extract(S12) preparation for Racemase assay
The preparation of E. coli extracts for the racemase assay essentially followed the method described on extract protocol( Go to Protocol page )
As we wanted to exclude small molecules in the extract, especially amino acids in this case, from the extract, we dialyzed the extract using S12 Buffer after run-off reaction according to Jewett's method[Silverman et.al 2019].The run-off reaction was performed by incubating the extracts in 15 ml tubes in a shaker at 80 min, 37°C, 220 rpm, shaded with aluminum foil.
Reaction of the extract with beta-chloro-L-alanine
The extract was incubated with 40 mM beta-chloro-L-alanine at 37°C for 80 min. The reaction solution was then dialyzed in S12 Buffer four times to exclude β-chloro L-alanine.
Measurement of fluorescence values during the reaction
Measurements of cell-free expressed GFP fluorescence values were performed by real-time PCR(Step One Plus Real-Time PCR System, Applied Biosystems, Mx3005P, Stratagene California) and the measured fluorescence values were quantitatively calibrated by FITC.
Reference
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