Technical Note pubs.acs.org/ac
Complementary Cell-Free Translational Assay for Quantification of Amino Acids Yeon-Jae Jang,† Kyung-Ho Lee,† Tae Hyeon Yoo,*,‡ and Dong-Myung Kim*,† †
Department of Chemical Engineering and Applied Chemistry, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134, Korea ‡ Department of Molecular Science and Technology, Ajou University, 206 Worldcup-ro, Yeongtong-gu, Suwon 16499, Korea S Supporting Information *
ABSTRACT: In this study, we present a simple and economical method that enables rapid quantification of amino acids based on their polymerization into a signalgenerating protein. This method harnesses amino aciddeficient cell-free protein synthesis systems that generate fluorescence signals in response to exogenous amino acids. When premixed with assay samples containing the amino acids in question, incubation of the cell-free synthesis reaction mixture rapidly resulted in the production of sfGFP, the fluorescence intensity of which was linearly proportional to the concentration of the amino acids. The assay method achieved a limit of detection as low as ∼100 nM and was successfully applied to the quantification of disease-related amino acids in biological samples. Compared with standard methods in current use that require chemical derivatization of amino acids and chromatographic equipment, the complementation assay method developed in this work enables the direct translation of amino acid titer into measurable biofluorescence intensity in a much shorter period, providing a more affordable and flexible option for the quantification of amino acids.
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integrity.15,16 On the basis of this, it was envisioned that a cellfree synthesis system could be developed into a simple biological assay platform by selective removal of certain components from the reaction mixture, which makes signal generation via protein synthesis dependent on the exogenous addition of the missing components.17 A reaction mixture for cell-free synthesis devoid of specific constituents is essentially analogous to a short electrical circuit. Similar to reconnecting a circuit, an incompetent cell-free synthesis reaction mixture can be activated to produce protein outputs upon complementation with the missing components. We attempted to extend this concept of a complementary translational assay to measure the concentration of amino acids. A reaction mixture for cell-free synthesis of superfolder green fluorescence protein (sfGFP) was prepared without the amino acids that were to be quantified. Due to the lack of a building block for protein synthesis, incubation of this incomplete reaction mixture did not produce a significant fluorescence signal. When premixed with assay samples containing the amino acids of interest, however, incubation resulted in the production of sfGFP-associated fluorescence, the intensity of which was linearly correlated with the amino acid concentration
mino acid analysis is important for a diverse range of areas, including food, environment, pharmaceuticals, and diagnostics.1−4 Current methods for amino acid analysis are based on liquid chromatographic separation with or without their derivatization,5−7 which involves complicated procedures, a long processing time, and costly laboratory setup. As an alternative approach, a biological method using auxotrophic Escherichia coli strains was recently reported.8,9 However, even though this demonstrated that the biological mechanism of protein synthesis could be implemented for assaying amino acids, the method has limitations, including a high background signal and the requirement for the construction of an auxotrophic strain for each amino acid to be analyzed. More importantly, maintenance of cell viability remains a challenging issue when developing cell-based assay methods as practical tools. Cell-free protein synthesis has emerged as a powerful approach that can overcome many of the limitations associated with cell-based gene expression methods.10,11 Unlike methods using living cells in which protein synthesis takes place within the highly complex and ordered cellular structure,12 cell-free synthesis works based on in vitro utilization of the machinery of protein biogenesis in a homogeneous reaction mixture.13,14 Due to being an open system, in principle, the constituents of cellfree synthesis can be individually manipulated without being limited by the requirement for maintaining cell viability and © XXXX American Chemical Society
Received: May 23, 2017 Accepted: August 4, 2017 Published: August 4, 2017 A
DOI: 10.1021/acs.analchem.7b01956 Anal. Chem. XXXX, XXX, XXX−XXX
Technical Note
Analytical Chemistry in the assay sample (Figure 1). This method was validated by measuring the concentration of amino acids in fetal bovine
serum (FBS). The presented method offers a rapid and economical alternative to currently used assay procedures that require substantial equipment and complicated sample treatment. We believe that the method can be further developed as a versatile assay platform for various applications, including onsite amino acid analysis and point-of-care diagnostic purposes.
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EXPERIMENTAL SECTION Construction of Cell-Free Protein Synthesis Systems. E. coli strain BL21-Star (DE3) cells were cultivated in 3 L of 2 × YTPG media at 37 °C. To induce the expression of T7 RNA polymerase, 1 mM isopropyl-thiogalactopyranoside (IPTG) was added to the culture broth when the OD600 reached 0.6. Cells were harvested in mid log phase (OD600 ∼ 4.5) and washed 3 times in 20 mL of wash buffer consisting of 10 mM TRIS−acetate pH 8.2, 14 mM magnesium acetate, 80 mM potassium acetate, 1 mM dithiothreitol (DTT), and 0.05% (v/ v) 2-mercaptoethanol (2-ME) per g of wet cells. Washed cells (10 g) were resuspended in 12.7 mL of lysis buffer (wash buffer without 2-ME) and lysed in a French pressure cell (Thermo Fisher Scientific, Waltham, MA) at a constant pressure of 20 000 psi. The cell lysate was centrifuged twice at 12 000g for 30 min to recover the supernatant (S12 extract). To remove residual amino acids, the S12 extract was centrifuged in a
Figure 1. Schematic illustration of the complementary cell-free translational assay for quantifying amino acids.
Figure 2. Standard curves for concentration of arginine, isoleucine, leucine, lysine, methionine, phenylalanine, tyrosine, and valine vs sfGFP fluorescence intensity. The inset image shows the concentration-dependent increase in sfGFP fluorescence in a microtiter plate. B
DOI: 10.1021/acs.analchem.7b01956 Anal. Chem. XXXX, XXX, XXX−XXX
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used to quantify the amino acids in biological samples. FBS was used for this purpose as a surrogate for clinical human samples. Five microliters of FBS was diluted 4-fold in water, and 10 μL of the diluted sample was added to incomplete cell-free synthesis mixtures (complete reaction mixtures lacking one of the 8 target amino acids). After a 1 h incubation of the resulting mixture, 15 μL was withdrawn and diluted in 200 μL of phosphate buffered saline (PBS), and the fluorescence intensity was measured. In the initial experiments, as shown in Figure 3A, cell-free synthesis reactions were not significantly activated
Vivaspin centrifugal concentrator (Sartorius Stedim Biotech GmbH, Göttingen, Germany) with a 50 000 Da molecular weight cutoff membrane.17 After addition of 18 mL of wash buffer to 2 mL of S12 extract, the concentrator was centrifuged at 2000g to reduce the volume of the diluted extract to the original volume (2 mL). This step was repeated three times. After the final concentration step, aliquots of S12 extract were flash frozen and stored at −80 °C. Amino Acid Analysis. Translational amino acid analysis reactions were conducted in 30 μL of cell-free synthesis reaction mixtures consisting of 57 mM HEPES-KOH, pH 8.2; 1.2 mM ATP; 0.85 mM each of GTP, UTP, and CTP; 80 mM ammonium acetate; 12 mM magnesium acetate; 80 mM potassium acetate; 34 μg/mL 1,5-formyl-5,6,7,8-tetrahydrofolic acid; 2 mM each of amino acids; 2% polyethylene glycol 8000; 3.2 U/mL creatine kinase; 67 mM creatine phosphate; 24% (v/ v) filtrated S12 extract; and 6.7 μg/mL plasmid pK7sfGFP in a 1.5 mL microtube. For measuring amino acids in assay samples, an incomplete reaction mixture devoid of the target amino acid was prepared in a volume of 20 μL. After they were mixed with 10 μL of assay sample, cell-free synthesis reactions were initiated by placing the microtube in a water bath set at 30 °C for 1 h, and the sfGFP fluorescence intensity signal was measured to be compared with a standard curve for determination of the amino acid concentration. Depending on experiments, assay samples were heat-treated for 10 min at 80 °C. Protein aggregates formed during heat treatment were removed using a centrifugal ultracentrifugation device. The results of the translational assay were verified using a Hitachi L-8900 amino acid analyzer (Hitachi High-Technologies, Tokyo, Japan) following the manufacturer’s protocols. SPSS version 22.0 software (SPSS Inc., Chicago, IL) was used for all statistical analyses. P > 0.05 was considered to be statistically indifferent.
Figure 3. (A) Cell-free synthesis of sfGFP after complementation of incomplete reaction mixtures with FBS. NC, negative control reaction without template DNA; PC, positive control with 2 mM each of the 20 amino acids; RM, reaction mixture. Gray bars, reactions without the addition of FBS; black bars, reactions with the addition of FBS. (B) Concentration of the eight amino acids measured by the standard amino acid analyzer.
by the addition of FBS, and the sfGFP fluorescence signal from all eight cell-free synthesis reactions was only comparable to that from a negative control reaction conducted in the absence of sfGFP DNA. Meanwhile, a separate HPLC assay showed that FBS contained 35−420 μM of the 8 target amino acids (Figure 3B). Because these concentrations were within or exceeded the range used for the preparation of the standard curves, it was presumed that FBS used in this experiment contained certain components that interfere with the cell-free protein synthesis reactions. Indeed, the translational efficiency of a complete cell-free protein synthesis reaction mixture was dramatically lowered by the addition of FBS in a volumedependent manner, indicating the presence of inhibitory components (Figure 4A). Although not specifically confirmed, it appears that the inhibitory effect of FBS can be at least in part attributed to nuclease activity because analysis of RNA extracted during the cell-free synthesis reactions revealed severe degradation of mRNA and rRNA in the presence of FBS (Figure 4B). In line with this assumption, the inhibitory effect was substantially reduced when FBS was heat-treated before its addition to cellfree synthesis reactions. Although a significant reduction in protein synthesis was still observed if large volumes of heattreated FBS were added (Figure 4A), it was also found that the inhibitory effect of FBS was eliminated almost completely when protein aggregates formed during the heat treatment were removed by ultrafiltration, and the degradation of RNA species was not observed in reactions containing heat-treated and filtrated FBS (Figure 4C). These results suggest that a portion of the heat-denatured RNases (and other deteriorating enzymes) refold during protein synthesis reactions.
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RESULTS AND DISCUSSION Cell-Free Protein Synthesis Systems Dependent on Exogenous Addition of Amino Acids. We selected eight amino acids as the initial assay targets: arginine, isoleucine, leucine, lysine, methionine, phenylalanine, tyrosine, and valine, each of which is related to different amino acid metabolism disorders (Table S1). As summarized in Figure 1, our method works by complementing the reaction mixture for cell-free protein synthesis containing 19 amino acids with the missing amino acid in the assay sample. For example, incubation of a cell-free synthesis reaction mixture devoid of lysine will only make truncated protein fragments and fail to generate a sfGFP fluorescence signal from the template DNA because ribosomes will stall at lysine codons of mRNA. Upon the addition of an assay sample containing lysine, however, translation can proceed to completion to produce full-length sfGFP and hence a fluorescence signal. We predicted that the intensity of the sfGFP fluorescence signal would be proportional to the lysine titer in the assay sample. Using standard solutions of varying amino acid concentration, we first prepared linear standard curves of amino acid concentration against sfGFP fluorescence intensity for the eight selected amino acids (Figure 2). The results showed that the sfGFP fluorescence was linear over a relatively wide range of amino acid concentration (up to 100 μM) for all 8 amino acids tested, and the limit of detection (LOD) of the amino acids ranged from 130 to 980 nM. Determination of Amino Acid Concentrations in FBS. We next examined whether the developed method could be C
DOI: 10.1021/acs.analchem.7b01956 Anal. Chem. XXXX, XXX, XXX−XXX
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performance of translational assays. For example, we found that four amino acids (aspartic acid, asparagine, glutamic acid, and glutamine) failed to give a titer-dependent sfGFP signal during the translational assay (Figure S1). Incubation of an incomplete cell-free synthesis reaction mixture without aspartic acid generated an intense sfGFP signal, and addition of exogenous aspartic acid was unable to further increase the fluorescence signal. Similar results were obtained with reaction mixtures devoid of asparagine, glutamic acid, and glutamine. These results indicate enzymatic interconversion of aspartic acid/ asparagine and glutamic acid/glutamine by the corresponding enzymes (glutamate synthetase, glutamine synthase, asparagine synthetase, and asparaginase) present in the cell extract. A straightforward solution for such issues resulting from cell-free metabolism unrelated to protein synthesis would be to employ the PURE cell-free synthesis system that is reconstituted with individually purified translational machinery components.18 In principle, changes in the amino acid concentration in the PURE system should be entirely dependent upon translation reactions. Indeed, translational assays of these four amino acids using the PURE system revealed a linear correlation between the sfGFP fluorescence signal and the amino acid concentration (Figure S3), whereas quantification was not possible using the extractbased cell-free synthesis system. However, given the high cost and complicated preparation steps of the PURE system, we believe it is more desirable to further engineer extract-based cell-free synthesis systems. A possible approach would be to prepare the S12 extract using E. coli strains in which the genes for the problematic enzymes are knocked out. Calhoun and Swartz previously showed that amino acids in a cell-free synthesis reaction mixture can be insulated from the cellular metabolism by selective removal of related enzymatic activities from the cell extract.19 Although not used in this study, a similar approach could be taken to develop affordable cell-free platforms for amino acid assays.
Figure 4. Inhibitory effects of FBS on the efficiency of cell-free protein synthesis. (A) Varying volumes of untreated (dark bars), heat-treated (gray bars), or heat-treated and filtrated (blank bars) FBS were added to standard reaction mixtures containing all components required for protein synthesis. The fluorescence intensity signal from cell-free synthesized sfGFP was measured after a 1 h incubation at 30 °C. (B) RNA species were extracted and electrophoresed after a 20 min incubation of cell-free synthesis reaction mixtures in the presence of untreated FBS. (C) RNA species were extracted and electrophoresed after a 20 min incubation of cell-free synthesis reaction mixtures in the presence of heat-treated and filtrated FBS.
Upon the addition of heat-treated and filtrated FBS prepared as described above, translational activity was restored in each of the eight incomplete cell-free synthesis reaction mixtures, generating a sfGFP fluorescence intensity signal proportional to the addition volume (Figure 5A). Importantly, the concen-
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CONCLUSIONS In this study, we developed a method for rapid and accurate measurements of amino acid concentrations based on the basic biological principle that amino acids are the building blocks of a functional protein. Taking advantage of the facile maneuverability of the cell-free protein synthesis system, the presence of amino acids of interest was directly translated into the measurable signal of a fluorescence protein. For the most of the 20 amino acids, this method could precisely measure their titers in a biological sample within a matter of hours without any chemical treatment or chromatographic separation steps. We believe that the presented method can be further developed into ready-to-use kits for amino acid analysis.
Figure 5. (A) Generation of sfGFP fluorescence in response to the addition of pretreated FBS (open circle, Arg; open diamonds, Ile; filled diamonds, Leu; open squares, Lys; open triangles, Met; filled circles, Phe; filled squares, Tyr; filled diamonds, Val. (B) Comparison of amino acid concentration measured by the complementary cell-free translational assay (blank bars) and the amino acid analyzer (gray bars). Labeled numbers indicate the percent recovery of the complementary translational assay. *p > 0.05.
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tration of amino acids obtained by comparing the resultant sfGFP fluorescence with the standard curves was almost identical to the actual concentration determined by the standard HPLC assay (Figure 5B). Furthermore, the translational assay was able to quantify most of the other amino acids (Figures S1 and S2). Cell-Free Metabolism Interferes with the Analysis of a Few Amino Acids. A cell-free synthesis system prepared with crude cell extract contains most of the cellular enzymes and can therefore metabolize biological molecules. It is thus not surprising that the concentrations of certain amino acids indicated translation-independent changes, which can affect the
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b01956. List of amino acid-related diseases; standard curves for quantification of Ala, Asp, Asn, Cys, Glu, Gln, His, Pro, Ser, Thr, and Trp; comparison between the complementary cell-free translational assay and standard HPLC assay; and standard curves for quantification of Asp, Asn, Glu, and Gln by the PURE system (PDF) D
DOI: 10.1021/acs.analchem.7b01956 Anal. Chem. XXXX, XXX, XXX−XXX
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Dong-Myung Kim: 0000-0001-7875-0694 Author Contributions
T.H.Y. and D.M.K. designed the experiments; Y.J.J. and K.H.L. performed the experiments, and D.M.K. supervised the work. Y.J.J., T.H.Y., and D.M.K. wrote the manuscript. All authors have given approval of the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by National Research Foundation of Korea (Grants 2015M3D3A1A01064878 and 2014M3C1A3051473).
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