Previously, we have developed a system for cell-free protein synthesis operated at high temperatures using a lysate of thermococcus kodakaraensis. Her... Sequences - Chemistry - Protein engineering - Biochemistry - Purification - Archaea - Genomics - Bioinformatics - Chemical industry - USA Councils - genetics - molecular biophysics - proteins - multiple gene products - single polycistronic mRNA - thermococcus kodakaraensis-based translation system - acetyl-CoA synthetase III - intracellular enzyme complex - C-terminus of TK0943 protein - TK0943-HA proteins
Synthesis of multiple gene products from a single polycistronic mRNA using the Thermococcus kodakaraensis-based translation system Tamotsu Kanail, Takashi Endohl and Tadayuki Imanaka2
1) Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan. 2) Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Noji Higashi, Shiga 525-8577, Japan. Abstract: Previously, we have developed a system for cell-free protein synthesis operated at high temperatures using a lysate of Thermococcus kodakaraensis. Here, we tested the synthesis of multiple gene products from a single mRNA. The selected target proteins were TK0944 and TK0943 proteins, the a-subunit and the ,-subunit of acetyl-CoA synthetase III (ACS III) forming an intracellular enzyme complex composed of two a- and two ,-subunits (a2P2). To facilitate the detection and purification of synthesized protein, a His-tag was attached to the N-terminus of TK0944 protein (His-TK0944) and an HA-tag was attached to the C-terminus of TK0943 protein (TK0943-HA). By His-tag purification, only His-TK0944 protein was detected by SDS-PAGE analysis. On the other hand, TK0943-HA protein was detected in the fraction after cell-free protein synthesis. These results indicated that His-TK0944 and TK0943-HA proteins were simultaneously synthesized from a single polycistronic mRNA by using the T. kodakaraensis translation system. 1. INTRODUCTION An operon is a genetic unit consisting of a cluster of structural genes and the operator and promoter sequences, and is transcribed as a single unit to produce one polycistronic mRNA. Operons occur primarily in prokaryotes (Bacteria and Archaea). The first operon described was the /ac-operon in Escherichia coli, by Jacob and Monod in 1961 (6), which is required for the transport and metabolism of lactose. In general, structural genes in the same operon encode proteins sharing a common physiological task. In the case of the lac operon, three structural genes (lacZ, lacY, and lacA) encode 3-galactosidase, lactose permease, and thiogalactoside acetyltransferase. Up to now, we have succeeded in the synthesis of a single gene product (ChiAA4 and tGFP) using the T. kodakaraensis cell-free protein synthesis system (2-4). To further develop the T. kodakaraensis cell-free protein synthesis system for a broader range of applications, synthesis of multiple gene products is one of the important topics to address. Like the other members in Archaea, T. kodakaraensis contains operons on its genome (5) suggesting the presence of polycistronic mRNA.AAGT
Here, we have tested whether multiple gene products could be synthesized from a single mRNA using the T. kodakaraensis cell-free system. 2. MATERIALS AND METHODS Chemicals - Sulfur, Tris-acetate, ammonium acetate, polyethyleneglycol 8000 (PEG8000), and potassium phosphoenolpyruvate were purchased from Wako Pure Chemical Industries (Osaka, Japan). ATP, GTP, CTP, UTP,
20 amino acids, CoA and hydroxylamine were from Sigma (St. Louis, MO, USA). RNase inhibitor was RNAsecureT from Ambion (Austin, TX, USA). All the other reagents were obtained from Nacalai Tesque (Kyoto, Japan). Plasmid construction - A 220 bp-DNA fragment containing the T7 promoter region and the ribosome-binding site of the T kodakaraensis glutamate dehydrogenase gene (RBS) was amplified from pTRC1 (4) by PCR using the two primers, T7RBS-N following (5'AAAAGAATTCACTCTAGCTAGAGGATCTCGATCCC -3', underlined sequences correspond to EcoRI site) and T7RBS-C (5'- AAAAGTCGACTTCCCGTGAGGTTGT AGTACTCAAC -3', underlined sequences correspond to SalI site). After treatment with EcoRI and SalI, the amplified fragment was inserted into the respective sites of pUCI 18, to make pUC-t7rbs. A 2,223 bp-DNA fragment containing acetyl-CoA synthetase III operon genes (TK0944 and TK0943) was amplified with genomic DNA of T kodakaraensis KODI by PCR using the following two primers, ACS-N (5'AAAACATATGCACCACCACCACCACCACTCAGAGA AAATCGTCGAA-3 ', underlined sequences correspond to NdeI site) and ACS-C (5'- AAAAGTCGACTC AGGCGTAGTCCGGAACGTCGTACGGGTACTCTTTC TTTTCTGGAGC-3', underlined sequences correspond to SalI site). Nucleotide sequences of these primers were arranged to fuse a hexahistidine-tag (His-tag) at the amino-terminus of TK0944 protein (His-TK0944) and a hemagglutinin tag (HA-tag) at the carboxyl-terminus of TK0943 protein (TK0943-HA) (Fig. 1). After treatmentAwith NdeI and SalI, the amplified fragment was inserted into the respective sites of pUC-t7rbs, to make pTACS1 A 61 bp-DNA fragment containing an artificial stem-loop structure, pHP1O (1, 3), was synthesized using the following two primers, Php-SLN1 (5'AC GCTGATGAAATA GCT -3', underlined sequences correspond to SalI site) and
V C XC
AA GA(AGA .
Fig. 1 (A) Schematic drawing of plasmid pTACS1-SL used for preparation of mRNA coding for His-TK0944 and TK0943-HA. (B) Possible structure of a heterotetramer composed of His-TK0944 and TK0943-HA.
(5'- AAAAGTCGACTGCAGCTGAATAGA GCTCACTCT -3', underlined sequences correspond to Sall site). The synthesized fragments were treated with SalI, and inserted into the respective sites of pTACS1, to make pTACS1-SL (Fig. lA). mRNA preparation - A 2469 bp-DNA fragment containing the T7 promoter region, RBS, and TK0944-TK0943 genes was amplified by PCR using pTACS1-SL as a template and the two RVS (5'following primers, ACACTTTATGCTTCCGGCTC -3') and Php-SLC1. This fragment was used to prepare mRNA by the T7 RiboMAXTm Express RNA system (Promega, Madison, WI, USA). The synthesized mRNA was suspended in RNase-free water and stored at -80 °C until use. Reaction conditions for cell-free protein synthesis - T. kodakaraensis lysate used for cell-free protein synthesis (S30 extract) was prepared as described previously (2) using T kodakaraensis KHR1 strain (Aphr). Reactions for protein synthesis were performed at 65 °C for 60 min in a 30 F>L mixture containing mRNA (12 rig), T kodakaraensis S30 extract (16 mg/mL), magnesium acetate (4.0 mM), potassium acetate (250 mM), ammonium acetate (80 mM), Tris-acetate (56 mM, pH 8.2), ATP (3.0 mM), GTP (1.5 mM), CTP (1.5 mM), UTP (1.5 mM), potassium phosphoenolpyruvate (10 mM), PEG8000 (2 %, w/v), spermidine (0.2 mM), 20 amino acids (2.0 mM, each), and RNase inhibitor (RNAsecure-T) (4.0 , v/v). Purification of the synthesized protein - After reactions of protein synthesis, the synthesized protein was purified with the TALONspinTM column (Takara Bio, Kyoto, Japan) for the purification of polyhistidine-tagged proteins. The purified fractions were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis
(SDS-PAGE, 12.5 % acrylamide concentration) and visualized with silver staining. Western blot analysis - After cell-free protein synthesis, 0.5 FiL of the reaction mixture was analyzed by SDS-PAGE (12.5 % acrylamide concentration). Western blot analysis was performed according to the procedure described previously (4) using rabbit anti-HA tag antibodies (Sigma) or mouse anti-His tag antibodies (Sigma). Acetyl-CoA synthetase assay - Acetyl-CoA-forming activities were measured by a modified hydroxamate method (7). Reactions were performed in a mixture (500 FiL) composed of protein mixture, 2 mM CoA, 20 mM ATP, 100 mM hydroxylamine, 5 mM MgCl2, and 50 mM acetate (pH 6.5) in 140 mM MES-NaOH buffer (pH 6.5). After incubation at 75 °C for 30 min, 350 FiL of 20% trichloroacetic acid and 150 FiL of 1 M FeCl3 were added, and formation of the iron-acetylhydroxymate complex derived from acetyl-CoA was determined spectrophotometrically at 520 nm. 3. RESULTS
Cell-free synthesis of the T. kodakaraensis acetyl-CoA synthetase III gene operon - Acetyl-CoA synthetase catalyzes the formation of acetate from acetyl-CoA and couples this reaction with the synthesis of ATP from ADP and inorganic phosphate. Acetyl-CoA synthetase III of T kodakaraensis (ACSIII) is a heterotetrameric enzyme composed of two oc- and two n-subunits (ut212) (T. Awano, A. Wilming, H. Atomi and T. Imanaka, unpublished data). Genes encoding these subunits constitute a single operon (TK0944-TK0943); where TK0944 encodes the oc-subunit and TK0943 encodes the n-subunit.
66 | His-TK0944
* nonspecific band
TK4J943-HA (282 kA)
Fig. 3 Detection of His-TK0944 and TK0943-HA by Western blot analysis using the reaction mixtures in the presence (+mRNA) or absence (-mRNA) of mRNA. Detection was performed using polyclonal antibodies against anti-His tag (upper panel) or anti-HA-tag
with His-TK0944, this protein should also be present in the purified fraction. However, the protein could not be detected. The TK0943-HA protein simply may not have been synthesized in the cell-free system, or the interaction between these two proteins may not be strong enough for the two to maintain contact during the purification procedures. To determine whether or not the TK0943-HA protein was synthesized, Western blot analysis was performed for the reaction mixtures using anti-HA-tag antibodies. As a result, a band corresponding to the molecular weight of TK0943-HA (28.2 kDa) was clearly detected together with His-TK0944 (52.7 kDa) (Fig. 3), indicating successful synthesis of both gene products (His-TK0944 and TK0943-HA) from a polycistronic mRNA by the T.
* oTK0943-HA (28.2 kDa)
14.4 Fig. 2 Purification of His-TK0944 from the reaction mixtures in the presence (+mRNA) or absence (-mRNA) of mRNA coding for His-TK0944 and TK0943-HA. Purified fractions were separated by
kodakaraensis cell-free translation system. As the synthesis of both ACS subunits were confirmed, ACS activities in the reaction mixtures were determined. ACS activities in the mixtures with or without the presence
SDS-PAGE and visualized by the silver staining procedure. Asterisks indicate the protein bands absorbed nonspecifically to the TALONspin
The ACSIII operon was selected as a target to be synthesized by the T. kodakaraensis cell-free translation system. For detection and purification of the proteins synthesized, template genes were modified to attach a His-tag to the amino-terminus of TK0944 protein (His-TK0944), and a HA-tag to the carboxyl-terminus of TK0943 protein (TK0943-HA) (Fig. 1). With a polycistronic mRNA coding for His-TK0944 and TK0943-HA, cell-free translation was performed at 65°C for 60 min, and the reaction products were purified using the TALONspin
column that selectively attach proteins with His-tag. When the purified fraction was analyzed by SDS-PAGE, a protein corresponding to the molecular weight of His-TK0944 (52.7 kDa) was clearly detected in a mRNA-dependent manner (Fig. 2), suggesting a successful synthesis of the first gene product. As for the second gene product, TK0943-HA protein, if this was synthesized and formed a stable complex
of mRNA were examined for ATP-dependent acetyl-CoA formation by the hydroxamate method. However, even in the mixture without mRNA, considerable ACS activities were detected (Abs. 0.568 ± 0.014), and no significant increase in activities could be detected by the presence of mRNA (Abs. 0.564 ± 0.017). 4. DISCUSSION In this study, an attempt was tested to synthesize two proteins (His-TK0944 and TK0943-HA) that potentially form a heterooligomeric complex, from a single polycistronic mRNA using the T. kodakaraensis cell-free translation system. Successful synthesis of both proteins was confirmed, but the complex formation of these proteins was not detected. There may be several explanations for this. The first one is that tag regions fused to these proteins might
interfere with complex formation. The crystal structure of acetyl-CoA synthetase II from Pyrococcus horikoshii has been determined for dimeric uc-subunits (PH0766: Protein Data Bank code 2CSU) and a monomeric n-subunit (PH1788: Protein Data Bank code 1WR2), independently. From these structures, Shikata et al. illustrated how these subunits are arranged (7). According to their prediction, a His-tag attached to the amino-terminus of TK0944 would be located outside of the complex, while the location of a HA-tag attached to the carboxyl-terminus of TK0943 is not clear and may inhibit complex formation. Therefore, the author also tested the synthesis of His-TK0944 protein with the wild-type TK0943 protein. However, in this case also, no complex formation was observed when His-TK0944 proteins were purified using the His-tag (data not shown), showing that the fusion of the HA-tag is not the major reason for the inability to form a complex. The second reason may be the limited affinity between TK0944 protein and TK0943 protein. Recently, assembly of the n-subunit protein (TK0943) with multiple cc-subunits (TK0944, TK1880, TK0139, and TK2127) was suggested in vivo (7). As the reaction mixture contains multiple uc-subunits in a large amount originating from the cell-free (S30) extract, it can be presumed that the synthesized TK0943-HA protein does not bind exclusively to the synthesized His-TK0944 protein. REFERENCES
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