Design and preparation of recombinant uvsY with or without His-tag
The His-tag is known to facilitate the purification of recombinant proteins. However, it sometimes decreases the solubility and activity of proteins . We previously expressed recombinant uvsX, uvsY, and gp32 as N- and C-terminal His-tagged proteins with a thrombin recognition site. In uvsX and gp32, the tags were removed by thrombin treatment. Meanwhile, thrombin treatment of the N- and C-terminal His-tagged uvsY (uvsY-NChis) resulted in precipitation . Fortunately, untreated uvsY-NChis was functional in the RPA reaction , indicating that the His-tag of uvsY does not abolish its function in RPA. However, it is possible that the uncleaved His-tag decreases the function of uvsY. To address this issue, we designed three new forms of uvsY, i.e., untagged uvsY (uvsY-Δhis), N-terminal His-tagged uvsY (uvsY-Nhis), and C-terminal His-tagged uvsY (uvsY-Chis). Figure 1 shows the E. coli expression plasmids for these three uvsYs and uvsY-NChis.
These four genes were expressed in E. coli BL21(DE3) cells. Purification was based on the procedure we previously described , but with several modifications. First, polyethyleneimine treatment of the soluble fraction of the cells, which was originally included to remove nucleic acids, was excluded. This prevented the viscosity of the solution from becoming so high that the flow rate of the successive Ni2+ affinity column chromatography was reduced. Second, ammonium sulfate fractionation was used instead. The ammonium sulfate concentrations at which uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis precipitated were 80%, 60%, 80%, and 40% saturation, respectively, indicating that the uvsY-NChis was the least soluble. From a 2 L culture, 25, 11, 11, and 9 mg of uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis were obtained.
Figure 2 shows the results of the SDS-PAGE analysis of the active fractions at each purification stage and the purified enzyme preparations. The purified uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis preparations yielded single bands with molecular masses of 16, 17, 17, and 22 kDa, respectively. The molecular masses of these uvsYs calculated from the amino acid sequences were 15,952, 17,419, 17,159, and 19,847 Da, respectively (Fig. 1), indicating that the two molecular masses were considerably different for uvsY-NChis, but were almost similar for uvsY-Δhis, uvsY-Nhis, and uvsY-Chis. These results suggested that when both the N- and C-terminal His-tags were present, the structure of uvsY was considerably altered.
Comparison of the solubility of uvsY with or without His-tag
The remaining soluble protein concentration was determined after thermal treatment at 42ºC. The natural logarithm of the soluble fraction was plotted against the incubation time (Fig. 3). The soluble fraction of uvsY-Δhis were stable. The soluble fraction of uvsY-Nhis decreased to 15% at 60 min. The soluble fractions of uvsY-Chis and uvsY-NChis decreased more rapidly than did uvsY-Nhis, and decreased to less than 5% at 60 min. These results indicated that the solubility was in the order of uvsY-Δhis > uvsY-Nhis > uvsY-Chis ≈ uvsY-NChis, suggesting that the presence of His-tag, especially C-terminal His-tag, reduced the solubility of uvsY.
Comparison of the optimal concentration of uvsY with or without His-tag in RPA
The effect of each uvsY concentration on the RPA reaction efficiency was examined. For this purpose, the RPA detection system for SARS-CoV-2 (Supplementary Figure 2), which we established previously , was used. Figure 4 shows the analysis of the products in the RPA reaction with each uvsY using agarose gel electrophoresis. The uvsY concentrations at which amplified DNA band was observed were 10–20 ng/µL for uvsY-Δhis, 10–40 ng/µL for uvsY-Nhis, 10–100 ng/µL for uvsY-Chis, and 40–100 ng/µL for uvsY-NChis. Non-specific bands were observed at 10–100 ng/µL uvsY-Δhis.
Our previous results indicated that uvsY concentrations that are too low or excessive are detrimental for the reaction . These findings were also evident for uvsX, gp32, and ATP . Thus, our results suggested that the specific activity of uvsY-Nhis was higher than those of the other three uvsY. We set 20, 20, 80, and 60 ng/µL as the optimal concentrations of uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively, and conducted the subsequent experiments.
It was first reported that by gel-shift assay, uvsX, uvsY, and gp32 form a ternary complex with a single-stranded DNA (ssDNA) . The presence of the ternary complex was also observed using surface plasmon resonance and isothermal titration calorimetry . However, binding of uvsY to ssDNA lessens the subsequent binding of the ssDNA to gp32 . Thus, uvsY and gp32 bind to ssDNA competitively. In the RPA process, this competition should be adjusted to achieve a high reaction efficiency by optimizing the concentrations of uvsX, uvsY, gp32, and ATP. If the binding of uvsY to DNA primer is not strong enough, the binding of uvsX to DNA primer will also not be strong enough. Thus, the DNA primer cannot invade double-stranded DNA, preventing it from binding to the target sequence. In contrast, if the binding of uvsY to DNA primer is too strong, the binding of uvsX to the DNA primer will be also too strong, and uvsX will remain occupied even after the elongation starts. This will prevent another nucleoprotein from binding to the target sequence and initiating the elongation.
Comparison of sensitivity and speed of RPA reaction using uvsY with or without His-tag
For comparison of sensitivity, RPA reaction was carried out using each uvsY with 60−6 ×107 copies of standard DNA at 41°C for 30 min. In the analysis of the products in the subsequent electrophoresis, the minimal initial copy numbers of standard DNA from which the amplified products were observed were 6 ×105, 60, 600, and 600 copies for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively (Fig. 5). Several non-specific bands were observed at 0−6 ×103 copies for the RPA with uvsY-Δhis (lanes 1−4 in Fig. 5A),
For comparison of speed, RPA reaction was carried out using each uvsY with 6,000 copies of standard DNA at 41°C for 10−60 min. The minimal reaction time at which the amplified products were observed were 20, 20, 30, and 20 min for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively (Fig. 6). More importantly, the RPA with uvsY-Nhis exhibited clearer bands than that with either of other three uvsYs. These results indicated that the reaction efficiency of RPA with uvsY-Nhis was higher than that with either of the other three.
The first crystallographic analysis of uvsY reported that uvsY exists as a hexamer . A more recent crystallographic analysis revealed that it exists as a heptamer and that one uvsY molecule consists of four α-helices (H1–H4: H1, E5–Y14; H2, L21–S65; H3, K80–S88; and H4, K91–E134) . When viewed from the top of the heptamer, H4 is located inside, whereas H1, H2, and H3 are located outside. In one heptamer, seven N-terminal residues are located apart from each other and seven C-terminal residues are located close together. In this study, the presence of a C-terminal His-tag of uvsY reduced its function in RPA (Figs. 4–6). It was previously described that cleavage of the His-tag by thrombin rendered uvsY-NChis insoluble . These results might be explained as follows. In the heptameric assembly, C-terminal peptides containing His-tag and thrombin recognition sequence in close proximity to each other alter the uvsY structure unfavorably, leading to decreased activity and precipitation by thrombin treatment. The results of solubility test (Fig. 3) supports this hypothesis. uvsY-Δhis exhibited higher solubility than other three uvsYs (Fig. 3). As for its low activity (Figs. 4–6), we presume that the preparation contained some impurities, such as nucleic acid, that inhibited the RPA reaction. Such impurities can be removed using Ni2+ affinity chromatography.
In conclusion, the reaction efficiency of RPA with N-terminal tagged uvsY was higher than that with untagged uvsY, C-terminal tagged uvsY, or N- and C-terminal tagged uvsY. Our results enhance the flexibility in fabricating RPA reagents for point-of-care use.