Results and Discussion
Expression and purification of Dopa-free bMAP and Dopa-bMAP without overexpression of any AARS. Dopa incorporation into bMAP was performed using pQE80-bMAP containing the gene of dMAP (dfp-3) according to a residue-specific incorporation protocol (Johnson et al., 2010; Link and Tirrell, 2005). Briefly, the pQE80-bMAP plasmid was transformed into AY12 Tyr auxotrophic E. coli strain generating AY12[pQE80-bMAP]. An overnight culture of the AY12[pQE80-bMAP]E. coli cells in LB media was inoculated into M9 minimal media containing 20 natural amino acids. When the cell density reached a set point, a media shift into Tyr-deficient M9 minimal media supplemented with Dopa was performed. Expression of bMAP was induced by the addition of isopropyl-D-thiogalatopyranoside (IPTG). The cell lysates before induction (BI) and after induction (AI) were subjected to SDS-PAGE analysis. The visible molecular weight of Dopa-incorporated bMAP on an SDS-PAGE gel appeared to be about 10 kDa (Fig. 2A, B), which is greater than the calculated molecular weight from amino acid sequence (about 7 kDa). Such a band shift was made likely by the high pI value of bMAP (10.24) and was consistent with band shifts reported previously (Yang et al., 2014). The purification of Dopa-bMAP was performed as described previously (Yang et al., 2014). The nitroblue tetrazolium (NBT) staining was reported to be a specific detection method of proteins containing quinone by redox-cycling (Paz et al., 1991). With NBT staining, bMAP bands in the after induction sample and purified sample were clearly observed in a blue-purple color, whereas no bMAP band in the before induction sample was detected (Fig. 2B). These results demonstrated that the Dopa were successfully incorporated into bMAP (Yang et al., 2014). In order to obtain Dopa-free bMAP samples, AY12[pQE80-bMAP] cells were cultured in LB media and bMAP was expressed using 1 mM IPTG. Dopa incorporation into bMAP was performed at 30 °C. The purification of Dopa-free bMAP was performed via metal affinity chromatography using a hexahistidine tag according to the manufacturer’s protocol (Qiagen).
To confirm incorporation of Dopa into Tyr residues of bMAP, Dopa-bMAP samples and Dopa-free bMAP were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Bruker Daltonics). bMAP has 10 Tyr residues. The mass of the major peak of Dopa-free bMAP was 6,652 Da (Fig. 3A), which is almost identical to the theoretical mass of Dopa-free bMAP (6,654 Da). The mass difference between Dopa and Tyr is 16 Da. The masses of the major peaks of Dopa-bMAP_Control were 6,811; 6,796; 6,780; and 6,765 Da, which matched well with the theoretical masses of bMAP samples containing 10, 9, 8, and 7 Dopa residues (6,814; 6,798; 6,782; and 6,766 Da) with less than 1% error, respectively. Since the average number of Dopa in the Dopa-bMAP sample was 9.1, the Dopa incorporation yield was 91%, consistent with the yields in the literature (Yang et al., 2014).
Expression and purification of Dopa-bMAP coexpressed with Dopa-specific AARS. In order to enhance the production yield of Dopa-bMAP, we explored the coexpression of Dopa-specific AARS. We chose MjTyrRS-Dopa as a Dopa-specific AARS. The original plasmid prepared for site-specific incorporation of Dopa into an amber codon has the gene forM. jannascii tyrosyl-tRNA amber suppressor (MjtRNATyrCUA). In this study, since Dopa should be incorporated into Tyr residues (UAU codon), the anticodon of amber suppressor was mutated into AUA-generating MjtRNATyrAUA using site-directed mutagenesis. In the final construction of pEVOL-Dopa plasmid, the gene of MjTyrRS-Dopa is under control of an inducible ara promoter and the gene of MjtRNATyrAUA is under control of a constitutive proK promoter.
Deficiency of Tyr in minimal culture media would not allow translation of functional MjTyrRS-Dopa. All 20 natural amino acids in minimal culture media are required to produce functional MjTyrRS-Dopa inE. coli cells. Therefore, MjTyrRS-Dopa should be expressed in the presence of all 20 natural amino acids in minimal media before media shift to Tyr-deficient minimal media. In this study, when the optical density at 600 nm (OD600) of cultured cells reached 0.7, arabinose was added to culture media to induce MjTyrRS-Dopa. After 30-min expression of MjTyrRS-Dopa, media shift was performed. Then bMAP expression was induced by the addition of IPTG and Dopa. Dopa-incorporated bMAP coexpressed with MjTyrRS-Dopa (Dopa-bMAP_MjTyrRS) and purification of Dopa-bMAP with coexpression were performed similar to preparation of Dopa-bMAP expressed without coexpression of any AARS, except the arabinose addition and 30-min incubation prior to media shift. The protein band of Dopa-bMAP_MjTyrRS was observed to be similar to that of Dopa-bMAP_Control (Fig. 2A, B). The spectrum of MALDI-TOF MS analysis of Dopa-bMAP_MjTyrRS exhibited a pattern similar to that of Dopa-bMAP_Control (Fig. 3C). The masses of the major peaks of Dopa-bMAP_MjTyrRS were 6,812; 6,796; 6,781; and 6,765 Da, which correspond with Dopa-bMAP samples containing 10, 9, 8, and 7 Dopa residues, respectively. The Dopa incorporation yield of Dopa-bMAP_MjTyrRS was 93%, which is comparable to that of Dopa-bMAP_Control.
Expression and purification of Dopa-bMAP coexpressed withE. coli TyrRS. As an alternative strategy to enhance production yield of Dopa-bMAP, we explored coexpression of E. coli TyrRS. The gene of E. coli TyrRS (EcTyrRS) was amplified from E. coli genomic DNA and was subcloned into pEVOL-Dopa to replace the MjTyrRS-Dopa gene, generating pEVOL-EcTyrRS. Therefore, the gene of EcTyrRS was also under control of the ara promoter; the amino acid sequence of EcTyrRS is shown in Supporting Information Table 1. Both pEVOL-EcTyrRS and pQE80-bMAP plasmids were transformed into AY12 cells to generate AY12[pEVOL-EcTyrRS][pQE80-bMAP] cells. Expression and purification of Dopa-bMAP in the presence of EcTyrRS (Dopa-bMAP_EcTyrRS) was performed similarly to Dopa-bMAP_MjTyrRS.
SDS-PAGE analysis demonstrated that the molecular weight of Dopa-bMAP_EcTyrRS was very similar to the molecular weight of Dopa-bMAP_Control and Dopa-bMAP_MjTyrRS (Fig. 2A, B). The spectrum of MALDI-TOF MS analysis of Dopa-bMAP_EcTyrRS exhibited a pattern similar to that of Dopa-bMAP_Control (Fig. 3D). The masses of the major peaks of Dopa-bMAP_EcTyrRS were 6,814; 6,799; and 6,783 Da, which correspond to Dopa-bMAP samples containing 10, 9, and 8 Dopa residues, respectively. The Dopa incorporation yield of Dopa-bMAP_EcTyrRS was about 90%, which is comparable to that of Dopa-bMAP_Control. The characterization results of Dopa-bMAP_MjTyrRS and Dopa-bMAP_EcTyrRS led to a conclusion that they have properties comparable to those of Dopa-bMAP_Control.
Improvement of production yield of Dopa-bMAP with coexpression of MjTyrRS or EcTyrRS. To compare the production yield of purified Dopa-bMAP samples, the concentrations of purified Dopa-bMAP samples were calculated by measuring the absorbance at 280 nm and applying the Beer-Lambert law. The production yield of Dopa-bMAP without coexpression of any AARS (Dopa-bMAP_Control) was 4.18 ± 0.02 mg/L. With coexpression of MjTyrRS and EcTyrRS, the production yield of Dopa-bMAP was 6.56 ± 0.04 and 7.11 ± 0.05 mg/L, which is greater than that of Dopa-bMAP_Control by 57% and 70%, respectively (Fig 4). Considering that the Dopa incorporation yields of three Dopa-bMAP samples (Dopa-bMAP_Control, Dopa-bMAP_MjTyrRS, and Dopa-bMAP_EcTyrRS) were comparable, these results successfully demonstrated that coexpression of Dopa-recognizing AARS (MjTyrRS and EcTyrRS) is an effective strategy to enhance the production yield of bMAP with a high Dopa incorporation yield (greater than 90%).