Materials and Methods
Materials. Ni-nitrilotriacetic acid (NTA) agarose was purchased from Qiagen (Valencia, CA, USA). 0.20 µm syringe filters were purchased from Advantec (Dublin, CA, USA). Zeba spin desalting columns were purchased from Thermo Fisher Scientific (Waltham, MA, USA). PD-10 desalting columns were obtained from GE Healthcare (Piscataway, NJ, USA). All other chemicals and reagents were purchased from Sigma-Aldrich (Saint Louis, MO, USA) and used without further purification unless otherwise indicated.
Plasmid construction for overexpression of AARS and bMAP. The engineered pair of M. jannaschii tyrosyl tRNA synthetase specific for Dopa (MjTyrRS-Dopa) and tyrosyl-tRNA amber suppressor (MjtRNATyrCUA) were originally developed by Dr. Schultz and the colleagues (Alfonta et al., 2003). The plasmid-encoding MjTyrRS-Dopa and MjtRNATyrCUA (pSPEL 143 plasmid) were obtained from Dr. Tae Hyeon Yoo at Ajou University in South Korea. Based on the previous work enhancing the enzymatic activity of MjTyrRS (Amiram et al., 2015), site-directed mutagenesis was performed at the tRNA anticodon binding interface of MjTyrRS-Dopa, using pSPEL 143 as a template and primers (CGC769GGC(t1)–F: 5’-AAATTTCTCCGGGCCCTTAATCGTCAGTGGGTATTC-3’; CGC769GGC(t1)–R: 5’-GAATACCCACTGACGATTAAGGGCCCGGAGAAATTT-3’). Then the anticodon of MjtRNATyrCUA was mutated into AUA by site-directed PCR mutagenesis using primers (tRNAc35a-F: 5’-TGCCATGCGGATTTATAGTCCGCCGTTCTGC-3’; tRNAc35a-R: 5’-GCAGAACGGCGGACTATAAATCCGCATGGCA-3’), generating pEvol-Dopa plasmid. The E.coli TyrRS (EcTyrRS) gene was amplified by PCR using genomic DNA of Top10 E. coli cells as a template and primers (F: 5’-ATGGCAAGCAGTAACTTG-3’; R: 5’-TTATTTCCAGCAAATCAGACAG-3’). The EcTyrRS gene fragment digested by Bgl II and Sal I was cloned between Bgl II and Sal I sites of pEvol-Dopa, generating pEvol-EcTyrRS. The fp-3 gene encoding MAP with an N-term histidine tag (Yang et al., 2014) was cloned into pQE80 plasmid (Qiagen) to generate pQE80-bMAP plasmid.
Preparation of Dopa-incorporated bioengineered MAP samples. For expression of bMAP without incorporation of Dopa (Dopa-free bMAP), pQE80-bMAP plasmid was transformed into TOP10 cells. The transformants were cultured in LB media. When OD600 of the cell culture reached 1.0, 1 mM IPTG was added to induce expression of Dopa-free bMAP. For expression of Dopa-bMAP using overexpressed MjTyrRS-Dopa/MjtRNATyrAUA or EcTyrRS, pQE80-bMAP and either pEVOL-Dopa or pEVOL-EcTyrRS plasmid were cotransformed into JW2581 E. coli cells (a tyrosine-auxotrophicE. coli strain obtained from Yale Genetic Stock Center) to generate JW[pQE80-bMAP/pEVOL-Dopa] or JW[pQE80-bMAP/pEVOL-EcTyrRS] expression cells, respectively. For expression of Dopa-incorporated bMAP (Dopa-bMAP), cotransformed cells were inoculated into M9 minimal medium (100 mL of M9 salt [67.8 g of Na2HPO4, 30.0 g of KH2PO4, 5.0 g of NaCl, and 10.0 g of NH4Cl in 1 L distilled water (DW)], 20 mL of 20% glucose, 1 mL of 2 M MgSO4, 1 mL of 0.1 M CaCl2, and 1 mg of thiamine-hydrochloric acid in 1 L DW) with 20 natural amino acids (40 mg/L), ampicillin (100 μg/mL), and chloramphenicol (35 μg/mL), and were incubated at 37 ºC with 210 rpm shaking overnight. The overnight culture was transferred to fresh M9 minimal medium with 20 natural amino acids and was cultured at 37 ºC with 210 rpm shaking. When the optical density at 600 nm (OD600) reached 0.7, L-(+)-arabinose was added to the culture at the final concentration 0.2%. When OD600reached 1.0, a medium shift was performed. To remove residual tyrosine in the medium, cell pellets were collected by centrifugation at 6,000 rpm for 10 min and the supernatant was decanted. The cell pellets were resuspended in ice-cold 0.9% NaCl solution. This procedure was performed three times. Then the cell pellets were resuspended in M9 minimal medium with 19 natural amino acids deficient of tyrosine. After the culture was incubated at 30 ºC with 210 rpm shaking for 10 min, 1 mM IPTG and 1 mM Dopa were added to the culture in order to induce bMAP-Dopa. The cells were cultured at 30 ºC with 210 rpm shaking for 15 hr and then harvested by centrifugation at 6,000 rpm for 10 min at 4 ºC. The cell pellets were stored at −80 ºC until required. For expression of bMAP without overexpression of any AARS (Dopa-bMAP_Control), pQE80-bMAP plasmid was transformed into JW2581 E. coli cells to generate JW[pQE80-rMAP] expression cells. Then the expression of Dopa-bMAP_Control was achieved by the same procedure except that neither chloramphenicol nor L-(+)-arabinose was added to the culture.
To purify bMAP samples, cell pellets were resuspended in lysis buffer (100 mM NaH2PO4, 10 mM Tris-Cl, 10 mM ascorbic acid, and 8 M urea, pH 6.9) and sonicated for 20 min (cycled 3 sec on and 7 sec off). The cell lysate was centrifuged at 10,000 rpm for 30 min. The supernatant of cell lysate was mixed with Ni-NTA agarose beads (Qiagen) for 30 min at 18 ºC and then loaded onto a column. The column was washed with wash buffer (100 mM NaH2PO4, 10 mM Tris-Cl, 10 mM ascorbic acid, and 8 M urea, pH 6.9). The bMAP samples were eluted with 0.5 M HCl. To exchange the buffer condition, a PD-10 desalting column (GE Healthcare) was used. SDS-PAGE analysis was performed to confirm the expression of Dopa-bMAP samples. For NBT staining of Dopa-bMAP, the resolved protein samples on SDS-PAGE gel were transferred to nitrocellulose blotting membranes (BioTrace™ NT, Pall Life Science, Pensacola, FL) at 100 V for 2 h using a pH 8.3 transfer buffer containing 25 mM Tris, 190 mM glycine, and 20% methanol. Then the NBT staining of Dopa-bMAP was achieved by the similar procedure as previously reported (Paz et al., 1991).
The concentration of purified bMAP samples was calculated using molar absorbance at 280 nm and the Beer-Lambert law. The molar extinction coefficient of bMAP at 280 nm was calculated using the following equation (Pace et al., 1995):
\begin{equation} \varepsilon_{280}=\left(5500\times n_{\text{Trp}}\right)+\left(1490\times n_{\text{Tyr}}\right)+\left(125\times n_{\text{Cys}}\right)+\left(2630\times n_{\text{DOPA}}\right)\nonumber \\ \end{equation}
where 5,500; 1,490; 125; and 2,630 are the molar extinction coefficients of tryptophan, tyrosine, cysteine, and Dopa, respectively. The\(\varepsilon_{280}\) of Dopa-bMAP was calculated to be 42,800 M-1cm-1. The absorbance of purified proteins was measured using a Synergy microplate reader (BioTek, Winooski, VT, USA).
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis of purified bMAP samples.Purified bMAP samples were desalted with ZipTip pipette tips (Merck Millipore, Burlington, MA, USA) and diluted in α-cyano-4-hydroxycinnamic acid matrix solution in a buffer (30:70 [v/v] of acetonitrile: 0.1% TFA aqueous solution). A small quantity of sample matrix mixture was spotted on the polished steel target plate (Bruker Daltonics, Billerica, MA, USA). The sample was analyzed with an autoflex MALDI-TOF/TOF mass spectrometer (Bruker Daltonics).