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).