Preparation of purified wild type and mutant GADβ enzymes
Wild type and mutant GADβ enzymes were expressed in the E. coli BL21(DE3) strain
harboring the plasmid pET28b gadB or pET28b gadB E89Q/Δ465-466 (Tho Ho et al., 2013).
Cells were grown at 37°C in 500 mL of LB medium containing 50 μg/mL of kanamycin, and
GAD expression was induced by adding 1 mM of IPTG when OD600 reached 2.5. After being
grown further at a 37°C for 2 h, cells were harvested and washed with wash buffer (10 mM
Tris-acetate, pH 8.2, 14 mM Mg(OAc)2, 60mM K(OAc), 1 mM dithiothreitol). After being
washed three times in excessive volume of wash buffer, the cell pellet was resuspended in 50
mL of wash buffer and lysed in a French Pressure Cell by single passage at 12,000 psi. After a
centrifugation (30,000 RCF, 4°C, 30 min), GADβ in the supernatant of the lysate was recovered
using Ni-NTA agarose beads (Qiagen, Valencia, CA) according to the manufacturer’s protocol.
Isolated GADβ was concentrated in buffer B by VIVA-spin 20 ultrafiltration membranes with
10 kDa MWCO membranes (Sartorius, Göttingen, Germany) and stored at -80°C prior to use.
The concentrations of the purified GADβ were determined by Bradford method, using
Coomassie Protein Assay Kit (Thermo Scientific, Waltham, MA).
Supporting Tables
Table S1. Primers used in this study. F, forward primers; R, reverse primers. Each number
of primer was correspond to the number of oligonucleotide fragments in α-2,3-sialyltransferase.
T7 15UP and GTB were used as 1st-sense primer and 4th-antisense primer of mutational PCR,
respectively. They were also used as sense and antisense primer for overlap-extension PCR and
amplification of template for the cell-free reaction.
Name of
primers
T7 15UP
Nucleotide sequences (5’ 3’)
TCGATCCCGCGAAATTAATACGACTCACTATAGG
1-R
AAAGATAAATTTAGCTTGTTGCACTTC
2-F
GAAGTGCAACAAGCTAAATTTATCTTTNNSGGCACG
2-R
AGGATGCCCTTTAAAGTAGATTTT
3-F
AAAATCTACTTTAAAGGGCATCCTNNSGGTGGTGAAATTAATGACTACATTCTGA
3-R
TGAACTTGCAACACCACCCAC
4-F
GTGGGTGGTGTTGCAAGTTCANNSTATTTC
GTB
CAAAAAACCCCTCAAGACCCGTTTA
Table S2. Relative activity of selected mutant STs compared with the wild type enzyme. Each
of enzymes were synthesized in the GADβ implemented cell-free system and assayed in a
microtiter plate.
T265
R313
L357]
Relative activity
T
S
S
2.929
T
W
S
2.821
S
V
T
2.786
S
S
V
2.786
T
R
S
2.786
T
G
T
2.750
T
T
S
2.679
T
Q
S
2.679
S
N
V
2.607
-
-
-
1.000
Supporting Figures
Figure S1. Cell-free expression and screening of variant α-2,3-sialyltransferases. The
nucleotide sequences of the hot spot residues of α-2,3-sialyltransferase are randomized by PCR
with the primers having NNS (NNG or NNC) sequence. Amplified PCR products are cloned
into a plasmid and transformed into DH5α E.coli strain. Individual variant genes separated in
the colonies are amplified and expressed in cell-free protein synthesis reactions. Activities of
the synthesized variant enzymes are directly analyzed in an automated liquid handling system.
Figure S2. Enzyme assay of cell-free synthesized ST. Left, colorimetric enzyme assay was
conducted with 0.1 ng (open circles), 0.2 ng (hatched circles), 0.3 ng (grey circles) and 0.4 ng
(black circles) of purified ST. Right, same amounts of ST enzymes synthesized in standard cellfree synthesis reactions were assayed without purifications. Assay reactions were conducted in
100 µL. Error bars represent standard deviations from three independent samples.
Figure S3. Glucose fueled cell-free protein synthesis reactions with various concentrations of
HEPES-KOH buffer. Relative expression level of sfGFP and pH during the course of cell-free
synthesis reaction were represented red solid lines and black dashed lines, respectively. 57 mM
(circles), 118 mM (triangles), 179 mM (squares), 240 mM (diamonds) and 300 mM (asterisks)
of HEPES-KOH buffers were used in the presence of 80 mM glucose. Error bars represent the
standard deviations between two separate reactions. Error bars represent standard deviations
from three independent samples.
Figure S4. Mechanism of amino acid decarboxylase-mediated acid resistance.
Figure S5. Effect of GABA on the efficiency of cell-free protein synthesis. Varying amounts
of GABA was added to the standard reaction mixture for cell-free protein synthesis of sfGFP.
Expression levels of sfGFP were determined by measuring the fluorescence of reaction samples
after 3 h incubation. Error bars represent the standard deviations between two separate
reactions. Error bars represent standard deviations from three independent samples.
Figure S6. The use of wild type and mutant GADβ to maintain pH during glucose-fueled cellfree protein synthesis. Control reaction without GADβ (circles) was conducted in the presence
of 57 mM of HEPES-KOH buffer. 0.4 mg/mL of wild type GADβ (triangles) or GADβ
E89Q/Δ465-466 (squares) was added into the reaction mixture to examine their effect on the
changes in pH (black dashed lines) and protein synthesis (red solid lines). Error bars represent
the standard deviations between two separate reactions. Error bars represent standard
deviations from three independent samples.
Figure S7. Replacement of chemical buffer with the engineered GADβ. Reaction mixture for
cell-free protein synthesis was prepared in the absence of any chemical buffers. Prepared
reaction mixture was supplied with purified GADβ E89Q/Δ465-466 enzyme at the indicated
concentrations. sfGFP fluorescence of the reaction mixture was measured after 3 h incubation.
Error bars represent the standard deviations from three independent samples.
Figure S8. Changes in glutamate concentration in the reaction mixture containing GADβ
E89Q/Δ465-466. Reaction samples were taken at the indicated time points and the residual
glutamate concentration was measured using an amino acid analyzer (Hitachi L-8900) after 5%
TCA-precipitation of proteins in the samples. Glutamate in the reaction mixture was found to
be depleted almost completely after 6 h incubation of the reaction mixture. Error bars represent
the standard deviations between two separate reactions. Error bars represent standard
deviations from three independent samples.
References for Supporting Information
Thu Ho NA, Hou CY, Kim WH, Kang TJ. 2013. Expanding the active pH range of Escherichia
coli glutamate decarboxylase by breaking the cooperativeness. J Biosci Bioeng 115:154158.