Supplementary Data
Construction of expression vectors
The plasmid pGFPuv (Clonetech) has restriction enzyme sites in order as NcoI, AgeI,
XhoI, and NotI. The genes encoding His6-tag and Z cloned in pET28a (Novagen)
(Schwartz et al., 1999) were amplified using PCR and then ligated to pGFPuv using
NcoI/AgeI site. This step was performed to introduce His6-tag gene having NcoI/NheI
site to pGFPuv. The resulting plasmid, pGFPuv-L was inserted by GST gene from
pET31a (Novagen) using NheI/AgeI site and EGFP gene from pEGFP-c2 (Clonetech)
using XhoI/NotI site. The duplex DNA of FS1 (Programmed -1 ribosomal frameshifting
cis-element No.1) was inserted to the resulting plasmid using AgeI/XhoI site. The
resulting gene fragment containing His6-tag-GST-FS1-EGFP was inserted to pET28a at
NcoI/NotI site and named as pGST-EGFP-FS1. The plasmids pGST-EGFP-FS2, 3, 4
were constructed by following the same method for pGST-EGFP-FS1 using FS2, FS3,
and FS4, respectively, instead of FS1.
Plasmids pGST, pEGFP, and pGST-EGFP-PLUS were made by following the method
of pGST-EGFP-FS1 without EGFP-FS1, GST-FS1, and FS1, respectively.
1
Assessment of metabolic burden
We have compared four different expression systems, pGST encoding GST only,
pGST-EGFP-FS1 having frameshifting (FS) sequence between GST and EGFP genes,
pGST-EGFP-PLUS without FS sequence, and pEGFP encoding EGFP only. First, the
maximum specific growth rates after induction were not significantly different for four
types of heterologous protein expression systems (Table S2). The maximal GST activity
and green fluorescence was 19.59 units/OD=1 of GST-only system and 212.62 of
EGFP-only, respectively (Table S2). If we assume that cells use the same resources and
energy to express foreign proteins, GST activity and green fluorescence for GST-EGFP
fusion system should be about 9 units of GST and 100 of EGFP, respectively. However,
the fact that only 24% (4.64/19.5) of GST activity and 25% (52.98/212.62) of EGFP
were detected by using fusion system means approximately 75% of activity loss was
caused by co-expression of EGFP. The other hand, 74% (14.42/19.59) of GST was
expressed as the active form with FS system. This means that only 1/4 of resource was
consumed to express the EGFP and metabolic burden could be reduced by using FS
system.
Determination of co-linearity between protein concentration and fluorescent
2
intensity in 384-well format
The concentrations of purified proteins, Trx, GST, MBP and NusA having their
intact and EGFP fused forms, were determined by measuring the absorbance at 280 nm.
Each protein solution was diluted with 10 mM PBS buffer to adjust to 0, 25, 50, 100,
150, 200 and 250 mg/L and then 100 L of each sample was loaded into 384 micro-well
plate to read green fluorescent intensity. From 0 to 25 mg/L of purified EGFP and from
0 to 1000 mg/L of BSA were also tested as positive and negative control, respectively.
REFERENCES
Schwartz T, Rould MA, Lowenhaupt K, Herbert A, Rich A. 1999. Crystal structure of
the Z domain of the human editing enzyme ADAR1 bound to left-handed Z-DNA.
Science 284:1841-1845.
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal Biochem
72:248-254.
3
Tables
Table S1. Sequence of the expression vector pGST-EGFP-FS1. Complete sequence of
pGST-EGFP-FS1 is shown with GST gene, -1RFS cis-element and EGFP gene in italic.
Table S2. Maximum values for four types of expression systems.
4
Figures
Figure S1. Schematic map of pGST-EGFP-FS1. pET28a vector (Novagen) has been
modified to encode GST and EGFP. The implanting cis-elements for programmed -1
ribosomal frameshifting, FS1, between the two genes, result in 5.2% of the GST being
fused to EGFP. The product also includes a N-terminal His6-tag for protein purification.
Figure S2. Visualization of protein expressed cells of: (a) pGST-EGFP-FS1; (b) pGST;
(c) pEGFP; and (d) pGST-EGFP-PLUS. Cells were cultivated in a 250 mL Erlenmeyer
flask with 25 mL of LB medium with protein expression induced by addition of ITPG at
OD600 = 0.5. The cells harvested after 5.5 h after induction were visualized using
confocal microscope (LSM 510 META, Carl Zeiss Co., Ltd., Germany). Left: green
fluorescence image; Middle: phase contrast image; Right: mixed image.
Figure S3. Comparison of protein amounts expressed from pGST encoding GST-only
(lane 1), pGST-EGFP-FS1 having frameshifting sequence (lane 2), pGST-EGFP-PLUS
without frameshifting sequence (lane 3), and pEGFP encoding EGFP only (lane 4).
Figure S4. (a) Time course data of MBP concentration () and EGFP fluorescence
5
intensity (). (b) Visualized MBP using 12% SDS-PAGE by running the whole extracts
obtained from samples shown in (a). Lane M: size marker (myosin, 250 kDa;
phosphorylase, 148 kDa; BSA, 98 kDa; glutamic dehydrogenase, 64 kDA; alcohol
dehydrogenase, 50 kDa; carbonic anhydrase, 36 kDa; myoglobin red, 22 kDa;
lysozyme, 16 kDa). Lane 1: Before induction. Lane 2: After 0.5 h of induction. Lane 3:
After 1.0 h of induction. Lane 4: After 1.5 h of induction. Lane 5: After 2.0 h of
induction. Lane 6: After 3.0 h of induction. Lane 7: After 4.0 h of induction. Lane 8:
After 7.0 h of induction. Lane 9: After 19.5 h of induction. The size marker was
obtained from Invitrogen (Carlsbad, CA). Cells were cultivated in 5-L jar fermentor
with 2 L LB medium with protein expression induced by addition of IPTG. Samples
were taken at 0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 7.0, and 19.5 h after induction. The
concentration of MBP was calculated from data acquired by the scanning of SDS-PAGE
plate using the program Scion image (Scion Corp., Frederic, Maryland, USA).while
total cellular protein concentration obtained by the Bradford method (Bradford, 1977)
using bovine serum albumin as a standard.
Figure S5. Co-linearity between protein concentration and fluorescent intensity
The concentrations and green fluorescent intensities of purified proteins, (a) Trx, (b)
6
GST, (c) MBP and (d) NusA having their intact and EGFP fused forms, were
determined. From 0 to 25 mg/L of purified (e) EGFP and from 0 to 1000 mg/L of (f)
BSA were also tested as positive and negative control, respectively.
Figure S6. Fluorescence images of purified proteins on chip surface. Purified
proteins were spotted on the slide glass and analyzed by measuring green fluorescence
and the shape of spots using fluorescence microscope. A linear relationship was found
between fluorescence intensity and the amount of spotted purified GST-EGFP/GST
protein (Fig. 4.). EGFP and BSA were used as positive and negative controls,
respectively.
7
Table S1. Sequence of the expression vector pGST-EGFP-FS1. Complete sequence of
pGST-EGFP-FS1 is shown with GST gene, -1RFS cis-element and EGFP gene in italic.
1
85
169
253
337
421
505
589
673
757
841
925
1009
1093
1177
1261
1345
1429
1513
1597
1681
1765
1849
1933
2017
2101
2185
2269
2353
2437
2521
2605
2689
2773
2857
2941
3025
3109
3193
3277
3361
3445
3529
3613
3697
3781
3865
3949
4033
4117
TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAG
CGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGG
GCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCC
ATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAAC
ACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTA
ACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCC
TATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAA
ACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGC
AGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGT
CAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCC
AGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCT
GAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCG
CATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACC
ATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCAT
CTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTG
TCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAG
AGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACC
AAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTT
CTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTT
TTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAG
AACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACC
GGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAG
CGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGG
TATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC
GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCG
GCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGT
ATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAG
CGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTG
ATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGC
TGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAG
GTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGC
CTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGT
TTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGAT
GCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCG
GGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCC
TGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCAT
TCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACC
AGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGAT
AATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGC
AAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTAC
GAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAA
GGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTT
CCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGG
TGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCA
CGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGT
ATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGG
CAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTT
CCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTA
ATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGA
AAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGG
CATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTT
CGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCG
ACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGG
GAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGG
AAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCT
CTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGAC
TCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCG
CCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCT
8
4201
4285
4369
4453
4537
4621
4705
4789
4873
4957
5041
5125
5209
5293
5377
5461
5545
5629
5713
5797
5881
5965
6049
6133
6217
6301
6385
6469
6553
6637
6721
6805
TCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAG
AGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAA
TTTTGTTTAACTTTAAGAAGGAGATATAccatgggcagcagccatcatcatcatcatcacagcagcggcctggtgccgcgcggc
agccatatggctagcatgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatat
cttgaagaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggag
tttcccaatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagcac
aacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttggatattagatacggtgtttcg
agaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaagctacctgaaatgctgaaaatgttcgaagat
cgtttatgtcataaaacatatttaaatggtgatcatgtaacccatcctgacttcatgttgtatgacgctcttgatgttgtttta
tacatggacccaatgtgcctggatgcgttcccaaaattagtttgttttaaaaaacgtattgaagctatcccacaaattgataag
tacttgaaatccagcaagtatatagcatggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaatcg
gatggttcaaccggtgctttaaactagctcgcggcaccgtccgcgtcaacaaacggctcgagaaaatggtgagcaagggcgagg
agctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcg
agggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtga
ccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccg
aaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgaca
ccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactaca
acagccacaacgtcaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaaca
tcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtga
acttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggcc
ccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcc
tgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtcctaatgaattccaactgagcggccgc
ACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTG
AGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT
9
Table S2. Maximum values for four types of expression systems.
max after induction
(h-1)
Max. GST activity
Max. green
fluorescence
GST-only
1.34
19.59±2.24
0.69±0.32
GST-EGFP (with
FS)
1.38
14.42±1.33
10.60±0.96
GST-EGFP
(without FS,
fused)
1.33
4.64±0.05
52.98±1.18
EGFP-only
1.29
0.38±0.49
212.62±13.93
Expression system
10
Fig. S1
NcoI
T7 promoter
His6-tag
NheI
GST
AgeI
XhoI
pGST-EGFP-FS1
(6884 bp)
EGFP
Kanr
NotI
11
FS: -1 RFS elements
Fig. S2
a
b
c
d
12
Fig. S3
13
60
21
MBP
18
EGFP
50
15
40
30
20
70
60
50
40
30
20
10
0
12
9
slope = 3.27
R2 = 0.97
0
10
3
6 9 12 15 18 21
Green fluorescence
0
0
5
10
Time after induction (h)
14
15
6
3
0
20
Green fluorescence (arbitrary/OD600=1)
70
MBP concentration
Concentration of MBP (mg/L/OD600=1)
Fig. S4a
Fig. S4b
250 kDa
148 kDa
98 kDa
64 kDa
M 1 2 3 4 5 6 7 8 9
50 kDa
MBP
36 kDa
22 kDa
16 kDa
15
Green fluorescence (arbitrary units)
Green fluorescence (arbitrary units)
Fig. S5.
35
Trx
30
25
20
15
y = 0.1318x
R2 = 0.9893
10
5
0
0
50
100
150
200
50
GST
40
30
20
y = 0.1763x
R2 = 0.993
10
0
250
0
50
50
MBP
40
30
20
y = 0.1641x
R2 = 0.9713
10
0
0
50
100
150
200
250
y = 8.348x
R2 = 0.9803
100
50
0
10
15
250
NusA
30
20
y = 0.1312x
R2 = 0.9895
10
0
0
Green fluorescence (arbitrary units)
Green fluorescence (arbitrary units)
150
5
200
50
100
150
200
250
Concentration (mg/L)
EGFP
0
150
40
Concentration (mg/L)
200
100
Concentration (mg/L)
Green fluorescence (arbitrary units)
Green fluorescence (arbitrary units)
Concentration (mg/L)
20
25
0.03
BSA
0.02
0.01
0.00
-0.01
-0.02
0
Concentration (mg/L)
200
400
600
Concentration (mg/L)
16
800
1000
Fig. S6.
17