Supplementary data:
Figure S1: Time required for the respective in vitro translation reaction to reach 90% of the maximal
fluorescence plotted against the molecular weight of the product.
1
Figure S2: Expression levels calculated as mass concentration of the individual in vitro translation
product plotted against the protein sizes.
mg/mL Lysate protein content
30
25
20
E. coli
15
WGE
10
HeLa
LTE
5
0
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
mg/mL Produced protein
Figure S3: Productivity of cell-free systems quantified as the amount of protein produced per
reaction in relation to the total protein concentration of the lysate. The total protein
concentration of the lysates was assessed using Bradford method using the BSA as a standard.
The total yield of the in vitro translated products was calculated based on total GFP fluorescence
using purified GFP as a standard.
2
Figure S4: SDS-PAGE analysis of the test library expressed in E. coli CF
3
Figure S5: SDS-PAGE analysis of the test library expressed in WGE
4
Figure S6: SDS-PAGE analysis of the test library expressed in HeLa
5
Figure S7: SDS-PAGE analysis of the test library expressed in LTE
6
Figure S8: integrity of recombinant proteins calculated as the percentage of full-length products
relative to the total signal plotted against the product size.
Comparison of expression level analysis performed by assessing GFP fluorescence or
Western blot
The GATA1 and STAT3 ORFs tagged with N-terminal eGFP constructs were
expressed in all four cell-free systems and the N-terminally tagged eGFP SREBF2
only in HeLa and E. coli. From each reaction 1:2 and 1:4 dilutions were prepared
and mixed 1:1 with SDS-PAGE loading buffer. After incubation of the samples at 72°
C for 150 seconds 4 μL were loaded on 17-well NuPAGE® Novex® 4-12% Bis-Tris
Protein Gels, 1.0 mm (Life Technologies) in duplicates. The gels were subjected to
Western blotting onto a Osmonics NitroBind™ Nitrocellulose Transfer Membrane (GE
LifeSciences) in Tris-Glycine transfer buffer using a XCell II™ Blot Module (Life
Technologies). The membranes were blocked overnight with 1x Casein Blocking
7
Buffer (Sigma-Aldrich) in 1xPBS and 0.1% (v/v) Tween-20 (Sigma-Aldrich). The
detection of the eGFP and the eGFP-tagged constructs was performed with 1:2000
dilution of GFP (D5.1) XP® Rabbit mAb (Cell Signaling) primary antibody and 1:2500
IRDye® 800CW Goat anti-Rabbit IgG (LI-COR) secondary antibody. Both antibodies
were diluted in 1x Casein Blocking Buffer. The fluorescent secondary antibody was
detected by scanning the blot with the Odyssey Infrared Imager (LI-COR) at 800
nm. The bands from acquired images of the western blots were quantified using
ImageJ image analysis software. Dilutions of purified eGFP (150, 100, 75, 50, 25 and
10 μg/mL) were prepared, loaded on to gels and blotted as described above in order
to produce a calibration curve. A 50 μg/mL eGFP dilution was included on every
individual gel and Western blot in order to correlate the data among blots. The
interpolation of the measured signals was performed using the GraphPad Prism 6
software.
12000
Protein in nM
10000
8000
6000
4000
2000
0
GATA1
WGE
STAT3
GATA1
STAT3
GATA1
LTE
STAT3
SREBF2
HeLa
eGFP Fluorescence
GATA1
STAT3
SREBF2
E. coli
Western Blot
Figure S9: Comparison of protein concentration measurement by eGFP fluorescence intensity and
by Western blotting. The difference in measured protein concentrations for the eGFP constructs
produced in the eukaryotic cell-free systems between the both techniques is within the acceptable
experimental error significant. The proteins expressed in the E. coli cell-free system display lower
GFP fluorescence compared to the GFP amounts estimated by the Western blotting.
Table S1: List of human genes used for benchmarking. The position of the construct in the library
plate, accession number, gene symbol, product size including GFP as well as description of the gene
8
Library
position
Accession
number
A03
Symbol
kDa
Description
(incl. GFP)
E-Cad
34.28
Cadherin-1 cytosolic domain (155-709)
A04
BC032811
TFF1
39.43
Trefoil factor 1
A05
BC014392
S100A1
40.826
S-100 protein alpha chain
A06
BC013306
TCEB2
43.412
Transcription elongation factor B (SIII)
A07
BC117491
BATF3
44.748
Basic leucine zipper transcriptional factor ATF-like 3
A08
BC022371
BTF3L4
47.551
Transcription factor BTF3 homolog 4
A09
BC032462
VPS29
47.725
Vacuolar protein sorting 29 homolog
A10
BC066257
IL2
47.908
Interleukin-2
A11
BC009507
ISG15
48.195
ISG15 ubiquitin-like modifier
A12
BC000901
CIRBP
48.928
Cold-inducible RNA-binding protein
B01
BC070256
IFNG
49.628
Interferon gamma
B02
BC006322
ATF3
50.856
Cyclic AMP-dependent transcription factor ATF-3
B03
BC070031
RAP2A
50.895
RAP2A, member of RAS oncogene family
B04
BC008062
BTF3
52.448
RNA polymerase B transcription factor 3
B05
BC029891
TFEC
52.93
Transcription factor EC
B06
BC015511
IL6
53.998
Interleukin-6
B07
BC028148
TNF
55.924
Tumor necrosis factor
B08
BC001359
YWHAB
58.362
Tyr 3-monooxygenase/Trp 5-monooxygenase
activation protein
B09
BC126366
TFAM
59.377
Transcription factor A, mitochondrial
B10
BC029619
ATF1
59.513
Cyclic AMP-dependent transcription factor ATF-1
B11
BC008678
IL1B
61.028
Interleukin-1 beta
B12
BC021000
GTF2B
65.113
Transcription initiation factor IIB
C01
BC009797
GATA1
65.71
GATA binding protein 1
C02
BC049821
USF2
67.235
Upstream transcription factor 2
C03
BC060807
CREBZF
67.414
CREB/ATF bZIP transcription factor
C04
BC035993
ANXA1
68.994
Annexin A1
C05
BC010576
EBNA1BP2
69.006
Transcription factor AP-4
C06
BC010576
TFAP4
69.006
Transcription factor AP-4
C07
BC103951
SP6
70.12
Transcription factor Sp6
9
C08
BC106755
CAMK1
71.617
Calcium/calmodulin-dependent protein kinase type
1
C09
BC107156
GTF2A1
71.794
Transcription initiation factor IIA subunit 1
C10
BC000433
MAPK13
72.37
Mitogen-activated protein kinase 13
C11
BC003596
TP53
73.966
Tumor protein p53
C12
BC035716
IRF9
73.976
Interferon regulatory factor 9
D01
BC022242
TGFB1
74.621
Transforming growth factor beta-1
D02
BC065021
GTF2H2D
74.732
General transcription factor IIH subunit 2-like
protein
D03
BC037308
YY1
74.993
Transcriptional repressor protein YY1
D04
BC126361
TFDP3
75.247
Transcription factor Dp family member 3
D05
BC011685
TFDP1
75.35
Transcription factor Dp-1
D06
BC053532
CSNK2A1
75.424
Casein kinase II subunit alpha
D07
BC020493
CALR
78.422
Calreticulin
D08
BC016368
PSMC1
79.465
26S protease regulatory subunit 4
D09
BC021113
TFDP2
79.516
Transcription factor Dp-2
D10
NM_005513
GTF2E1
79.732
General transcription factor IIE, polypeptide 1a
D11
CR456430
CYP2D6
80.333
Cytochrome P450
D12
BC012886
RUVBL1
80.508
TATA box-binding protein-interacting protein
E01
BC037225
TFAP2B
80.754
Transcription factor AP-2 beta
E02
BC005145
GDI2
80.943
Rab GDP dissociation inhibitor beta
E03
BC004935
GTF2H4
82.466
General transcription factor IIH
E04
BC025699
SMAD2
82.586
SMAD family member 2
E05
BC025991
GTF2A1L
82.724
General transcription factor II A, 1-like factor
E06
BC032448
TFEB
83.145
Transcription factor EB
WAVE 2
84.564
Wiskott-Aldrich syndrome protein family member 2
PPARG
84.961
Peroxisome proliferator-activated receptor gamma
CTTN
85.28
Src substrate cortactin
E07
E08
BC006811
E09
E10
BC000301
HDAC1
85.383
Histone deacetylase 1
E11
BC000479
AKT1
85.966
RAC-alpha serine/threonine-protein kinase
E12
NM_000766
CYP2A13
86.967
Cytochrome P450
F01
BC069418
CYP3A4
87.623
Cytochrome P450 3A4
F02
BC028383
BMPR1A
90.478
Bone morphogenetic protein receptor type-1A
10
F03
BC032496
FYN
91.042
Tyrosine-protein kinase Fyn
F04
BC014445
EHD2
91.441
EH-domain containing 2
F05
BC000365
GTF2H1
92.311
General transcription factor IIH, polypeptide 1
F06
BC016680
SP2
95.18
Transcription factor Sp2
F07
BC110580
TCF3
98.79
Transcription factor E2-alpha
F08
BC016027
DDX5
99.428
Probable ATP-dependent RNA helicase DDX5
F09
BC132690
RRN3
104.387
RNA polymerase I-specific transcription initiation
factor RRN3
F10
BC050556
TCF12
106.125
Transcription factor 12
F11
BC009349
TCF25
106.947
Transcription factor 25
F12
BC109273
PRKCA
107.03
Protein kinase C alpha type
G01
BC002704
STAT1
113.322
Signal transducer and activator of transcription 1
G02
BC036022
TCEB3B
114.228
Transcription elongation factor B polypeptide 3B
(elongin A2)
G03
BC012863
FOXM
114.563
Forkhead box M1
CTNNB1
115.28
Catenin beta-1
G04
G05
BC014482
STAT3
118.348
Signal transducer and activator of transcription 3
G06
BC014482
STAT3
118.348
Signal transducer and activator of transcription 3
G07
BC027036
STAT5A
120.927
Signal transducer and activator of transcription 5A
G08
BC075852
STAT6
124.415
Signal transducer and activator of transcription 6
G09
BC001965
RAD54B
133.247
DNA repair and recombination protein RAD54B
G10
BC036065
WWP1
135.482
NEDD4-like E3 ubiquitin-protein ligase WWP1
G11
BC051765
NFKB1
135.707
Nuclear factor of kappa light polypeptide gene
enhancer
G12
BC052985
BMPR2
145.481
Bone morphogenetic protein receptor type-2
H01
BC008800
POLD1
153.911
DNA polymerase delta catalytic subunit
H02
CT841522
SREBF2
153.967
Helix-loop-helix domain
H03
BC032224
PDGFRB
154.248
Platelet-derived growth factor receptor beta
H04
BC012151
NFX1
154.674
Nuclear transcription factor, X-box binding 1
H05
BC011617
PC
159.914
Pyruvate carboxylase
H06
BC014243
TYK2
163.93
Non-receptor tyrosine-protein kinase TYK2
H07
BC032680
ZFYVE9
186.683
Zinc finger FYVE domain-containing protein 9
11
Table S 2: Published records of recombinant production of proteins used in the current
benchmarking in in vivo and in vitro expression systems
Library
position
Symbol
A03
E-Cad
A04
TFF1
+1
A05
S100A1
+2
A06
TCEB2
+3
A07
BATF3
A08
BTF3L4
A09
VPS29
A10
IL2
A11
ISG15
A12
CIRBP
B01
IFNG
B02
ATF3
B03
RAP2A
+7
B04
BTF3
+8
B05
TFEC
B06
IL6
+9
B07
TNF
+10
B08
YWHAB
+11
B09
TFAM
+12
B10
ATF1
B11
IL1B
+13
B12
GTF2B
+14
C01
GATA1
C02
USF2
C03
CREBZF
+16
C04
ANXA1
+17
C05
EBNA1BP2
+18
C06
TFAP4
E. coli
Other
cell cultures
HeLa
Cell-free
+4
+5
+6
+15
12
C07
SP6
C08
CAMK1
C09
GTF2A1
C10
MAPK13
+20
C11
TP53
+21
C12
IRF9
D01
TGFB1
D02
GTF2H2D
D03
YY1
D04
TFDP3
+23
D05
TFDP1
+24
D06
CSNK2A1
+26,27
+28
D07
CALR
+29
+30
D08
PSMC1
D09
TFDP2
+32
D10
GTF2E1
+33
D11
CYP2D6
+35
D12
RUVBL1
+36
E01
TFAP2B
E02
GDI2
E03
GTF2H4
E04
SMAD2
+40
E05
GTF2A1L
+42
E06
TFEB
+43
E07
WAVE 2
+44
E08
PPARG
+45
Bosc; Cos-146
E09
CTTN
+47,48
HEK29349,50
E10
HDAC1
E11
AKT1
+52
E12
CYP2A13
+53,54
F01
CYP3A4
+55
+19
+22
+25
+31
+34
+37
HEK29338
+39
+41
+44
HEK293F51
COS-756
13
F02
BMPR1A
+57
F03
FYN
+58
F04
EHD2
F05
GTF2H1
F06
SP2
F07
TCF3
+61
F08
DDX5
+63
F09
RRN3
F10
TCF12
F11
TCF25
F12
PRKCA
G01
STAT1
G02
TCEB3B
G03
FOXM
+70
+71
G04
CTNNB1
+72
+73
G05
STAT3
HEK293T74
G06
STAT3
HEK293T74
G07
STAT5A
+75
G08
STAT6
+77
G09
RAD54B
+80,81
HCT11682Cos7,BALL181
G10
WWP1
+83,84
HEK293T, MCF785
G11
NFKB1
+86
G12
BMPR2
H01
POLD1
H02
SREBF2
H03
PDGFRB
H04
NFX1
H05
PC
H06
TYK2
H07
ZFYVE9
Bosc23, SYF59
+60
+39
+62
+64
HEK 293T, NIH 3T365
+66
Cos-767
+68
Cos-769
MDCK, MCF-7, CHO76
+78
Sf979
+87
HEK29388
+89
+90
HEK29391,92, M-1992
+93
HEK293, CHO, Sf994
HEK29395,96, HFK96
+97
HT-1080, HEK293T98,
Sf2199
+100
BBCE101
14
+98
Table S 3: Expression levels of individual constructs obtained in different cell-free systems (in μg/mL)
Library
position
A03
A04
A05
A06
A07
A08
A09
A10
A11
A12
B01
B02
B03
B04
B05
B06
B07
B08
B09
B10
B11
B12
C01
C02
C03
C04
C05
C06
C07
C08
C09
C10
C11
C12
D01
D02
D03
D04
D05
D06
D07
D08
D09
D10
D11
kDa
(incl.
GFP)
34.280
39.430
40.826
43.412
44.748
47.551
47.725
47.908
48.195
48.928
49.628
50.856
50.895
52.448
52.930
53.998
55.924
58.362
59.377
59.513
61.028
65.113
65.710
67.235
67.414
68.994
69.006
69.006
70.120
71.617
71.794
72.370
73.966
73.976
74.621
74.732
74.993
75.247
75.350
75.424
78.422
79.465
79.516
79.732
80.333
E. coli
CF
WGE
HeLa
LTE
229
206
207
152
365
261
302
152
266
403
198
17
153
412
431
246
346
540
482
393
440
508
579
2
439
372
458
571
336
379
463
312
484
585
413
2
515
214
300
374
195
666
570
408
633
253
159
149
279
267
269
215
153
182
128
239
174
165
183
272
209
144
424
213
181
168
200
323
212
231
247
350
236
178
326
366
199
190
546
217
341
270
249
279
418
265
400
330
41
140
8
8
8
7
16
12
14
7
13
20
10
1
8
22
23
13
19
32
29
23
27
33
38
0
30
26
32
39
24
27
33
23
36
43
31
0
39
16
23
28
15
53
45
33
51
98
41
94
65
81
101
93
81
119
131
80
126
89
56
102
61
53
166
104
124
108
144
58
125
65
66
24
127
102
128
134
62
178
195
62
89
120
87
110
0
88
126
51
141
97
Library
position
D12
E01
E02
E03
E04
E05
E06
E07
E08
E09
E10
E11
E12
F01
F02
F03
F04
F05
F06
F07
F08
F09
F10
F11
F12
G01
G02
G03
G04
G05
G06
G07
G08
G09
G10
G11
G12
H01
H02
H03
H04
H05
H06
H07
15
kDa
(incl.
GFP)
80.508
80.754
80.943
82.466
82.586
82.724
83.145
84.564
84.961
85.280
85.383
85.966
86.967
87.623
90.478
91.042
91.441
92.311
95.180
98.790
99.428
104.387
106.125
106.947
107.030
113.322
114.228
114.563
115.280
118.348
118.348
120.927
124.415
133.247
135.482
135.707
145.481
153.911
153.967
154.248
154.674
159.914
163.930
186.683
E. coli
CF
WGE
HeLa
LTE
331
546
6
559
384
487
536
331
396
412
373
474
493
417
4
483
297
446
519
923
662
863
839
562
703
1064
110
902
836
942
890
1032
820
749
535
875
638
53
187
414
490
689
176
1320
423
344
236
336
337
419
341
326
261
424
323
245
281
268
248
399
470
238
345
295
295
425
320
317
434
458
344
455
536
423
463
532
299
504
388
398
531
371
2
239
590
266
115
484
27
44
0
46
32
40
45
28
34
35
32
41
43
37
0
44
27
41
49
91
66
90
89
60
75
121
13
103
96
111
105
125
102
100
72
119
93
8
29
64
76
110
29
246
167
138
59
67
73
145
194
155
100
119
129
129
90
85
73
94
103
86
197
92
178
112
181
161
118
0
116
126
146
158
175
139
138
178
182
223
113
60
79
39
126
44
85
83
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