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 References: 1. 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