Supporting Information

Átírás

1 Supporting Information Cell-free GFP simulations Cell-free simulations of degfp production were consistent with experimental measurements (Fig. S1). Dual emmission GFP was produced under a P70a promoter in an E. coli extract for eight hours using maltose and 3-phosphoglycerate (3PG) as a carbon and energy source (R 2 = 0.84, Fig. S1A). Uncertainty in experimental factors such as the concentration of RNA polymerase, ribosomes, transcription and translation elongation rates, as well as the upper bounds on oxygen, maltose, and 3pg consumption rates, did not qualitatively alter the performance of the model (blue region, 95% confidence estimate of 100 sets). However, these simulations were only conducted at a single plasmid concentration of 5 nm. Thus, it was unclear if the model could capture cell-free protein synthesis for a range of plasmid concentrations. Simulations of the cell-free degfp titer for a range of plasmid concentrations were consistent with experimental measurements (Fig. S1B). The titer at each plasmid concentration was calculated by multiplying the degfp synthesis flux by the active time of production, approximately 8 hours. The mean of the ensemble (calculated by sampling the uncertainty in the model parameters) captured the saturation of degfp production as a function of plasmid concentration (R 2 = 0.97). However, while the mean and 95% confidence estimate of the ensemble were consistent with measured degfp levels, the model under predicted the degfp titer at the saturating plasmid concentration of 5 nm. These results showed that the sequence specific template reactions, metabolic network, and literature parameters were sufficient to predict protein production under different promoters. Nutrient consumption TXTL 2.0 A robust analysis of maltose and 3pg consumption was performed for TXTL 2.0 E. coli extract that determined oxidative phosphorylation was not necessary to meet degfp protein productivity constraints (Fig. S2). An ensemble of N = 20 ssfba solutions was calculated by S-1

2 randomly sampling the concentration of RNA polymerase, ribosomes, transcription and translation elongation rates within literature values. Each solution met the transcription and translation constraints determined from the ssfba modeling framework. Since maltose and 3pg consumption rates are not reported for TXTL 2.0, we sampled a range of 3pg consumption rates to determine if constraints could be met with and without oxidative phosphorylation. Solutions were found for both cases, thus it is not conclusive whether or not oxidative phosphorylation is occurring in TXTL 2.0 s cell extract. However, with oxidative phosphorylation, TXTL 2.0 would be more efficient with lower consumption rates by up to 3.8 fold compared to without oxidative phosphorylation. S-2

3 A 95 % CI of Ensemble Mean of Ensemble Experimental Data B Time (h) Plasmid Concentration (nm) Figure S1: Sequence specific flux balance analysis of degfp under a P70a promoter in TXTL 2.0 E. coli extract. A. degfp production for 8 h using maltose and 3PG as a carbon and energy source (R 2 = 0.84). Error bars denote a 10% deviation from the nominal value. B. Predicted versus measured degfp concentration as a function of plasmid concentration in TXTL 2.0 (R 2 = 0.97). Error bars denote the standard deviation of experimental measurements. The blue region denotes the 95% CI over an ensemble of N = 100 sets, the black line denotes the mean of the ensemble, and dots denote experimental measurements. Without OxPhos Activity With OxPhos Activity Mean 3PG Uptake (mm/h) Figure S2: Robust analysis of maltose and 3PG consumption for TXTL 2.0 E. coli extract with and without oxidative phosphorylation activity that meet the transcription and translation constraints. Each dot represents the mean of an ensemble of N = 20 ssfba solutions, black dots are solutions without oxidative phosphorylation and grey dots are solutions with oxidative phosphorylation. S-3

4 Supporting Information Dataset S2 Bone Morphogenetic Protein 10 (BMP10) ATGGGTTCTCTGGTTCTGCCGCTGAGCGCCGTCTTCTGCCTGGTGGCTCGTCTGGCTTCTG GCAGCCCCATCATGGGCCTTGAGCAGTCGCCCCTGGAAGAAGACATGCCCTTCTTTGATGA TATCTTCACAGAACAAGATGGTATTGACTTCAACACACTGCTGCAGAGCATGAAGGACGAGT TTCTCAAGACGTTGAATCTGTCAGACATTCCCCCACAGGACACAGGCAGAGTGGATCCGCC GGAGTACATGCTGGAGCTCTACAACAAATTCGCCACGGACCGGACCTCCATGCCATCTGCT AACATCATCCGGAGCTTCAAGAATGAAGATCTGTTTTCTCAACCAGTCAGTTTCAATGGGATC CGGAAGTATCCTCTCCTCTTCAACGTGTCCATCCCTCACCACGAAGAGGTCGTCATGGCTGA ACTGAGGTTGTACACGCTGGTGCAGAGAGATCGTTTGATGTATGATGGTGTGGACCGTAAAA TAATCATCTTTGAGGTTCTAGAGAGTGCCGATGGTAGCGAGGATGAGAGGAGCATGCTGGT CTTGGTATCAACAGAGATCTACGGAACCAACAGTGAGTGGGAGACATTTGACATCACGGATG CCACCAGACGTTGGCAAAAGTCAGGCCCATCAACCCACCAGCTGGAGATCCACATCGAAAG CAGACAAAACCAAGCTGAGGACACCGGAAGGGGACAACTGGAAATAGATATGAGTGCTCAG AATAAGCACGACCCTTTGCTTGTTGTGTTTTCTGATGACCAAAGCGGTGACAAGGAGCAGAA AGAAGAGCTGAATGAACTGATCTCCCACGAGCAGGATCTGGACCTGGGCACCGATGGTTTC TTTGGTGGGCCTGATGAAGAGGCTCTCCTGCAGATGAGGTCAAACATGATCGATGACTCTAC CGCTCGGATCAGGAGGAACGCCAAGGGGAACTACTGCAAGAAGACTCCACTGTACATCGAC TTCAAGGAGATCGGCTGGGACTCCTGGATCATCGCGCCTCCTGGTTACGAGGCCTATGAGT GCCGCGGTGTGTGCAACTACCCTCTGGCGGAGCACCTCACACCTACGAAACACGCAATTAT CCAGGCCTTGGTCCACCTCAAGAATTCCCAGAAAGCCTCCAAAGCCTGCTGCGTGCCCACG AAGCTGGATCCCATCTCCATCCTCTATTTAGATAAAGGTGTTGTCACCTATAAGTTTAAATAC GAAGGAATGGCCGTGTCTGAATGTGGCTGTAGATAG MGSLVLTLCALFCLAAYLVSGSPIMNLEQSPLEEDMSLFGDVFSEQDGVDFNTLLQSMKD EFLKTLNLSDIPTQDSAKVDPPEYMLELYNKFATDRTSMPSANIIRSFKNEDLFSQPVSF NGLRKYPLLFNVSIPHHEEVIMAELRLYTLVQRDRMIYDGVDRKITIFEVLESKGDNEGE RNMLVLVSGEIYGTNSEWETFDVTDAIRRWQKSGSSTHQLEVHIESKHDEAEDASSGRLE IDTSAQNKHNPLLIVFSDDQSSDKERKEELNEMISHEQLPELDNLGLDSFSSGPGEEALL QMRSNIIYDSTARIRRNAKGNYCKRTPLYIDFKEIGWDSWIIAPPGYEAYECRGVCNYPL AEHLTPTKHAIIQALVHLKNSQKASKACCVPTKLEPISILYLDKGVVTYKFKYEGMAVSECGCR Chloramphenicol Acetyltransferase (CAT) ATGGAAAAAAAAATTACCGGCTATACCACCGTGGATATTAGCCAGTGGCATCGCAAAGAACA TTTTGAAGCGTTTCAGAGCGTGGCGCAGTGCACCTATAACCAGACCGTGCAGCTGGATATTA CCGCGTTTCTGAAAACCGTGAAAAAAAACAAACATAAATTTTATCCGGCGTTTATTCATATTCT GGCGCGCCTGATGAACGCGCATCCGGAATTTCGCATGGCGATGAAAGATGGCGAACTGGT GATTTGGGATAGCGTGCATCCGTGCTATACCGTGTTTCATGAACAGACCGAAACCTTTAGCA GCCTGTGGAGCGAATATCATGATGATTTTCGCCAGTTTCTGCATATTTATAGCCAGGATGTG GCGTGCTATGGCGAAAACCTGGCGTATTTTCCGAAAGGCTTTATTGAAAACATGTTTTTTGTG AGCGCGAACCCGTGGGTGAGCTTTACCAGCTTTGATCTGAACGTGGCGAACATGGATAACT TTTTTGCGCCGGTGTTTACCATGGGCAAATATTATACCCAGGGCGATAAAGTGCTGATGCCG CTGGCGATTCAGGTGCATCATGCGGTGTGCGATGGCTTTCATGTGGGCCGCATGCTGAACG AACTGCAGCAGTATTGCGATGAATGGCAGGGCGGCGCG MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFLKTVKKNKHKFYPAFI HILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSSLWSEYHDDFRQFLHIY SQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANMDNFFAPVFTMGKYYTQG DKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA S-4

5 Caspase 9 (CASP9) ATGGACGAAGCGGATCGGCGGCTCCTGCGGCGGTGCCGGCTGCGGCTGGTGGAAGAGCT GCAGGTGGACCAGCTCTGGGACGCCCTGCTGAGCCGCGAGCTGTTCAGGCCCCATATGAT CGAGGACATCCAGCGGGCAGGCTCTGGATCTCGGCGGGATCAGGCCAGGCAGCTGATCAT AGATCTGGAGACTCGAGGGAGTCAGGCTCTTCCTTTGTTCATCTCCTGCTTAGAGGACACAG GCCAGGACATGCTGGCTTCGTTTCTGCGAACTAACAGGCAAGCAGCAAAGTTGTCGAAGCC AACCCTAGAAAACCTTACCCCAGTGGTGCTCAGACCAGAGATTCGCAAACCAGAGGTTCTCA GACCGGAAACACCCAGACCAGTGGACATTGGTTCTGGAGGATTTGGTGATGTCGAGCAGAA AGACCATGGGTTTGAGGTGGCCTCCACTTCCCCTGAAGACGAGTCCCCTGGCAGTAACCCC GAGCCAGATGCCACCCCGTTCCAGGAAGGTTTGAGGACCTTCGACCAGCTGGACGCCATAT CTAGTTTGCCCACACCCAGTGACATCTTTGTGTCCTACTCTACTTTCCCAGGTTTTGTTTCCT GGAGGGACCCCAAGAGTGGCTCCTGGTACGTTGAGACCCTGGACGACATCTTTGAGCAGTG GGCTCACTCTGAAGACCTGCAGTCCCTCCTGCTTAGGGTCGCTAATGCTGTTTCGGTGAAA GGGATTTATAAACAGATGCCTGGTTGCTTTAATTTCCTCCGGAAAAAACTTTTCTTTAAAACAT CATAA MDEADRRLLRRCRLRLVEELQVDQLWDALLSRELFRPHMIEDIQRAGSGSRRDQARQLII DLETRGSQALPLFISCLEDTGQDMLASFLRTNRQAAKLSKPTLENLTPVVLRPEIRKPEV LRPETPRPVDIGSGGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTR TGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQ ASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVAS TSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSG SWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS Dual Emission Green Fluorescent Protein (degfp) ATGGAGCTTTTCACTGGCGTTGTTCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCC ACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGA AGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGAC CTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAG TCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACT ACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGA AGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAA CAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAG ATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACC CCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCC CTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCC GCCGGGATCTAA MELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQ CFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKED GNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDN HYLSTQSALSKDPNEKRDHMVLLEFVTAAGI Prothrombin (FII) S-5

6 ATGGCGCACGTCCGAGGCTTGCAGCTGCCTGGCTGCCTGGCCCTGGCTGCCCTGTGTAGC CTTGTGCACAGCCAGCATGTGTTCCTGGCTCCTCAGCAAGCACGGTCGCTGCTCCAGCGGG TCCGGCGAGCCAACACCTTCTTGGAGGAGGTGCGCAAGGGCAACCTAGAGCGAGAGTGCG TGGAGGAGACGTGCAGCTACGAGGAGGCCTTCGAGGCTCTGGAGTCCTCCACGGCTACGG ATGTGTTCTGGGCCAAGTACACAGCTTGTGAGACAGCGAGGACGCCTCGAGATAAGCTTGC TGCATGTCTGGAAGGTAACTGTGCTGAGGGTCTGGGTACGAACTACCGAGGGCATGTGAAC ATCACCCGGTCAGGCATTGAGTGCCAGCTATGGAGGAGTCGCTACCCACATAAGCCTGAAA TCAACTCCACTACCCATCCTGGGGCCGACCTACAGGAGAATTTCTGCCGCAACCCCGACAG CAGCACCACGGGACCCTGGTGCTACACTACAGACCCCACCGTGAGGAGGCAGGAATGCAG CATCCCTGTCTGTGGCCAGGATCAAGTCACTGTAGCGATGACTCCACGCTCCGAAGGCTCC AGTGTGAATCTGTCACCTCCATTGGAGCAGTGTGTCCCTGATCGGGGGCAGCAGTACCAGG GGCGCCTGGCGGTGACCACACATGGGCTCCCCTGCCTGGCCTGGGCCAGCGCACAGGCC AAGGCCCTGAGCAAGCACCAGGACTTCAACTCAGCTGTGCAGCTGGTGGAGAACTTCTGCC GCAACCCAGACGGGGATGAGGAGGGCGTGTGGTGCTATGTGGCCGGGAAGCCTGGCGAC TTTGGGTACTGCGACCTCAACTATTGTGAGGAGGCCGTGGAGGAGGAGACAGGAGATGGG CTGGATGAGGACTCAGACAGGGCCATCGAAGGGCGTACCGCCACCAGTGAGTACCAGACT TTCTTCAATCCGAGGACCTTTGGCTCGGGAGAGGCAGACTGTGGGCTGCGACCTCTGTTCG AGAAGAAGTCGCTGGAGGACAAAACCGAAAGAGAGCTCCTGGAATCCTACATCGACGGGCG CATTGTGGAGGGCTCGGATGCAGAGATCGGCATGTCACCTTGGCAGGTGATGCTTTTCCGG AAGAGTCCCCAGGAGCTGCTGTGTGGGGCCAGCCTCATCAGTGACCGCTGGGTCCTCACC GCCGCCCACTGCCTCCTGTACCCGCCCTGGGACAAGAACTTCACCGAGAATGACCTTCTGG TGCGCATTGGCAAGCACTCCCGCACAAGGTACGAGCGAAACATTGAAAAGATATCCATGTTG GAAAAGATCTACATCCACCCCAGGTACAACTGGCGGGAGAACCTGGACCGGGACATTGCCC TGATGAAGCTGAAGAAGCCTGTTGCCTTCAGTGACTACATTCACCCTGTGTGTCTGCCCGAC AGGGAGACGGCAGCCAGCTTGCTCCAGGCTGGATACAAGGGGCGGGTGACAGGCTGGGG CAACCTGAAGGAGACGTGGACAGCCAACGTTGGTAAGGGGCAGCCCAGTGTCCTGCAGGT GGTGAACCTGCCCATTGTGGAGCGGCCGGTCTGCAAGGACTCCACCCGGATCCGCATCAC TGACAACATGTTCTGTGCTGGTTACAAGCCTGATGAAGGGAAACGAGGGGATGCCTGTGAA GGTGACAGTGGGGGACCCTTTGTCATGAAGAGCCCCTTTAACAACCGCTGGTATCAAATGG GCATCGTCTCATGGGGTGAAGGCTGTGACCGGGATGGGAAATATGGCTTCTACACACATGT GTTCCGCCTGAAGAAGTGGATACAGAAGGTCATTGATCAGTTTGGAGAGTAG MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRANTFLEEVRKGNLEREC VEETCSYEEAFEALESSTATDVFWAKYTACETARTPRDKLAACLEGNCAEGLGTNYRGHV NITRSGIECQLWRSRYPHKPEINSTTHPGADLQENFCRNPDSSTTGPWCYTTDPTVRRQE CSIPVCGQDQVTVAMTPRSEGSSVNLSPPLEQCVPDRGQQYQGRLAVTTHGLPCLAWASA QAKALSKHQDFNSAVQLVENFCRNPDGDEEGVWCYVAGKPGDFGYCDLNYCEEAVEEETG DGLDEDSDRAIEGRTATSEYQTFFNPRTFGSGEADCGLRPLFEKKSLEDKTERELLESYI DGRIVEGSDAEIGMSPWQVMLFRKSPQELLCGASLISDRWVLTAAHCLLYPPWDKNFTEN DLLVRIGKHSRTRYERNIEKISMLEKIYIHPRYNWRENLDRDIALMKLKKPVAFSDYIHP VCLPDRETAASLLQAGYKGRVTGWGNLKETWTANVGKGQPSVLQVVNLPIVERPVCKDST RIRITDNMFCAGYKPDEGKRGDACEGDSGGPFVMKSPFNNRWYQMGIVSWGEGCDRDGKY GFYTHVFRLKKWIQKVIDQFGE Coagulation Factor 10 (FX) S-6

7 ATGGGGCGCCCACTGCACCTCGTCCTGCTCAGTGCCTCCCTGGCTGGCCTCCTGCTGCTC GGGGAAAGTCTGTTCATCCGCAGGGAGCAGGCCAACAACATCCTGGCGAGGGTCACGAGG GCCAATTCCTTTCTTGAAGAGATGAAGAAAGGACACCTCGAAAGAGAGTGCATGGAAGAGA CCTGCTCATACGAAGAGGCCCGCGAGGTCTTTGAGGACAGCGACAAGACGAATGAATTCTG GAATAAATACAAAGATGGCGACCAGTGTGAGACCAGTCCTTGCCAGAACCAGGGCAAATGT AAAGACGGCCTCGGGGAATACACCTGCACCTGTTTAGAAGGATTCGAAGGCAAAAACTGTG AATTATTCACACGGAAGCTCTGCAGCCTGGACAACGGGGACTGTGACCAGTTCTGCCACGA GGAACAGAACTCTGTGGTGTGCTCCTGCGCCCGCGGGTACACCCTGGCTGACAACGGCAA GGCCTGCATTCCCACAGGGCCCTACCCCTGTGGGAAACAGACCCTGGAACGCAGGAAGAG GTCAGTGGCCCAGGCCACCAGCAGCAGCGGGGAGGCCCCTGACAGCATCACATGGAAGCC ATATGATGCAGCCGACCTGGACCCCACCGAGAACCCCTTCGACCTGCTTGACTTCAACCAG ACGCAGCCTGAGAGGGGCGACAACAACCTCACCAGGATCGTGGGAGGCCAGGAATGCAAG GACGGGGAGTGTCCCTGGCAGGCCCTGCTCATCAATGAGGAAAACGAGGGTTTCTGTGGT GGAACTATTCTGAGCGAGTTCTACATCCTAACGGCAGCCCACTGTCTCTACCAAGCCAAGAG ATTCAAGGTGAGGGTAGGGGACCGGAACACGGAGCAGGAGGAGGGCGGTGAGGCGGTGC ACGAGGTGGAGGTGGTCATCAAGCACAACCGGTTCACAAAGGAGACCTATGACTTCGACAT CGCCGTGCTCCGGCTCAAGACCCCCATCACCTTCCGCATGAACGTGGCGCCTGCCTGCCTC CCCGAGCGTGACTGGGCCGAGTCCACGCTGATGACGCAGAAGACGGGGATTGTGAGCGGC TTCGGGCGCACCCACGAGAAGGGCCGGCAGTCCACCAGGCTCAAGATGCTGGAGGTGCCC TACGTGGACCGCAACAGCTGCAAGCTGTCCAGCAGCTTCATCATCACCCAGAACATGTTCTG TGCCGGCTACGACACCAAGCAGGAGGATGCCTGCCAGGGGGACAGCGGGGGCCCGCACG TCACCCGCTTCAAGGACACCTACTTCGTGACAGGCATCGTCAGCTGGGGAGAGGGCTGTGC CCGTAAGGGGAAGTACGGGATCTACACCAAGGTCACCGCCTTCCTCAAGTGGATCGACAGG TCCATGAAAACCAGGGGCTTGCCCAAGGCCAAGAGCCATGCCCCGGAGGTCATAACGTCCT CTCCATTAAAGTGA MGRPLHLVLLSASLAGLLLLGESLFIRREQANNILARVTRANSFLEEMKKGHLERECMEE TCSYEEAREVFEDSDKTNEFWNKYKDGDQCETSPCQNQGKCKDGLGEYTCTCLEGFEGKN CELFTRKLCSLDNGDCDQFCHEEQNSVVCSCARGYTLADNGKACIPTGPYPCGKQTLERR KRSVAQATSSSGEAPDSITWKPYDAADLDPTENPFDLLDFNQTQPERGDNNLTRIVGGQE CKDGECPWQALLINEENEGFCGGTILSEFYILTAAHCLYQAKRFKVRVGDRNTEQEEGGE AVHEVEVVIKHNRFTKETYDFDIAVLRLKTPITFRMNVAPACLPERDWAESTLMTQKTGI VSGFGRTHEKGRQSTRLKMLEVPYVDRNSCKLSSSFIITQNMFCAGYDTKQEDACQGDSG GPHVTRFKDTYFVTGIVSWGEGCARKGKYGIYTKVTAFLKWIDRSMKTRGLPKAKSHAPEVITSS PLK Fibroblast Growth Factor 21 (FGF21) ATGGACTCGGACGAGACCGGGTTCGAGCACTCAGGACTGTGGGTTTCTGTGCTGGCTGGTC TGCTGGGAGCCTGCCAGGCACACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGGGGG CCAAGTCCGGCAGCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGA GATCAGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTGCA GCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTTCCTGT GCCAGCGGCCAGATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCCTGCAGCTT CCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCG CTGCACCTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGACCAGCTCGC TTCCTGCCACTACCAGGCCTGCCCCCCGCACTCCCGGAGCCACCCGGAATCCTGGCCCCC CAGCCCCCCGATGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCAGGGCCGA AGCCCCAGCTACGCTTCCTGA S-7

8 MDSDETGFEHSGLWVSVLAGLLLGACQAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAH LEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEA CSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGI LAPQPPDVGSSDPLSMVGPSQGRSPSYAS Single Chain Variable Fragment R4 (scfvr4) ATGGCGGAAGTGCAGCTGGTGGAAAGCGGCGGCAGCCTGGTGAAACCGGGCGGCAGCCT GCGCCTGAGCTGCGCGGCGAGCGGCTTTACCTTTAGCAACTATAGCATGAACTGGGTGCGC CAGGCGCCGGGCAAAGGCCTGGAATGGATTAGCAGCATTAGCGGCAGCAGCCGCTATATTT ATTATGCGGATTTTGTGAAAGGCCGCTTTACCATTAGCCGCGATAACGCGACCAACAGCCTG TATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTGCGTGCGCAGCAGCA TTACCACCTTTGGCGGCGGCATGGATGTGTGGGGCCGCGGCACCCTGGTGACCGTGAGCA GCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGAGCGTGCT GACCCAGCCGGCGAGCGTGAGCGGCAGCCCGGGCCAGAGCATTACCATTAGCTGCGCGG GCACCAGCAGCGATGTGGGCGGCTATAACTATGTGAGCTGGTATCAGCAGCATCCGGGCAA AGCGCCGAAACTGATGATTTATGAAGATAGCAAACGCCCGAGCGGCGTGAGCAACCGCTTT AGCGGCAGCAAAAGCGGCAACACCGCGAGCCTGACCATTAGCGGCCTGCAGGCGGAAGAT GAAGCGGATTATTATTGCAGCAGCTATACCACCCGCAGCACCCGCGTGTTTGGCGGCGGCA CCAAACTGGCGGTGCTGGGCGCGGCGGCGGAACAGAAACTGATTAGCGAAGAAGATCTGA ACGGCGCGGCGCATCATCATCATCATCAT MAEVQLVESGGSLVKPGGSLRLSCAASGFTFSNYSMNWVRQAPGKGLEWISSISGSSRYIYYAD FVKGRFTISRDNATNSLYLQMNSLRAEDTAVYCVRSSITTFGGGMDVWGRGTLVTVSSGGGGS GGGGSGGGGSQSVLTQPASVSGSPGQSITISCAGTSSDVGGYNYVSWYQQHPGKAPKLMIYE DSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTTRSTRVFGGGTKLAVLGAAAEQ KLISEEDLNGAAHHHHHH Maltose Binding Protein (MBP) ATGAAAATGAATAAAAGTCTCATCGTCCTCTGTTTATCAGCAGGGTTACTGGCAAGCGCG CCTGGAATTAGCCTTGCCGATGTTAACTACGTACCGCAAAACACCAGCGACGCGCCAGCC ATTCCATCTGCTGCGCTGCAACAACTCACCTGGACACCGGTCGATCAATCTAAAACCCAG ACCACCCAACTGGCGACCGGCGGCCAACAACTGAACGTTCCCGGCATCAGTGGTCCGGTT GCTGCGTACAGCGTCCCGGCAAACATTGGCGAACTGACCCTGACGCTGACCAGCGAAGTG AACAAACAAACCAGCGTTTTTGCGCCGAACGTGCTGATTCTTGATCAGAACATGACCCCA TCAGCCTTCTTCCCCAGCAGTTATTTCACCTACCAGGAACCAGGCGTGATGAGTGCAGAT CGGCTGGAAGGCGTTATGCGCCTGACACCGGCGTTGGGGCAGCAAAAACTTTATGTTCTG GTCTTTACCACGGAAAAAGATCTCCAGCAGACGACCCAACTGCTCGACCCGGCTAAAGCC TATGCCAAGGGCGTCGGTAACTCGATCCCGGATATCCCCGATCCGGTTGCTCGTCATACC ACCGATGGCTTACTGAAACTGAAAGTGAAAACGAACTCCAGCTCCAGCGTGTTGGTAGGA CCTTTATTTGGTTCTTCCGCTCCAGCTCCGGTTACGGTAGGTAACACGGCGGCACCAGCT GTGGCTGCACCCGCTCCGGCACCGGTGAAGAAAAGCGAGCCGATGCTCAACGACACGGAA AGTTATTTTAATACCGCGATCAAAAACGCTGTCGCGAAAGGTGATGTTGATAAGGCGTTA AAACTGCTTGATGAAGCTGAACGCCTGGGATCGACATCTGCCCGTTCCACCTTTATCAGC AGTGTAAAAGGCAAGGGGTAA MKMNKSLIVLCLSAGLLASAPGISLADVNYVPQNTSDAPAIPSAALQQLTWTPVDQSKTQTTQLA TGGQQLNVPGISGPVAAYSVPANIGELTLTLTSEVNKQTSVFAPNVLILDQNMTPSAFFPSSYFTY QEPGVMSADRLEGVMRLTPALGQQKLYVLVFTTEKDLQQTTQLLDPAKAYAKGVGNSIPDIPDP VARHTTDGLLKLKVKTNSSSSVLVGPLFGSSAPAPVTVGNTAAPAVAAPAPAPVKKSEPMLNDT ESYFNTAIKNAVAKGDVDKALKLLDEAERLGSTSARSTFISSVKGKG S-8

9 S-9