insect
Contents
1. E ep Tactin Superflow 1 Triton X 100 1 Tween 0 3 CHAPS 2 Igepal CA 630 1M NaC1l Anti FLAGP M2 Agarose from mouse 5 Triton X 100 5 Tween20 0 1 CHAPS 0 1 Tgepal CA 630 1 M NaCl 50kDa 70kDa2. V N 5 ATGNNNNNNNNNNNNNNN 3 N 15 30 mer 2X 4 T 4X G C 55 60 2 PCR 5 C 5 GGGGTACCTTANNNNNNNNNN 3 N Kpnl stop 25 40 mer 2X A T 4X G C 55 60 2 3 Kpnl GG 2
3. 3mL 3mL mRNA 100zL 400L mRNA 1 5mL 400L mRNA mRNA 40zL 3M 950zL 15 000rpm 4C 20 70 mRNA 20 100 mL mRNA 2mg mL 5 3 mRNA mRNA mRNA
4. 1 m mRNA 320 ug 4 mM Methionine 20 ulL Reaction Buffer 300 L Insect Cell Extract 500 L to 1000 gL 12 II 005 25 5 15 000 rpm 15 4 mRNA Nick columns szrep tag Strep Tactin Superflow QIAGEN 30001 30003 50 1 1mL mRNA Nick columns
5. Stop PCR Terminal transferase Fidelity PCR 2uL 005 KOD plus TOYOBO KOD 201 cDNA 5 ng 96 C 2 min 10 x Buffer 5 LL 96 C 15 sec dNTP 2 mM each 5 LL 50 30 sec 30 25 mM MgS04 2 HE 68 C 1 min kbp N 10 uM 1 5 L 68 C 7 min C 10 M 5 RL 4 C co KOD plus I HE to 50 L 5 PCR 5
6. 50kDa 8 6 C 8 szrep tag G8 strep F 5 GGGAATTCGGTACCGGATCCGGTGGAGGTGGAGGTGGAGGTGGATGGAGCCATCCGCAGTTTGAAAAGTAATCTAGAGC 3 G8 strep R 5 GCTCTAGATTACTTTTCAAACTGCGGATGGCTCCATCCACCTCCACCTCCACCTCCACCGGATCCGGTACCGAATTCCC 3 pczoRI al
7. C EcoRI KpnI BamHI pcoRV 15 II 005 1 EcoRV OS i 2 RNase A mRNA 3 Transdirect 8 4 CR NG NACE NN NN 60C 1 mRNA
8. 0 DNA 2 3 PCR 5 nmRNA mRNA 70 6 Reaction Buffer 4 mM Methionine Insect Cell Extract 8 7 RNase 1unit L 8
9. kbp 0 7kbp TE 50 L 20pzL 50pzL 5 g PCR 005 IL mRNA mRNA RNA 3 mRNA NTP NTP mRNA nmRNA mRNA nmRNA NTP 1 mRNA 2
10. 1 5mL 5g mRNA DNA 7 PCR pTD1 20 A MO 2 pTD1 161 179 N pTD1 412 394 C 50C pTD1 161 179 5 GCAGATTGTACTGAGAGTG 3 pTD1 412 394
11. PCR EcoRV Kpnl PCR LB 1 5mL 37 GenElute PlasmidMiniprep Kit SIGMA PLN 70 0 5zL pTD1 PCR TOYOBO InsertCheck Ready Blue PIK 251 pTD1 161 179 5 GCAGATTGTACTGAGAGTG 3 pTD1 845 827 5 GGAAACAGCT
12. TOYOBO T4 Polynucleotide Kinase PNK 111 35uL Denaturation Buffer TOYOBO T4 Polynucleotide Kinase 90C 2 DNA 35 uL Blunt End Kinase Buffer 5 L 0 1M ATP 0 5 L 14 Polynucleotide Kinase 1 ulL 8 5 pL 50 wL 37C 1 90 2 85L C DNA 85 uL 10xBuffer 10 gL 30 50 U to 100 gL 37C 2 2 pTD1 EcoRV KpnIl
13. 1 Ezure T Suzuki T Higashide S Shintani E Endo K Kobayashi S Shikata M Ito M Tanimizu K and Nishimura 0 2006 Cel1 free protein synthesis system prepared from insect cells by freeze thawing Biotechnol Prog 22 1570 1577 2 Suzuki T Ito M Ezure T Kobayashi S Shikata M Tanimizu K and Nishimura 0 2006 Performance of expression vector pTD1 in insect cell free translation system Biosci Bioeng 102 69 71 3 Suzuki T Ito M Ezure T Shikata M Ando E Utsumi T Tsunasawa S and Nishimura 0 2006 N Terminal protein modifications in an insect cell free protein synthesis system and their identification by mass spectrometry Proteomics 6 4486 4495 4 Suzuki T Ito M Ezure T Shikata M Ando E Utsumi T Tsunasawa S and Nishimura 0 2007 Protein prenylation in an insect cell free protein synthesis system and identification of products by mass spectrometry Proteomics 7 1942 1950 17 005 Fig 1 mRNA 005 MW kDa 160 105 75 50 30 Fig 2 mRNA Transdirect Control DNA A
14. mRNA 20 x MOPS 1 aL 3 L 8 pnL 8 g to 20 ulL 65C 15 1 1 mRNA RNase DNA Transdirect Reaction Buffer 4 mM Methionine TInsect Cell Extract
15. 005 Transdirect insect ce77 J 2 gt 3 2008 4 14 2008 4 24 2008 4 24 Transdirect 4 frugiperda Ht E Sf21 Spodoptera Ht Ht Accession Number AB194742
16. bed volume 0 5mL 50mM Tris HC1 pH8 0 300mM NaCl Buffer A 5mL 0 5mL Buffer A 5 2 5mM Desthiobiotin SIGMA D1411 1G Buffer A 1 5mL 20 50rL 1 0 FLAGW tag Anti FLAGW M2 Agarose from mouse SIGMA A2220 50 1 1mL DTT 50mM Tris HC1 pH8 0 150mM NaC Buffer B PD 10 GE 17 0851 01 Buffer B 5mL
17. 1 1 2xQuick Ligation Reaction Buffer 10 ulL Quick T4 DNA Ligase 1 L to 20 gL 25C 5 4 0 3 DH5 o co77 DH5o Competent Cells 9057 2L Competent Cells 20pzL 30 005 42 1 5 SOC 180 Lt 37C 1 100 L LB 37 5
18. PD 10 1mL Buffer B 5 100g mL FLAG Peptide SIGMA F3290 Buffer B 2 5 ml 20 50L 1 1 B8 N C s ep tag FLAG tag 3 1m 10 20g g N C 13 II 005 5
19. pTD1 Sf21 MnNPV Malacosma neustria nucleopolyhedrovirus 5 mRNA T7 MCS polyA mRNA Transdirect K Mg 1 2 mL 30 50g
20. nmRNA 100L 300 600 g mRNA Epicentre Amp1iScribe T7 Flash Transcription Kit ASF3257 DNA 5 g 10 x Buffer 10 ulL ATP 9 ulL CTP 9 auL GTP 9 pL UTP 9 pL DTT 10 gL T7 RNA Polymerase 10 ulL RNase Free Water to 100 gL 37 30 Promega T7 RiboMAX Express Large Scale RNA Production System P1320 DNA 5 g 2xBuffer 50 wL Tl RNA Polymerase 10 ulL RNase Free Water to 100 gL 37 30 WY 4 II 005 2 mRNA mRNA NTP GE NICKM Columns 17 0855 01
21. Transdirect 10 3 4 005 NICKW Columns GE 17 0855 01 PD 10 GE 17 0851 01 TE PCR DNA LB LB SOC RNA MOPS Transdirect insect cell 292
22. 12 5 SDS PAGE Bio Rad FX pro 1 2 mRNA mRNA 19 005 MW 1 2 3 4 5 6 7 kDa 250 160 105 75 50 35 25 Fig 3 B N C strep tagR FLAGE tag Transdirect pTD1 mRNA 12 5 SDS PAGE CBB 1 2 3 5 4 7 N FLAG C FLAG N Strep C Strep 20
23. pTD1 EcoRI Xbal FLAGW tag G8 FLAG F 5 GGGAATTCGGTACC6GGATCC6GTGGAGGTGGAGGTGGAGGTGGAGACTACAAGGATGACGATGACAAGTAATCTAGAGC 3 G8 FLAG R 5 GCTCTAGATTACTTGTCATCGTCATCCTTGTAGTCTCCACCTCCACCTCCACCTCCACCGGATCCGGTACCGAATTCCC 3 14 005 pTD1 EcoRI Xbal pTD1 G8 strep 5 TAATACGACTCACTATAGGAGTAGTATTTTTATTCTTTCGTAAAAAAATTAGAAAAATAAAATATAA T7 Promoter 5 Polyhedrin Leader Sequence GATATCGAATTCGGTACCGGATCCIGGTGGAGGTGGAGGTGGAGGTGGAITGGAGCCATCCGCAGTTTGAAAAG coRV EcoRI Kpnl BamHl Gly x 8 spacer s ep tag TAATCTAGA stop Xbal pTD1 G8 FLAG 5 TAATACGACTCACTATAGGAGTAGTATTTTTATTCTTTCGTAAAAAAATTAGAAAAATAAAATATAAA T7 Promoter 5 Polyhedrin Leader Sequence GATATC GAATTCGGTACCGGATCCIGGTGGAGGTGGAGGTGGAGGTGGAIGACTACAAGGATGACGATGACAAG coRV coRI Kpnl gamHI Gly X 8 spacer FLAG tag TAATCTAGA stop Xbal N C
24. 005 5g pTD1 EcoRV plD1 5 ug DNA 10 L 10xBuffer 5 L coRV 30 U to 50 gL 37 1 1zL 3kbp 1 85zL DNA 85 uL 10 x Buffer 10 gL 30 50 U to 100 gL 37C 2 3 5zg 3 1 2 NEW ENGLAND Biolabs Quick Ligation Kit M2200S 10 g mL 2 L
25. 20kDa FluoroTect tRNA 20kDa RNase A 9 IV 10 Desthiobiotin Strep Tactin Superflow 5 10 16 005 11 Anti FLAGW M2 Agarose from mouse 5 10
26. 6 50gL 7 Reaction Buffer 15 L 4 mM Methionine 1 L 10 II 005 Insect Cell Extract 25 L mRNA 16 ug to 50 gL 25 5 SDS PAGE CBB Transdirect RI FluoroTect GreenLys in vitro Translation Labeling System L5001 50 L 1pL FluoroTect Reaction Buffer 15 L 4 mM Methionine 1 L Insect Cell Extrac
27. 30000 91 FluoroTect GreenLys in vitro Translation Labeling System L5001 Anti FLAGP M2 Agarose from mouse SIGMA A2220 Strep Tactin Superflow QIAGEN 30001 30003 FLAG Peptide SIGMA F3290 Desthiobiotin SIGMA D1411 1G 005 1 5 UTR 5 UTR pTD1 EcoRV Kpnl 90 EcoRV Kpnl EcoRV EcoRI 20 30 EcoRV BamHI lt 10 Smal Xbal 1 DNA 2
28. 5 ACAACGCACAGAATCTAGC S 8 mRNA mRNA PCR PCR Fidelity gmI ol Transdirect Ha RNase RN NT A 125g mL PCR PCR Fidelity KOD plus 005 Plasmid 1 ng u L 5 UL 10 x Buffer 5 LL dNTPs 2 mM each 5 UL 25 mM MgSO4 2 L pTD1 161 179 10 M ls5 ft pTD1 845 827 10 uM 1 5 tL KOD plus 1uL 29 L 50 L o 96 50 68 Oo OO 4 C 2 min 15 sec 30 sec 30 cycles 1 min 1 kbp 7 min Co 2rL
29. ATGACCATG 3 InsertCheck Ready Blue 1 mL 150 pmo1 PCR PCR 10x L PCR LB PCR PCR PCR 94 C 4 min 94 C 30 sec 50 C 5 sec 30 cycles 72 C x sec kbp 0 7kbp 2kbp 30sec 2kbp 1min 2 5kbp kbp 0 7kbp II 005 0 7kbp 6 LB mRNA
30. NNNN 3 C 5 GGGGTACC TTANNNNNNNNNN 3 Kpnl stop B FLAG tag N FLAGR tag N 5 ATGIGACTACAAGGATGACGATGACAAGINNNNNNNNNNNNNNN 3 C 5 GGGGTACC TTANNNNNNNNNN 3 pl stop C strep tagP C N 5 ATGNNNNNNNNNNNNNNN 3 strep tag C 5 GGGGTACC TTACTTTTCAAACTGCGGATGGCTCCANNNNNNNNNN 3 Kpnl stop D FLAGW tag C N 5 ATGNNNNNNNNNNNNNNN 3 FLAGR tag C 5 GGGGTACC TTACTTGTCATCGTCATCCTTGTAGTCINNNNNNNNNN 3 Kpnl stop PCR DNA pTD1 2 mRNA TI mRNA mRNA 100zL 500z g mRNA 3
31. t 25 wwL Fluorolect 1 L mRNA 16 ug to 50 L 25C 5 2L SDS PAGE 1oading buffer 4X 6L 70C 3 SDS PAGE 8 8B 2 V N C strep tag IBA GmbH FLAGW tag Sigma Aldrich Corp 9 1 cDNA pTD1 11 005 A srrep tag N strep tag N 5 ATGITGGAGCCATCCGCAGTTTGAAAAGNNNNNNNNNNN
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