大腸菌タンパク質発現系の ゴールドスタンダード pET 発現システム活用
Contents
1. pH 8 pBR322 10 mL oe B2. GILSP DNA e pET 0120 633 358 Web www merckmillipore jp pET pET System Manual 2 Novaden ColiRollers
3. GMP 25 mg mL 50 hg mL EE _20 Na 500X 1 A 0 22 um a 50 mg mL Oy 20C AE 2Na 1000 20 50 ug mL 022 um 30 udg mL Kan 905 0 20C 1000X 15 ng mL 0 22 um Kan 5 34 mg m 0 I 2 20 34 pg mL 2 022 um gt GS
4. 153 8927 1 8 1 5F WwWw merckmillipore jp On Line www merckmillipore jp jpts Tel 0120 633 358 Fax 03 5434 4859 BIM163 1506 12 5K M Merck KA Merck Millipore and the M mark are trademarks of Merck KGaA Darmstadt
5. DET pET 1IPTG T7 RNA T7RNA pET 3 0 9 9 Wi CE6 Novaden Merck Millipore is a business of MERCK MERCK MILLIPORE 3 Prrrrzt RNA T7 RNA IP RNA 2 T7 RNA 1 yy PET acUV5
6. em 2 lsopropyl 8 p 1 thiogalactopyranoside IPTG IN de T7 RNA IPTG 50 N end Rule Met N 2 10 Tobias et a 1991 2 X fIMet X 16 97 Hirel er al 1989 Lathop et al 1992 2 X Leu
7. Agel pET X Leu Wcol 2 G X Leu N 2 X Leu 2 IE Arg Arg GIn Glu N Leu Lys His Lys Met Phe Trp Tyr Phe Trp Tyr IPTG 1 mM pET IPTG acY7 IPTG
8. 9 tRNA Novagen Rosetta Rosetta pRARE tRNA tRNA Se EL Rosetta Rosetta2 ed Sa Class II Rosetta Rosetta2 AGA v CUA 0 039 0 006 0 037 0 008 AGG 0 022 0 003 CUC 0 104 0 08 CGG 0 098 0 008 a CUG 0 496 0 767 CGA3 0 065 0 011 CUU 0 104 0 056 CGU 0 378 0 643 UUA 0 131 0 055 CGC 0 398 0 33 UUG 0 129 0 034 GGA 0 109 0 02 CCA 0 191 0 153 Gy GGC 0 403 0 428 Pro CCC 0 124 0 016 GGG 0 151 0 044 CCG 0 525 0 719 GGU 0 337 0 508 CCU 0 159 0 112 EE RA EO EL 1 4290 Nakamura Y et al 2000 lle AUC 0 42 0 659 _ a 1 3 9 RE 2
9. pETcoco 1 40 IPTG CE6 T7 RNA T7 Novaden gs pET LB TB M9 M9ZB 0 4906 M9 M9ZB LB Miller noome C4 10 9 Yeast Extract 5 9 NaCl 10 g 1L 1N Na0H pH 7 5 A 4 M9 930 mL 20X M9 Salts M
10. 195 HenautA amp t Danchin A 1996 AUU 0 507 0 335 3 CGA CGA tRNA Rosetta Rosetta2 CGA tRNA Rosetta RosettaDE3 Competent Cells IPTG T7 RNA 6 tRNA 70954 3 70954 4 Rosetta 2 DE3 Competent Cells IPTG T7 RNA 7 tRNA 71397 3 71397 4 IPTG T7 RNA
11. 70837 3 70837 4 gcY7 IPTG Rosetta gami B DE3 Competent Cel MM T7 RNA 7x8 gor522 et 2 CU S cy7 IPTG 3 6 tRNA Hirel P H et al 1989 PNAS 86 21 p p 8247 8251 Lathrop B K et al 1992 Protein Expr Purif 3 6 p p 512 517 Kagawa N 2013 http kagawap450 blogspot jp 2013 01f iptg html Tobias J W er al 1991 Sc e7ce 254 5036 p p 1374 1377 N OV3 g e ne
12. IPTG 1 IPTG IPTG Kagawa N 2013 2 IPTG LacZ T7 0 4 mM gcY7 100 mM 6 238 mg 10 mD 20 2 Tlac 1 0 mM lacy1 T7 I7 gc 25 uM 1 0 mM acY7 Tuner DE3 Competent Cells acY IPTG pe en IPTG T7 RNA cY IPTG Origami B DE3 Competent Cells IPTG T7 RNA t x8 gor522
13. 2 TB Sambrook et al 1989 Deionized Water 900 mL Tryptone 12 g Yeast Extract 24 g Glycerol 4 mL 1L A 4 60 K Phosphate 100 mL 10X M9 Salts M9 NHACI 10 9 KH P0 30 9 Na zHP0 7H 0 7 60 9 1L GILSP pET EE EE T7 3 BL21 DE3 pET 15D BL21 PET 21a e US Patent number 4 952 496 2007 pET 16b pET 28a e US Patent number 5 693 489 2014 pET 19b BL21 DE3 pLysS pEI 3a e US Patent number 5 869 320 2004 pET 21a DEI 11a 3 2015 1 pET 21b Rosetta DE3 pET 30a pT pET 21d pET 32a pET 23b
14. Bacteriophage CE6 T7 RNA T7 TZjgc TL 020 38 000 DE3 Competent Cell Set 1 BL21 BLR HMS174 NovaBlue Tuner 0 2 mL Xx 5 71207 3 40 099 20 000 DE3 Competent Cell Set 2 0rigami 2 B Rosetta 2 RosettaBlue Rosetta gami B 0 2 mLX5 71208 3 38 699 19 000 DE3 pLysS Competent Cell Set 1 BL21 BLR HMS174 NovaBlue Tuner 0 2 mL X 4 71209 3 40 099 20 000 DE3 pLysS Competent Cell Set 2 0rigami 2 B Rosetta 2 RosettaBlue Rosetta gami B 0 2 mL X 4 71210 3 40 699 20 000 Origami 2 Competent Cell Set DE3 DE3 pLysS 00 71431 3 40 000 20 000 Origami B Competent Cell Set DE3 DE3 pLysS 70 70911 3 38 000 19 000 ROSEfta 2 COmpetent Cl Set DE3 DE3 plysS 2 71405 3 38 009 19 000 RosettaBlue Competent Cell Set DE3 DE3 pLysS I 71079 3 38 989 19 000 Rosetta gami 2 Competent Cell Set DE3 DE3 pLysS 0 71432 3 38 999 19
15. IPTG ge pET DE3 ge lacO 7ac V5 T7 RNA T7 gc 17 pET pET 3 T7 pLysS plysE T7 RNA pLysS 0 106 Triton X T7 pETcoco 0 20o6 1 1 pETcoco
16. SLGS033SS GV SLGV033RS DMS0 LG SLLG025SS 2 Met AUG 1 Leu 4 CUA CUC CUG CUU tRNA DNA Codon Usage Database http www kazusa or jp codon 9
17. Miller 25 g L 500 g sas y26 13 000 1L X 25 71752 4 2 160 069 50 000 r TI LB Miller 37 g 500 g 725 5 ya8 19 000 LB Lennox 20 g L 500 g 71751 5 2 24 999 12 000 TB 47 6 g L 500 g 71754 3 2 24 999 12 000 500 g 71280 3 2 38 999 19 000 500 g 71279 3 2 50 099 25 000 5 9 171254 5GM 2 24 008 12 000 SA 25 g 171254 25GM 2 78 099 39 000 250 mg 205805 250MG 2 20 099 10 000 5 9 420311 5GM 2 24 999 12 000 UE 25 g 420311 25GM 2 76 099 38 000 25 g 220551 25GM 2 24 099 12 000 0 100 g 220551 100GM 2 72 069 36 000 10 420322 1GM 2 14 099 7 000 5 g 420322 5GM 2 34 099 17 000 a Lac0 ac 10 9 220322 10GM 5 yp2 080 26 000 25 g 420322 25GM 2 125 069 62 500 7 CE6 4 0 2 mL 69390 3 2 000 10 500 Zac
18. Plating Beads X Gal Solution 40 mg mL BetaRed B Galactosidase Assay Kit 8 Galactosidase E coli gt 600 units mo LB Broth Miller LB Ager Miller LB Broth Lennox Terrific Broth Veggie Peptone Animal Free Veggie Yeast Extract Animal Free Ampicillin Sodium Salt Carbenicilin Disodium Salt Kanamycin Sulfate Streptomyces kanamyceticus Chloramphenicol IPTG Dioxane Free High Purity 98 OFF 2015 6 15 8 7 1 1 95 60 80 OS 2 6 689 3 000 SSM202 HG 74002 60 80 X 5 71013 4 2 20 099 10 000 LacZ 8 3 mL Om 2 34 999 17 000 LacZ 8 ONPG 10 500 70978 3 2 156 060 78 000 LacZ 8 5 mg 345788 5MG 2 62 099 31 000 1L X 25 71753 4 2 66 999 33 000 Ui Nn SS LB
19. B B lt DEI 3a pET 23d pET 22b pET 28b pET 28a 2NSS INESUSSOIE MA pET 30a e Genetics Institute Inc pET 32 pET 48 ET 50b DE ul OIAGEN GmbH HiseTag 1 GILSP 26 12 9 e IBA GmbH StrepeTag StrepeTactin 0 NN 0 2 GILSP 23 11 11 e GSTeTag a 2 e pET
20. Nakamura Y er al 2000 Nucleic Acids Res 28 1 p 292 3 T7 IPTG DE3 0 5 196 acUV5 T7 RNA DE3
21. xg goy522 Rosetta gami B DE3 Competent Cells 6 71136 3 71136 4 tRNA acY7 IPTG Rosetta gami 2 DE3 Competent Cells IPTG T7 RNA g go 522 5 a 4 7 1 tRNA Henaut A amp Danchin A 1996 In Escherichia coli and Salmonella typhimurium Cellular and Molecular Biology D Dp 2047 2066
22. 000 Rosetta gami B Competent Cell Set DE3 DE3 pLysS i MS 38 966 19 000 BL21 Competent Cell Set DE3 DE3 pLysS Ue 70232 3 38 000 19 000 HMS174 Competent Cell Set DE3 DE3 plysS AE 70234 3 38 899 19 000 Tuner Competent Cell Set DE3 DE3 pLysS ET 70726 3 38 699 19 000 Eaeepo0K 2 ZN mW 0 22 2 lt https www facebook com merckmilliporej https twitter com MerckMilliporeJ http www merckmillipore jp wm
23. 9 50 mL 20 Glucose 20 mL 1M MgS0 1 mL NaCl 0 5 9 100 mM K Phosphate KH P0 2 6 KHPO 125 4 g 1L pET pET Brookhaven National Laboratory Brookhaven Science Associates LB Lennox Tryptone 10 9 Yeast Extract 5 9 NaCl 5 9 4 1L 1N NaOH pH 7 5 4 M9ZB Studier et al 1990 N Z amine A Quest NZ 10 9 NaCl 5 9 4 1L vy 10X M9 Salts M9 100 mL 1 M MgS0 1 mL 4006 Glucose 10 mL 20X M9 Salts M9 0 2 6 20 g KH P0 60 g Na HP0 7H 0 7 120 g 1 L
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