Pfusion Purification Protocol

Pfusion Protein

MILDADYITE DGKPIIRIFK KENGEFKVEY DRNFRPYIYA LLKDDSQIDE VRKITAERHG KIVRIIDAEK 70

VRKKFLGRPI EVWRLYFEHP QDVPAIRDKI REHSAVIDIF EYDIPFAKRY LIDKGLIPME GDEELKLLAF 140

DIETLYHEGE EFAKGPIIMI SYADEEEAKV ITWKKIDLPY VEVVSSEREM IKRFLKVIRE KDPDVIITYN 210

GDSFDLPYLV KRAEKLGIKL PLGRDGSEPK MQRLGDMTAV EIKGRIHFDL YHVIRRTINL PTYTLEAVYE 280

AIFGKPKEKV YAHEIAEAWE TGKGLERVAK YSMEDAKVTY ELGREFFPME AQLSRLVGQP LWDVSRSSTG 350

NLVEWYLLRK AYERNELAPN KPDEREYERR LRESYAGGYV KEPEKGLWEG LVSLDFRSLY PSIIITHNVS 420

PDTLNREGCR EYDVAPEVGH KFCKDFPGFI PSLLKRLLDE RQEIKRKMKA SKDPIEKKML DYRQRAIKIL 490

ANSILPEEWV PLIKNGKVKI FRIGDFVDGL MKANQGKVKK TGDTEVLEVA GIHAFSFDRK SKKARVMAVK 560

AVIRHRYSGN VYRIVLNSGR KITITEGHSL FVYRNGDLVE ATGEDVKIGD LLAVPRSVNL PEKRERLNIV 630

ELLLNLSPEE TEDIILTIPV KGRKNFFKGM LRTLRWIFGE EKRVRTASRY LRHLENLGYI RLRKIGYDII 700

DKEGLEKYRT LYEKLVDVVR YNGNKREYLV EFNAVRDVIS LMPEEELKEW RIGTRNGFRM GTFVDIDEDF 770

AKLLGYYVSE GSARKWKNQT GGWSYTVRLY NENDEVLDDM EHLAKKFFGK VKRGKNYVEI PKKMAYIIFE 840

SLCGTLAENK RVPEVIFTSS KGVRWAFLEG YFIGDGDVHP SKRVRLSTKS ELLVNGLVLL LNSLGVSAIK 910

LGYDSGVYRV YVNEELKFTE YRKKKNVYHS HIVPKDILKE TFGKVFQKNI SYKKFRELVE NGKLDREKAK 980

RIEWLLNGDI VLDRVVEIKR EYYDGYVYDL SVDEDENFLA GFGFLYAHNS YYGYYGYAKA RWYCKECAES 1050

VTAWGREYIE FVRKELEEKF GFKVLYIDTD GLYATIPGAK PEEIKKKALE FVDYINAKLP GLLELEYEGF 1120

YVRGFFVTKK KYALIDEEGK IITRGLEIVR RDWSEIAKET QAKVLEAILK HGNVEEAVKI VKEVTEKLSK 1190

YEIPPEKLVI YEQITRPLHE YKAIGPHVAV AKRLAARGVK VRPGMVIGYI VLRGDGPISK RAILAEEFDL 1260

RKHKYDAEYY IENQVLPAVL RILEAFGYRK EDLRWQKTKQ TGLTAWLNIK KK

MATVKFKYKG EEKEVDISKI KKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKK GGGGGT

GTGGGGATVK FKYKGEEKEV DISKIKKVWR VGKMISFTYD EGGGKTGRGA VSEKDAPKEL LQMLEKQKKLEHHHHHH-

PI: 9.14

it binds to CM FF or SP FF, does not bind Q FF at pH 8.0

MW=169 kDa

Protocol 1

Materials
50% Ni-NTA agarose (Qiagen).
Econo-Pac columns (Bio-Rad).

5 ml HiTrap CM-FF.

10 ml HiTrap SP

Buffers

Buffer A, 50 mM Tris-HCl, pH7.5, 500 mM NaCl, 10 mM imidazole. Store at 4 oC.
Washing buffer:  the same as Buffer A.
Elution buffer (Buffer B): Buffer A with 250 mM imidazole. Store at 4 oC.

Buffer C: 0.2 M NaCl in 50 M phosphate buffer,pH 6.9

Buffer D: 25 mM Tris-HCl, pH 7.5

Buffer E: 25 mM Tris-HCl, pH 7.5, 50 mM NaCl

Buffer F: 25 mM Tris-HCl, pH 7.5, 1 M NaCl

Buffer G: 25 mM Tris-HCl, pH 7.5, 0.15 M NaCl

Dialysis Buffer I: 20 mM Tris-HCl (pH 7.4 at 25°C), 0.1 mM EDTA, 100 mM KCl.

Dialysis Buffer II: 20 mM Tris-HCl (pH 7.4 at 25°C), 0.1 mM EDTA, 100 mM KCl, 50% glycerol

Storage Buffer: 20 mM Tris-HCl (pH 7.4 at 25°C), 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, 0.5 M Betaine, 200 μg/ml BSA and 50 % glycerol
Storage Buffer Additives: 1 M DTT, 10 mg/ml BSA

These chemicals should be molecular biology grade (at least DNase-free): EDTA,  DTT,  KCl,  Betaine,  BSA and   glycerol

Working Storage Buffer (50 ml ): 2.92 g Betaine, 25 ml glycerol, 5 ml 1 M KCl, 10 ul 0.5 M EDTA, 25 ul 2 M DTT, 1 ml 10mg/ml BSA, 1 ml 1M Tris-HCl, pH7.4, water to 50 ml.

ethylene glycol and 1,2-propanediol can also be used to decrease http://www.biotechniques.com/BiotechniquesJournal/2009/September/Enhanced-amplification-of-GC-rich-DNA-with-two-organic-reagents/biotechniques-173811.html

PCR additives: 10 M betaine 5.5 µL (final 2.2 M), 1.5 µL ethylene glycol (final 1.075 M) and 1.5 µL 1,2-propanediol (final 0.816 M)  can be used for GC rich template.

Expression and Purification

1. Transform Nico(DE3)/pLysN with Pfusion expression plasmid .

2. Make 8 L  LB

3. Inoculate 200 ml LB medium supplemented with 1% (w/v) glucose, and 34 ug/ml chloramphenicol with the transformed E. coli expression strain and incubate with shaking overnight at 37 oC.

4. Inoculate LB medium supplemented with 1% (w/v) glucose, 50 ug/ml Kan, and 34 ug/ml chloramphenicol with the overnight culture in a ratio of 1:50 and incubate with vigorous shaking at 37 oC until an OD600= 1.2–1.5.

5. Induce expression by adding 1 M IPTG solution to a final concentration of 0.5 mM (add 0.5 ml of 1 M IPTG to 1 L) and continue incubation o/n

6. Pellet cells by centrifugation (6000 rpm, 4 min) and freeze the bacterial pellets at −20 oC, or -80 oC, or dry ice. The cell pellets can be stored at −20 oC for several days, but it is advisable to use them as soon as possible.

7. Thaw pellets in warm water and resuspend in 10 ml 1X buffer A per liter culture using a magnetic plastic stick.

8. Homogenize for 1 to 2 min, make sure not a lot of bubbles are genenated. 

9. Boil at 75 oc for 30 min.

10. Centrifuge for 30 min at 4 oC and 12000 rpm (do not run > 12000 rpm). Spin again at 30000 rpm for 25 min at 4 oC on ultracentrifuge.

12. Decant the supernatant, load to 2.5 cm column packed with ~ 10 ml Ni beads. 

13. Wash the Ni beads twice with 5 to 20 column volumes of washing buffer (10 mM imidazole) until no protein is detectable (using Braford).

14. Elution of the protein using Elution buffer, collect 3 ml fractions (until all protein is eluted, usually 10 to 15 fractions should be collected).

15. Load fractions (10 ml) containing Pfusion to TSK Gel G 3000 SW column (3~5 ml) equilibrated with cold Buffer C (0.2 M NaCl in 50 M phosphate buffer, pH 6.9).

16. FPLC Separation of proteins with 125 ml Buffer C.

17. Combine Pfusion protein fractions. Dilute 3 folds with Buffer D and load protein to 5 ml CM FF column, wash column with 10 CV of Buffer E.

or dilute 2 folds with Buffer D and load protein to 5 ml SP FF column, wash column with 10 CV of Buffer E.

18. Elute protein with 50 mM to 1 M NaCl linear gradient (Buffer E to Buffer F), 100 ml. Collect 3 ml fractions

19. Determine protein concentration using Bradford reagent.

20. Wash a dialysis tube thoroughly with double-distilled water.  Check if the dialysis tube is leaking.

21. Transfer protein solution to the dialysis tube and dialyse in 70 ml of Dialysis Buffer I for 4 hour in cold room. Dialyze protein again for 4 h in cold room.

22. Dialyse with 50 ml of Dialysis Buffer II in cold room overnight to concentrate protein. Change buffer and dialyse again with 50 ml of Dialysis Buffer II for 5~8 hour.

23. Determine protein concentration using  a nanodrop. Run SDS PAGE to check protein purity.  Mix 1 ul  with  5, 10, 20, 30, 40, 50 and 100 ul of Dialysis Buffer I, take 1 ul of mixed solution to run PCR.

24. Add 1 M DTT to adjust to 1 mM DTT final concentration, sterilize protein by filtration (0.22-um filter). Aliquot to 1.5 ml tube and store protein in −80 oC freezer.

25. Record QC data including protein name, protein concentration, plasmid name, clone name, competent cells, number of vials, enzyme activity, purification date, assay date etc., on Product database.