Paul.xu at 21:46, 4 December 2020

2020-12-04T21:46:51Z

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== Current Working Protocol ==

'''Columns''' 50 ml Ni-NTA agarose column 50 ml SP HP column

'''Plasmid:'''

'''MMLV''': pI/Mw: 9.08 / 79.6

*Add 0.001 mM  (1 uM) EDTA and 1 mM beta-ME to all buffers
*'''Step 11 to 19 should be completed within reasonable short time (Must be completed within one working day, can not stop in the middle)'''
*MMLV protein rapidly form aggreggate from  80 kDa to very large MW. Large aggregate will be precipitated in Step 25 and the yield will be very low if glycerol and arginine is not added
*Do not handle protein at room temperature. MMLV will form aggregate rapidly after exposing to room temperature for a only few minutes.
*Do not let protein expose to a buffer with low ionic stength ('''<200 mM NaCl)''' for a prolong time. Add arginine up to 0.5 M if low salt buffer is needed for purification.

=== '''Buffers''' ===

'''Buffer Q''': 25 mM Tris-HCl, pH 8.0, 1 mM beta-ME, 10% glycerol, 50 mM NaCl, <s>0.001 mM EDTA</s>.

'''Buffer Q2''': 25 mM Tris-HCl, pH 8.0, 1 mM beta-ME, 50 mM NaCl, <s>0.001 mM EDTA</s>.

'''Suspension Buffer''': 25 mM Tris-HCl, pH 8.0,    500 mM NaCl, 5% glycerol, 0.001 mM EDTA, 1 mM beta-ME.

'''Lysis Buffer''': 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5~10 mM imidazole, 5% glycerol,  1 mM beta-ME, 0.1% NP 40

'''Buffer A''', 25 mM Tris-HCl, pH 8.0,  500 mM NaCl,  0.5% NP40, '''10 mM '''imidazole, '''Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use.''' check pH ~8. Store at 4 oC or in cold room.

'''Buffer A2''', 25 mM Tris-HCl, pH 8.0, 10 mM imidazole, 5% glycerol. 1 mM beta-ME. Store at 4 oC or in cold room.

'''Ni Washing buffer:''' the same as Buffer A. Store at 4 oC or in cold room. '''Ni Elution buffer''':  50 mM Phosphate Buffer, '''pH 7.5''',  '''500 mM NaCl''', '''100 mM arginine,'''   '''150 mM imidazole''',  '''10% glycerol'''. Store at 4 oC or in cold room.

'''Dilution Buffer:'''  50 mM Phosphate Buffer,  pH 7.5 or pH7.0,   '''10% glycerol, '''1 mM beta-ME, '''0.001 mM EDTA'''. Store at 4 oC or in cold room.

 '''ETR Buffer''': '''200 mM Tris-HCl''', pH 7.0, 500 mM NaCl, 0.001 mM EDTA. Adjust to final concentration of 1 mM from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use. Store at 4 oC or in cold room.

'''SP Dilution Buffer'''''': '''50 mM Phosphate Buffer, pH 7.5 or 7.0,   10% glycerol, 1 mM beta-ME.

'''SP Washing Buffer  '''(No glycerol): 50 mM Phosphate Buffer, pH '''7.5''', '''50 mM NaCl''',  ''' '''  Adjust to final concentration of '''1 mM''' from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use. Store at 4 oC or in cold room.

'''SP Elution Buffer''' (No glycerol): 50 mM Tris-HCl, pH '''8. '''0,  '''1 M NaCl,  100 mM arginine, '''. '''Optionally, 10% Glycerol,''' Adjust to final concentration of '''1 mM''' from 14.3 M 2-Mercaptoethanol (in the fumehood) just before use, check pH to see if the buffer pH is ~8.0. Store at 4 oC or in cold room.

'''Dialysis Buffer A '''(No glycerol): 500 mM NaCl, 20 mM '''HEPES''',  pH 7.4, 100 mM Arginine,  15% Trehalose, 0.1 mM EDTA, add 1 mM DTT''' just before use. Store at 4 oC or in cold room.'''

'''Dialysis Buffer B '''(with glycerol): 500 mM NaCl, 20 mM '''HEPES''',  pH 7.4, 100 mM Arginine,  50% glycerol, 0.1 mM EDTA, add 1 mM DTT''' just before use. Store at 4 oC or in cold room.'''

'''Storage Buffer  '''(with glycerol): 100 mM NaCl, 20 mM '''HEPES''',  pH 7.4,  50% glycerol, add 1 mM DTT''' just before use. Store at 4 oC or in cold room.'''

'''1 M Arginine:'''  174.2g in 1L ddH2O. FIlter and store at 4 oC. Aliqute to 50 ml and store at -20 oC for long term storage. Store at 4 oC or in cold room. Arginine is very expensive.

''' 5X Reaction Buffer''': 250mM Tris-HCl (pH 8.3 @ 25°C), 375mM KCl, 15 mM MgCl2 and 50 mM DTT (Final 1X: 50mM Tris-HCl (pH 8.3 @ 25°C), 75mM KCl, 3mM MgCl2 and 10mM DTT)

'''5X Reaction Buffer (Thermo)''':  250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2,  10 mM DTT.

'''Storage Buffer (Promega): ''' 20mM Tris-HCl (pH 7.5), '''200''' mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.01% Nonidet® P-40 and 50% glycerol

'''Storage Buffer (Thermo): '''20 mM Tris-HCl (pH 7.5), '''100'''

'''Expression and Purification'''

 1. Transform  Nico21(DE3) star with the MMLV expression plasmid, for example, 2565 pET29b-MT-MMLV, 2861 pET30-MMLV-sso7d-GroEL-ES-TF grow in LB/Kanat 37 oC o/n.

Alternatively, spread transformed Nico21(DE3) star on LB/K plate, grow overnight at 37 oC

2. Make 20 L  LB Plus medium

500 g LB powder + 100 ml glycerol, add ddH2O to 20 L, aliquot  1 L to 2 L flask, autoclave.

or

100 g Yeast, 200 g NaCl, 200 g Peptone, + 100 ml glycerol, add ddH2O to 20 L, aliquot  1 L to 2 L flask, autoclave.

Use 8 L  for each purification.

3. Add 1 ml  of 1000X Kan to 1 L medium, or 200 ul 1000X Kan to 200 ml LB medium,  Inoculate 200 ml  or 1 L  LB  medium supplemented with 50 ug/ml '''Kan '''with the transformed E. coli expression strain and incubate with shaking overnight at 37 oC. 

Put 8 flasks of LB medium in 37 oC incubator, do not need to shake.

Next morning, add 1 ml of 1000X Kan to  each flask with 1 L LB medium

4. Inoculate 1L of  LB Plus medium supplemented with  50 ug/ml '''Kan''' with 20 ml of the overnight culture in a ratio of 1:50

i.e.: aliquote 1L or 200 ml of seed E coli culture to 8 L LB/K medium, '''no need''' to put lid onto flask again

Incubate with vigorous shaking at 37 oC until an OD600 > 1.0~1.5, about 2~3 hours

5. Induce MMLV 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 induce  5-8 hours at 37 oC with vigorous shaking .

6. Spin down cells,  6 min at 6000 rpm, RT., transfer pellet to one centrifuge tube. Weigh wet pellet, record weigh onto protein database [https://www.excellgen.com/protein/index.php [1]]. Add 100 ml Lysis Buffer to pellet and store at -80 oC o/n.

7. Thaw cell pellet, add ~400 ml '''cold Lysis Buffer.'''

8. add 10~50 mg lysozyme 

9.  Incubate at room temperature for 1~3 hour with continous stirring, or cold room overnight, until the cells are lysed, you will see liquid is very sticky

'''''The folling steps (10 to 19) should be completed in 1 working day.'''''

 '''10'''''.'''''Sonicate on ice,  2 X 2.5 min, until liquid is not sticky'''

11.  Spin again at 25,000 rpm for 30 min at 4 oC on ultracentrifuge.

If vaccum can not go down, or speed can not go up to > 3000 rpm

*stop centrifuge.
*Check if centrifuge tube is broken or missing a seal ring.
*Check if centrifuge caps are tighten

Note:

the vaccum pump could be damaged by evaporated salt and water if centrifuge tube is leaking.

Rotor should be   cooled down using dry ice. Remove residual water with clean paper or cloth if you want to cool down using dry ice.

12. Collect supernatant into a clean beaker, store at 4 oC.

13. Resuspend pellet with 200 ml of Lysis Buffer. Homogenize for 10 sec and spin at 30000 rpm again.

14. Combine supernatant and  load to a 50 ml Ni  column.

15. Wash column with 500~ 1000 ml '''Buffer A '''until OD280 drops to baseline.

16. Wash column with 1 CV of  '''Buffer A2'''.

17. Elute protein with  200 ml  of  '''Ni Elution Buffer (or Buffer B) on FPLC '''

18.  Dilute eluant with 2 volume of '''SP Dilution Buffer,''' load to SP FF or SP-HP columns using P1 pump + air bubble detector.

19. Wash  SP HP with 2 CV of '''SP Washing Buffer''' until OD280 reaches baseline,   use FPLC to monitor washing. 

20. Elute MMLV protein using 500 ml ('''10 CV) '''of linear gradient of '''SP Washing Buffer''' & '''SP Elution Buffer''' 

or Elute MMLV protein using  '''SP Elution Buffer without gradient'''

or   collect 10 ml fractions, switch to fraction size=0 when elution peak appears, collect protein using a beaker

'''add solid arginine immidiately to beaker if you see white aggregate.'''

MMLV will be eluted at ~ 0.5 M to 1.0 M NaCl at pH 7.5 ~8.0.

21.   Determine protein concentration (OD280) using nanodrop

22. Wash a dialysis tube thoroughly with 0.1 N NaOH then ddH2O,  Check if the dialysis tube is leaking.

23. Transfer MMLV protein solution to a dialysis tube. Wash residual protein from tubes using 0.25 M arginine and transfer to the dialysis tube. Adjust to '''2 mM DTT''' concentration from 2 M DTT (in a box labeled as AMP-DTT,  -20 oC fridge. For example, add 5 ul of 2 M DTT to 10 ml protein solution ( i.e., add 1:1000 dilution). Dialyse protein with  45~50 ml of '''Storage Buffer  '''(in 100 ml beaker) in fridge, '''mix occasionally for several times,''' then dialyse overnight to concentrate protein, Measure OD280. 

For freeze drying: use Dialysis Buffer A, otherwise use Dialysis B

24. Clean SP HP columns with 5 CV of 1.5 M NaCl + 0.1 M NaOH, wash with 10 CV of water (for longterm storage, wash with 20% ethanol).

25. Next morning, change '''Storage Buffer, '''dialyse for additional 3~6 hours, mix occasionally 

26. After dialysis, transfer protein to a clean 50 ml tube. Wash dialysis tube with 3 X 1 ml of '''1 M''' arginine, combine proteins.

27. Determine protein concentration using a nanodrop (OD280) or spectrophotometer. If concentration is too high for nanodrop, dilute 2 ul protein solution with water.   optionally: '''Remove aggregate by filtration (0.22-um low protein binding syringe filter). Aliquot to 1.5 ml tube  '''

28. Put vials immediately on ice. Record data on product databases, make sure to record all product information including protein name, protein concentration, plasmid name, clone name, competent cells, induction reagent (IPTG, autoinduce, L-arabinose), induction temperature and time, inducer concentration, weight of cell pellet, purification method, storage buffer,  number of vials, enzyme activity, endotoxin level, purification date, assay date and URL, etc.

29. Store protein in a box in −80 oC freezer. Record protein location on [[Proteins_in_Freezer|this page]]
[[Category:Protocols]]

MMLV Purification - Revision history

Paul.xu at 21:46, 4 December 2020