Editing MMLV Purification (section)

== Current Working Protocol ==

'''Columns'''<br/>50 ml Ni-NTA agarose column<br/>50 ml SP HP column



'''Plasmid:'''

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

*Add 0.001&nbsp;mM&nbsp; (1 uM) EDTA and 1 mM beta-ME to all buffers
*'''<font color="red">Step <u>11 to 19 </u>should be completed within reasonable short time (Must be completed within one working day, can not stop in the middle)</font>'''
*MMLV protein rapidly form aggreggate from&nbsp; 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.



=== <br/>'''Buffers''' ===

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

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

<u>'''Suspension Buffer'''</u>: 25 mM Tris-HCl, pH 8.0, &nbsp;&nbsp; 500 mM NaCl, 5% glycerol, 0.001 mM EDTA, 1 mM beta-ME.

<u>'''Lysis Buffer''':</u> 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5~10 mM imidazole, 5% glycerol,&nbsp; 1 mM beta-ME, 0.1% NP 40

'''<u>Buffer A</u>''', 25 mM Tris-HCl, pH 8.0,&nbsp; 500 mM NaCl,&nbsp;&nbsp;0.5% NP40,&nbsp;'''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.

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



'''<u>Ni Washing buffer</u>:''' the same as Buffer A. Store at 4 oC or in cold room.<br/>'''<u>Ni Elution buffer</u>''':&nbsp; 50 mM Phosphate Buffer, '''pH 7.5''',&nbsp;&nbsp;'''500 mM NaCl''', '''100 mM arginine,''' &nbsp; '''150 mM imidazole''',&nbsp; '''10% glycerol'''. Store at 4 <sup>o</sup>C or in cold room.

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

<br/>'''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.

<u>'''SP Dilution Buffer'''</u>''': '''50 mM Phosphate Buffer, pH 7.5 or 7.0, &nbsp; 10% glycerol, 1 mM beta-ME.

'''<u>SP Washing Buffer &nbsp;</u>'''(<font color="red">No glycerol</font>)<u>:</u> 50 mM Phosphate Buffer, pH '''7.5''', '''50 mM NaCl''',&nbsp; '''&nbsp;'''&nbsp; 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.

'''<u>SP Elution Buffer</u>'''&nbsp;(<font color="red">No glycerol</font>): 50 mM Tris-HCl, pH '''8.&nbsp;'''0,&nbsp;&nbsp;'''1 M NaCl,&nbsp; 100 mM arginine, '''.&nbsp;'''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.

'''<u>Dialysis Buffer A&nbsp;</u>'''(<font color="red">No glycerol</font>): 500 mM NaCl, 20 mM '''HEPES''',&nbsp; pH 7.4, 100 mM Arginine,&nbsp; <font color="red">15% Trehalose</font>, 0.1 mM EDTA, add 1 mM DTT'''&nbsp;just before use. Store at 4 oC or in cold room.'''

'''<u>Dialysis Buffer B&nbsp;</u>'''(<span style="color:#FF0000;">with&nbsp;</span><font color="red">glycerol</font>): 500 mM NaCl, 20 mM&nbsp;'''HEPES''',&nbsp; pH 7.4, 100 mM Arginine,&nbsp;&nbsp;<font color="red">50% glycerol</font>, 0.1 mM EDTA, add 1 mM DTT'''&nbsp;just before use. Store at 4 oC or in cold room.'''

'''<u>Storage Buffer &nbsp;</u>'''(<span style="color: rgb(255, 0, 0);">with&nbsp;</span><font color="red">glycerol</font>): 100 mM NaCl, 20 mM&nbsp;'''HEPES''',&nbsp; pH 7.4,&nbsp;&nbsp;<font color="red">50% glycerol</font>, add 1 mM DTT'''&nbsp;just before use. Store at 4 oC or in cold room.'''

'''<u>1 M Arginine:</u>'''&nbsp; 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.

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

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

'''Storage Buffer (Promega):&nbsp;'''&nbsp;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):&nbsp;'''20 mM Tris-HCl (pH 7.5), '''100'''



'''Expression and Purification'''

<br/>1. Transform&nbsp; 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&nbsp;Nico21(DE3) star on LB/K plate, grow&nbsp;overnight at 37 oC



2. Make 20 L&nbsp; LB Plus&nbsp;medium

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

Use 8 L&nbsp; for each purification.





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

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

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

4. Inoculate 1L of&nbsp; LB Plus medium supplemented with&nbsp;&nbsp;50 ug/ml '''Kan'''&nbsp;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 >&nbsp;1.0~1.5, about 2~3 hours

5. Induce MMLV&nbsp;expression by adding 1 M IPTG solution to a final concentration of&nbsp; 0.5 mM (add 0.5&nbsp; ml of 1 M IPTG to 1 L) and induce&nbsp; 5-8 hours at 37 oC with vigorous shaking .

6. Spin down cells,&nbsp; 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]].&nbsp;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&nbsp;

9.&nbsp; 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

'''''<font color="red">The folling steps (10 to 19) should be completed in 1 working day.</font>'''''

<br/>'''10'''''<b>.</b>'''''Sonicate on&nbsp;ice,&nbsp; 2 X 2.5 min, until liquid is not sticky'''



11.&nbsp; <u>Spin again at 25,000 rpm for 30 min at 4 oC on ultracentrifuge.</u>

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

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

Note:

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

Rotor should be&nbsp; &nbsp;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&nbsp; load to a 50 ml Ni&nbsp; column.

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

16. Wash column with 1 CV of&nbsp; '''Buffer A2'''.

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

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

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

20. Elute MMLV protein using 500 ml ('''10 CV)&nbsp;'''of linear gradient of&nbsp;'''SP Washing Buffer'''&nbsp;& '''<u>SP Elution Buffer</u>'''&nbsp;

or Elute MMLV protein using&nbsp;&nbsp;'''<u>SP Elution Buffer without gradient</u>'''

or&nbsp; &nbsp;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. &nbsp; Determine protein concentration (OD280) using nanodrop

22. Wash a dialysis tube thoroughly with 0.1 N NaOH then ddH2O,&nbsp; 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,&nbsp; -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&nbsp; 45~50 ml of '''<u>Storage Buffer&nbsp;</u> '''(in 100 ml beaker) in fridge,&nbsp;'''<u>mix occasionally for several times,</u>''' then dialyse&nbsp;overnight to concentrate protein, Measure OD280.&nbsp;

For freeze drying: use&nbsp;<u>Dialysis Buffer A, otherwise use Dialysis B</u>





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&nbsp;20% ethanol).

25. Next morning, change&nbsp;'''<u>Storage Buffer,&nbsp;</u>'''dialyse&nbsp;for additional 3~6 hours, mix occasionally&nbsp;

26. After dialysis, transfer protein to a clean 50 ml tube. Wash dialysis tube with 3&nbsp;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.&nbsp; &nbsp;optionally:&nbsp;'''Remove aggregate by&nbsp;filtration (0.22-um low protein binding syringe filter).&nbsp;Aliquot to 1.5 ml tube&nbsp;&nbsp;'''



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,&nbsp; number of vials, enzyme activity, endotoxin level, purification date, assay date and URL, etc.



29. Store protein in a box in&nbsp;−80 <sup>o</sup>C freezer. Record protein location on [[Proteins_in_Freezer|this page]]
[[Category:Protocols]]