T4 DNA Ligase 20121223
Method Development
[edit]Date: Dec, 2012
Protein eluted from Ni is very dirty:
Dilute protein from Ni column eluate to 50 mM NaCl. Load to 45 ml QFF column.
Wash with 100 ml 50 mM NaCl, then 50 ml 100 mM NaCl
Elute with 200~400 mM NaCl. Protein started to elute at 150 mM but the peak is tailing if 150 mM NaCl is used.
Should use 100 to 500 mM linear gradient.
This is another run using 4 5 ml Q FF in tandem. Looks like Q FF column is not good
So I concentrated protein from Q FF column using Pierce 9k spin column, load 4 ml to BioRad BioSil
TSK Gel 250 column. Run gel filtration. The peak is quite nice, but the protein is still not as pure as previous lot
The purity is about 90%. After dialysis vs 50% glycerol , I got ~ 3 ml, 13 mg/ml. I labeled it as 12 mg/ml
This gel shows the purity.
Protocol
[edit]Transform Nico21(DE3)-pLysN with pT7T3-T4 DNA ligase plasmid.
1. Grow E coli in 1 L LB/Kan/Chl at 37 oC until OD280 ~ 0.6-0.7. Total is 8 L
3. Induce with 0.3 mM IPTG o/n at 37 oC.
4. Spin down cells, resuspend in Buffer A (20 mM Tris-HCl, pH 7.5, 10 mM imidazole, 10% glycerol, 500 mM NaCl, 5 mM beta-ME).
Suspend 10 ml per g pellet. 30g --> 300 ml Buffer A.
5. Lyse cells by freezing and thawing then homogenization briefly.
6. Spin at 12000 rpm for 15 min, 4 oC.
7. Resuspend pellet in Buffer A (10 ml buffer A/g pellet). Homogenize and spin again. Pool supernatant.
8. Spin at 25000 rpm for 20 min, 4 oC.
9. Load clear supernatant to a 45 ml Ni column.
10. Wash with 500 ml Washing BufferI (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% Triton X-100 or NP-40, 5 mM beta-ME), then 300 ml Wash Buffer II (20 mM Tris-HCl, pH 7.5, 10 mM imidazole, 500 mM NaCl, 5 mM beta-ME). You can use peristaltic pump to wash the Ni column.
11. Elute with Buffer B (20 mM Tris-HCl, pH 7.5, 250 mM imidazole, 200 mM NaCl, 5 mM beta-ME). Collect 5 or 10 ml fractions. Discard the 1 CV (45 ml if Ni resin column is 45 ml).
12. Measure protein conc using Buffer B as blank. Combine fractions containing protein.
13. Equilibrate a 5 ml HiTrap Q FF column with Buffer B. Load protein solution to the column. T4 DNA ligase will not find and flow through should be collected. Flush out unbound protein using addition 10 ml of Buffer B.
After use, HiTrap Q FF column should be regenerated using 1.5M NaCl/0.1N NaOH, wash column with water and store the column in 20% ethanol.
14. Concentrate T4 DNA ligase using 2 Pierce 9k spin columns to 7~ 10 ml.
15. Load 3~ 4.5 ml to a Tosoh gel filtration BioRad Bio-Sil TSK250 column, elute with Buffer C (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM beta-ME).
Run > 2 times if you have more than 5 ml concentrated protein solution.
16. Collect 2.5 ml fractions. Determine protein conc in peak fractions. Run SDS-PAGE gel to check purity. Pool fractions containing T4 DNA ligase.
If protein is not pure enough, do step 17 to 23:
17. Equilibrate a 45 ml Q Sepharose FF XK26 column with 100 ml Buffer D (20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM beta-M).
18. Dilute protein solution with 0.5 to 1 volume of Buffer E (20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM beta-M). Load the diluted solution onto the Q column. Collect FT after 1 CV (45 ml), check protein concentration using buffer C as blank. Protein conc should be very low in FT.
19. Wash Q column with 100 ml (2 CV) of Buffer D.
20. Wash Q column with 50 ml (1 CV) of Buffer F (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM beta-M).
21. Connect XK column to FPLC. Elute protein with 200 ml linear gradient of Buffer D to Buffer F (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1 mM beta-ME), flow rate 2 ml per min. Collect 4 ml per fraction.
22. Measure protein concentration on fractions containing protein. Run SDS-PAGE on protein fractions. Protein purity should be at least 98%.
23. Pool fractions containing pure protein. Measure protein concentration.
24. Concentrate protein solution using two Pierce 9k spin columns. After concentration add DTT from 1 M or 2 M stock to protein solution to adjust final concentration of 1 mM. Dialyse protein solution vs Storage Buffer (20 mM Tris-HCl, 50 mM NaCl, 0.1 mM EDTA, 50% Glycerol, pH 7.5).
25. Take 2 ul and dilute to 2 ug/ul with Storage Buffer. Check enzyme activity using Fast T4 DNA Ligase Buffer
Specific activity: should be 300,000 U/mg.
Product for sale: dilute to 2 mg/ml (600,000 U/ml, 600U/ul) with Storage Buffer.
Fast T4 DNA Ligase Buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5 % PEG 6000, pH 7.6)
Make 5 X Buffer:
75g PEG 6000
50 ml of 1M Tris-HCl, pH 7.6
10 ml of 1 M MgCl2
18,3 MOhm ultrapure water to 200 ml
Autoclave, make sure buffer is not evaporated (measure the volume with autoclaved 100 ml measuring cylinder, add autoclave water to ~ 188.5ml if evaporated).
Add 2 ml of 0.5 M ATP, 0.5 ml of 2 M DTT.
If it is very viscous, dilute the buffer with water (add 50 ml autoclaved 18,3 MOhm ultrapure water) to make 4 X buffer.
Slow T4 DNA Ligase Buffer:
50 mM Tris-HCl
10 mM MgCl2
1 mM ATP
10 mM Dithiothreitol
pH 7.5 @ 25°C
Run gel to see if two fragments become one fragment after 1 hour ligation.
26. Create a record in protein database. Write detail description on every step.
27. Very Important: Do not dilute protein!!!.
Aliquote undiluted protein: 1 ml to 1.5 ml microcentrifuge tube. Label them and store undiluted protein in a freezer box at -80 oC.
28. Regenerate Q column using 100 ml CIP Solution (1.5 M NaCl, 0.1 N NaOH). Wash with 150 ml water and the flush with 60 ml 20% ethanol if the column will not be used in a few days.
Method Validation
[edit]8 L culture: induction with 0.3 mM IPTG, 30 oC, O/n. 30 g wet cells.
Lyse with 2 X 150 ml buffer A. Wash Ni beads with 400 ml Buffer A.
| Fraction | Protein, mg/ml | Vol, ml |
| 54 |
0.1 |
7.5 |
| 55 |
0.55 |
7.5 |
| 56 |
1.4 |
7.5 |
| 57 |
1.6 |
7.5 |
| 58 |
1.14 |
7.5 |
| 59 |
0.86 |
7.5 |
| 60 |
0.5 |
7.5 |
| 61 |
0.28 |
7.5 |
Take 10 ul to run RunBlue gel, marker is Invitrogen protein ladder SeeBlue® Plus2
M: 198, 98 (orange), 62, 49, 38, 28, 17 (pink), 14, 6, 3
MW: T4 ligase is 55292.65
PI= 6.14
Gel filtration, 2.5 ml/fraction
Take 10 ul to run gel
