Recombinant expression and purification of human EP300-KIX87 domain from Escherichia coli

Recombinant expression and purification of human EP300-KIX₈₇ domain from Escherichia coli

Materials and Reagents

1. pUC57-EP300-KIX₈₇ (synthesized by Genscript)
2. pMAL-c5e vector (NEB, Cat Number: N8110)
3. Restriction enzymes: BamH1 and Nde1 (NEB, Cat numbers: R0136S and R0111S)
4. Low melt Agarose (Biorad, Cat number: 1613111)
5. QIAquick Gel extraction Kit (Qiagen, Cat number: 28704)
6. Quick Ligase Kit (NEB, Cat Number: M2200S)
7. NEB turbo competent E. coli cells (NEB, Cat number: C2984H) and E. coli Rosetta 2 (DE3) cells (Millipore-Sigma, Cat Number: 71397-3)
8. Ampicillin Sodium salt (Millipore-Sigma, Cat number: A0166)
9. Terrific Broth (TB) (Fisher Scientific, Cat number: BP2468)
10. Isopropyl-β-D-thiogalactopyranoside (IPTG) (Fisher Scientific, Cat number: BP1755-10)
11. Amylose resin High Flow (New England Biolabs,Cat number: E8022L)
12. Amicon Ultra Centrifugal Filter (Millipore Sigma, Cat number: UFC903024)
13. 250 ml and 2 L Erlenmeyer culture flasks
14. Q-sepharose Fast Flow (Cytiva, Cat number: 17051010)
15. PD MiniTrap G-25 columns (Cytiva, Cat number: 28918007)
16. Pierce universal nuclease (ThermoFisher Scientific, Cat number: 88700)
17. cOmplete EDTA free Protease Inhibitor Cocktail (Roche, Cat number: 05056489001)
18. Tris Base (Fisher Scientific., Cat number: BP152)
19. NaCl (Fisher Scientific, Cat number: S271)
20. Ethylenediaminetetraacetic acid or EDTA (Fisher Scientific, Cat number: O2783)
21. Dithiothreitol or DTT (Fisher Scientific, Cat number: BP-172)
22. Maltose (Fisher Scientific, Cat number: M75)
23. HEPES (Millipore Sigma, Cat number: H3375)
24. Buffer A (See Recipes)
25. Buffer B (see Recipes)
26. Buffer C (see Recipes)
27. Buffer D (see Recipes)
28. Buffer E (see Recipes)
29. Buffer F (see Recipes)

Equipment 

1. Water Bath (Revolutionary Science)
2. Shaker incubator (Sanyo, orbital incubator)
3. Cell density meter (Ultrospec10 from GE healthcare)
4. Nanodrop 2000C (Thermo scientific)
5. High speed centrifuge (Beckman Coulter)
6. Emulsifex-C3 High pressure homogenizer
7. Benchtop centrifuge (Fisher Scientific)
8. NGC chromatography system (Biorad)

Procedure

1. Cloning in pMAL-c5e vector:

1. Restriction digestion of pUC57-EP300-KIX₈₇ with Nde1 and Bam H1 restriction enzymes.
2. Restriction digestion of pMAL-c5e vector with Nde1 and BamH1 restriction enzymes.
3. 1% agarose gel electrophoresis to separate the digested products.
4. Extract the digested products (EP300KIX₈₇ and pMAL-c5e) from agarose gel using gel extraction Kit.
5. Ligation reaction for vector (pMAL-c5e) and insert (p300-KIX₈₇) using quick ligase kit.
6. Transformed the ligated product into E. coli (NEB turbo) cells followed by colony picking on next day into 10 mL of TB media containing 100 mg/mL ampicillin.
7. Grow the cells at 37 °C for overnight at 260 rpm.
8. Plasmids isolation from colonies on transformed plate and sent for sequencing (Eurofin genomics).

Notes: 

           (1)         Human EP300-KIX₈₇ sequence

Synthesized ORF sequence

catatgggtatccgtaaacaatggcacgaagacatcacccaagacctgcgtaatcatctggttcacaaactggttcaagctatttttccgacgccggacccggccgcactgaaagaccgtcgcatggaaaacctggtggcgtatgcgcgtaaagttgaaggcgatatgtacgaaagcgcgaacaatcgcgcagaatattaccatctgctggcagaaaaaatctacaaaattcaaaaagaactggaagaaaaacgccgcacccgcctgtgaggatcc

*Underlines bases shows the cut sites for restriction enzymes.

Wild-type ORF sequence

ggaattcggaaacagtggcacgaagatattactcaggatcttcgaaatcatcttgttcacaaactcgtccaagccatatttcctacgccggatcctgctgctttaaaagacagacggatggaaaacctagttgcatatgctcggaaagttgaaggggacatgtatgaatctgcaaacaatcgagcggaatactaccaccttctagctgagaaaatctataagatccagaaagaactagaagaaaaacgaaggaccagactac

Human EP300-KIX₈₇ domain Protein sequence encoded by either ORF

MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGDDDDKVPHMGIRKQWHEDITQDLRNHLVHKLVQAIFPTPDPAALKDRRMENLVAYARKVEGDMYESANNRAEYYHLLAEKIYKIQKELEEKRRTRL*Blue highlighted amino acids are from vector containing MBP tag.

(2)        In the synthesized human EP300-KIX₈₇ ORF, codon usage is optimized for E. coli expression. NdeI and BamH1restriction sites are used for open reading frame (ORF) ligation into a pMAL-c5e vector (ampicillin-resistant). 

1. Overexpression of EP300-KIX₈₇:

1. Transform E. coli Rosetta2 (DE3) cells with pMAL-c5e vector containing the human EP300-KIX₈₇ domain open reading frame (residues 565-652).

1. Thaw one vial of competent cells on ice per transformation.
2. Add 50-100 ng of the plasmid in a volume of 25 mL to the cells and mix by gently tapping.
3. Incubate the vial(s) on ice for 30 min.
4. Heat shock the cells by incubating the vial(s) for exactly 45 s in a 42 °C water bath and then remove the vial(s) from the 42 °C water bath and quickly place on ice for 5 minutes.
5. Add 500 mL of pre-warmed SOC medium to the vial(s) and then incubate the vial(s) at 37 °C for 1 h.
6. Plate the transformation reaction onto LB plates containing ampicillin (100 mg/mL) for plasmid selection. Invert the plates and incubate at 37 °C overnight.

1. Collect 1 colony from transformed plate and inoculate into 25 mL of autoclaved TB medium (supplemented with 100 mg/mL ampicillin) in a 250 mL Erlenmeyer culture flask. Incubate overnight at 37 °C while shaking at 260 rpm.
2. Use ~5-10 mL of culture from Step 2 to inoculate 1 L of fresh TB medium containing 100 mg/mL ampicillin and grow in a 2 L Erlenmeyer culture flask at 37 °C until OD₆₀₀ = 0.8-1.0.
3. Cool the resulting culture to 25 °C and add IPTG to yield a final concentration of 1 mM.
4. After 12-16 h of induction with IPTG at 15 °C and stirring at 260 rpm, collect the cells by centrifuging the culture at 6,000 rpm for 60 mins.
5. Store the cell pallet in -80 °C until further use.

1. Purification of EP300-KIX₈₇:

1. Thaw frozen cells and suspend the cells in pre-chilled buffer A. 10 mL of Pierce universal nuclease and a tablet of protease inhibitor cocktail was also added to the suspended cell. Lyse the cells by emulsifex homogenizer at 5000-10000 psi with three rounds of cell lysis (in ice).
2. Transfer the lysate to a centrifuge tube and centrifuge at 35,000 rpm for 60 min at 4 °C before loading the resultant supernatant onto a 10 ml gravity flow column of containing amylose resin pre-equilibrated with buffer A.
3. Wash the resin with more than 10 resin-bed volumes using buffer B and then elute the protein with 50 ml of buffer C.
4. Dilute the eluted protein sample with 800 mL of Triple distilled water (TDW).
5. Equilibrate an anion exchange chromatography column (Q-Sepharose, 40 ml) with Buffer D using an NGC Liquid Chromatography System.
6. Load the diluted protein sample from Step 4 onto the pre-equilibrated anion exchange column, wash thoroughly using 120 mL of buffer D and elute using a 0 to 100% linear gradient of buffer E.
7. Eluted protein fractions were run on SDS PAGE to check the purity. The pure fractions were pooled together and concentrated using an Amicon Ultra-15 Centrifugal Filter (15 ml sample volume, 30 kDa cutoff). Sample concentration was checked using A₂₈₀ (extinction coefficient: 79300) on a nanodrop.
8. Sample aliquots were stored at -80 °C until further use.
9. Samples were buffer exchanged with Buffer F using PD MiniTrap G-25 columns for biochemical assays.

Recipes 

1. Buffer A 

20 mM Tris-HCl (pH 7.5)

50 mM NaCl

1 mM EDTA

1 mM DTT 

1. Buffer B

20 mM Tris-HCl (pH 7.5)

1 M NaCl

1 mM EDTA 

1 mM DTT

1. Buffer C

20 mM Tris-HCl (pH 7.5)

50 mM NaCl

1 mM EDTA

1 mM DTT

10 mM Maltose

1. Buffer D

50 mM Tris-HCl (pH 7.5)

2 mM DTT

1 mM EDTA

1. Buffer E

50 mM Tris-HCl (pH 7.5)

1 M NaCl

2 mM DTT

1 mM EDTA

1. Buffer F

50 mM HEPES (pH 7.5)

50 mM NaCl

1 mM DTT

**EP300-KIX as well CREBBP-KIX domain is very prone to degradation during recombinant overexpression and purification. So, the whole purification was carried out at 4 °C. Adding EDTA improves the stability of protein during purification.