Recombinant expression and purification of human EP300-KIX87 domain from Escherichia coli
Year: 2024
Type: article
Abstract: Recombinant expression and purification of human EP300-KIX87 domain from Escherichia coli Materials and Reagents pUC57-EP300-KIX87 (synthesized by Genscript)pMAL-c5e vector (NEB, Cat Number: N8110)Restriction enzymes: BamH1 and Nde1 (NEB, Cat numbers: R0136S and R0111S)Low melt Agarose (Biorad, Cat number: 1613111)QIAquick Gel extraction Kit (Qiagen, Cat number: 28704)Quick Ligase Kit (NEB, Cat Number: M2200S)NEB turbo competent E. coli cells (NEB, Cat number: C2984H) and E. coli Rosetta 2 (DE3) cells (Millipore-Sigma, Cat Number: 71397-3)Ampicillin Sodium salt (Millipore-Sigma, Cat number: A0166)Terrific Broth (TB) (Fisher Scientific, Cat number: BP2468)Isopropyl-β-D-thiogalactopyranoside (IPTG) (Fisher Scientific, Cat number: BP1755-10)Amylose resin High Flow (New England Biolabs,Cat number: E8022L)Amicon Ultra Centrifugal Filter (Millipore Sigma, Cat number: UFC903024)250 ml and 2 L Erlenmeyer culture flasksQ-sepharose Fast Flow (Cytiva, Cat number: 17051010)PD MiniTrap G-25 columns (Cytiva, Cat number: 28918007)Pierce universal nuclease (ThermoFisher Scientific, Cat number: 88700)cOmplete EDTA free Protease Inhibitor Cocktail (Roche, Cat number: 05056489001)Tris Base (Fisher Scientific., Cat number: BP152)NaCl (Fisher Scientific, Cat number: S271)Ethylenediaminetetraacetic acid or EDTA (Fisher Scientific, Cat number: O2783)Dithiothreitol or DTT (Fisher Scientific, Cat number: BP-172)Maltose (Fisher Scientific, Cat number: M75)HEPES (Millipore Sigma, Cat number: H3375)Buffer A (See Recipes)Buffer B (see Recipes)Buffer C (see Recipes)Buffer D (see Recipes)Buffer E (see Recipes)Buffer F (see Recipes) Equipment Water Bath (Revolutionary Science)Shaker incubator (Sanyo, orbital incubator)Cell density meter (Ultrospec10 from GE healthcare)Nanodrop 2000C (Thermo scientific)High speed centrifuge (Beckman Coulter)Emulsifex-C3 High pressure homogenizerBenchtop centrifuge (Fisher Scientific)NGC chromatography system (Biorad) Procedure Cloning in pMAL-c5e vector: Restriction digestion of pUC57-EP300-KIX87 with Nde1 and Bam H1 restriction enzymes.Restriction digestion of pMAL-c5e vector with Nde1 and BamH1 restriction enzymes.1% agarose gel electrophoresis to separate the digested products.Extract the digested products (EP300KIX87 and pMAL-c5e) from agarose gel using gel extraction Kit.Ligation reaction for vector (pMAL-c5e) and insert (p300-KIX87) using quick ligase kit.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.Grow the cells at 37 °C for overnight at 260 rpm.Plasmids isolation from colonies on transformed plate and sent for sequencing (Eurofin genomics). Notes: (1) Human EP300-KIX87 sequenceSynthesized ORF sequencecatatgggtatccgtaaacaatggcacgaagacatcacccaagacctgcgtaatcatctggttcacaaactggttcaagctatttttccgacgccggacccggccgcactgaaagaccgtcgcatggaaaacctggtggcgtatgcgcgtaaagttgaaggcgatatgtacgaaagcgcgaacaatcgcgcagaatattaccatctgctggcagaaaaaatctacaaaattcaaaaagaactggaagaaaaacgccgcacccgcctgtgaggatcc*Underlines bases shows the cut sites for restriction enzymes. Wild-type ORF sequenceggaattcggaaacagtggcacgaagatattactcaggatcttcgaaatcatcttgttcacaaactcgtccaagccatatttcctacgccggatcctgctgctttaaaagacagacggatggaaaacctagttgcatatgctcggaaagttgaaggggacatgtatgaatctgcaaacaatcgagcggaatactaccaccttctagctgagaaaatctataagatccagaaagaactagaagaaaaacgaaggaccagactac Human EP300-KIX87 domain Protein sequence encoded by either ORFMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGDDDDKVPHMGIRKQWHEDITQDLRNHLVHKLVQAIFPTPDPAALKDRRMENLVAYARKVEGDMYESANNRAEYYHLLAEKIYKIQKELEEKRRTRL*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). Overexpression of EP300-KIX87: Transform E. coli Rosetta2 (DE3) cells with pMAL-c5e vector containing the human EP300-KIX87 domain open reading frame (residues 565-652). Thaw one vial of competent cells on ice per transformation.Add 50-100 ng of the plasmid in a volume of 25 mL to the cells and mix by gently tapping.Incubate the vial(s) on ice for 30 min.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.Add 500 mL of pre-warmed SOC medium to the vial(s) and then incubate the vial(s) at 37 °C for 1 h.Plate the transformation reaction onto LB plates containing ampicillin (100 mg/mL) for plasmid selection. Invert the plates and incubate at 37 °C overnight. 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.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.Cool the resulting culture to 25 °C and add IPTG to yield a final concentration of 1 mM.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.Store the cell pallet in -80 °C until further use. Purification of EP300-KIX87: 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).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.Wash the resin with more than 10 resin-bed volumes using buffer B and then elute the protein with 50 ml of buffer C.Dilute the eluted protein sample with 800 mL of Triple distilled water (TDW).Equilibrate an anion exchange chromatography column (Q-Sepharose, 40 ml) with Buffer D using an NGC Liquid Chromatography System.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.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.Sample aliquots were stored at -80 °C until further use.Samples were buffer exchanged with Buffer F using PD MiniTrap G-25 columns for biochemical assays. Recipes Buffer A 20 mM Tris-HCl (pH 7.5)50 mM NaCl1 mM EDTA1 mM DTT Buffer B20 mM Tris-HCl (pH 7.5)1 M NaCl1 mM EDTA 1 mM DTTBuffer C20 mM Tris-HCl (pH 7.5)50 mM NaCl1 mM EDTA1 mM DTT10 mM MaltoseBuffer D50 mM Tris-HCl (pH 7.5)2 mM DTT1 mM EDTABuffer E50 mM Tris-HCl (pH 7.5)1 M NaCl2 mM DTT1 mM EDTABuffer F50 mM HEPES (pH 7.5)50 mM NaCl1 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.
Source: BIO-PROTOCOL
DOI: 10.21769/p2617
Authors: John H. Bushweller, Kwon-Sik Park, Ashish Kabra
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: diamond
OpenAlex ID: W4393089637