Protein expression, separation and purification Experimental protein expression, separation and purification can: (1) explore and study the function of genes and the mechanism of gene expression regulation; (2) study for structure and function; (3) act as a catalyst and nutrition Agent. Principle of the experimental method The plasmid carrying the target protein gene was overexpressed in E. coli BL21 at 37 ° C, induced by IPTG, and recombinant chloramphenicol acylase protein carrying 6 consecutive histidine residues was available. A chromatographic medium that solidifies nickel ions (Ni2 +) by covalently coupled nitrilotriacetic acid (NTA) is purified by metal affinity affinity chromatography (MCAC). The degree of protein purification can be analyzed by polyacrylamide gel electrophoresis. Experimental material: E. coli BL21 Reagent, kit: LB liquid culture medium Ampicillin Washing Buffer Elution Buffer IPTG Distilled water Tryptone yeast powder sodium chloride Instrument, consumables: shaker centrifuge chromatography column centrifuge tube pipette gun tip box beaker Glass rod experiment step 1. Reagent preparation 1. LB liquid medium: Trytone 10 g, yeast extract 5 g, NaCl 10 g, and make up to 1000 mL with distilled water. 2. Ampicillin: 100 mg / mL. 3. Loading buffer: 100 mM NaH2PO4, 10 mM Tris, 8M Urea, 10 mM 2-ME, pH 8.0. 4. Washing Buffer: 100 mM NaH2PO4, 10 mM Tris, 8 M Urea, pH 6.3.5 . Elution Buffer: 100 mM NaH2PO4, 10 mMTris, 8M Urea, 500 mM Imidazole, pH 8.0. 6. IPTG: 100 mM IPTG (isopropylthio-β-D-galactoside): 2.38g IPTG dissolved in 100ml In ddH2O, 0.22μm filter membrane is suction filtered and stored at -20 ℃. 2. Obtaining the target gene 1. Through the PCR method: using the cloned plasmid containing the target gene as a template, design a pair of primers according to the gene sequence (introducing different enzyme cleavage sites in the upstream and downstream primers respectively), and obtaining the desired gene by PCR cycle Fragment. 2. By RT-PCR method: TRIzol method is used to extract total RNA from cells or tissues, using mRNA as a template, reverse transcription to form the first strand of cDNA, and PCR cycle using the reverse transcription product as a template to obtain the product. 3. Construction of recombinant expression vector 1. Digestion of the vector: the expression plasmid is double-digested with restriction enzymes (the same sites as the primers), and the digested products are subjected to agarose electrophoresis, and the kit is recovered with gel The large fragment of the carrier is recovered by the melting method. 2. The PCR product is recovered after double digestion, and ligated into the vector under the action of T4 DNA ligase. 4. Obtain the expression strain containing the recombinant expression plasmid 1. Transform the ligation product into E. coli DH5α, select according to the marker of the recombinant vector (anti-Amp or blue and white spot), pick a single spot, and extract the plasmid in a small amount by the alkaline lysis method. Preliminary identification of double digestion. 2. Sequencing verifies that the insertion direction and reading frame of the target gene are correct, and proceeds to the next step. Otherwise, more clones should be screened and repeated subcloning or subcloning to different cleavage sites. 3. Use this recombinant plasmid DNA to transform competent cells expressing host bacteria. V. Induction of chloramphenicol acyltransferase recombinant protein 1. Inoculate E. coli BL21 strain containing recombinant chloramphenicol acyltransferase protein in 5 mL LB liquid medium (containing 100 ug / mL ampicillin), shaking at 37 ℃ Incubate overnight. 2. Dilute the overnight bacteria at a ratio of 1:50 or 1: 100, generally transfer 1 mL of the overnight culture in 100 mL (containing 100 ug / mL ampicillin) LB liquid medium, shake culture at 37 ℃ to OD600 = 0.6 -0.8 (preferably 0.6, about 3 hours). Take 10 ul samples for SDS-PAGE analysis. 3. In the control group, no inducer was added, and the experimental group was added IPTG to a final concentration of 0.5 mmol / l, and the cultivation was continued at 37 ° C for 1-3 hours. 4. Centrifuge at 12 000 rpm for 10 min, discard the supernatant, and store the bacterial pellet in a refrigerator at -20 ° C or -70 ° C. 6. Isolation and purification of chloramphenicol acyltransferase recombinant protein 1. Preparation of NTA chromatography column: add 1 mL of NTA medium to the chromatography column, and wash with 8 mL of deionized water and 8 mL of loading buffer respectively . 2. Denaturation and lysis of recombinant protein: freeze and thaw the bacterial pellet in an ice bath, add 5 mL of loading buffer, suck and resuspend with a pipette, rupture the bacterial with ultrasound, and gently mix the sample for 60 min with a shaker, etc. Centrifuge at 12000 rpm at 4 ° C for 30 min. Aspirate the supernatant into a clean container and discard the pellet. Take 10 ul of supernatant sample for SDS-PAGE analysis. 3. Put the supernatant sample on the Ni2 + -NTA column at a flow rate of 10-15 mL / h, collect the effluent, and take 10 ul of the sample for SDS-PAGE analysis. 4. Elution of impurities: Wash the column with Washing Buffer at a flow rate of 10-15 mL / h until OD280 = 0.01. Collect the eluent in steps, about 3-4 h. Take 10 ul of the sample at the beginning of elution for SDS -PAGE analysis. 5. Elute the target protein: wash the column with Elution Buffer, collect each 1 mL fraction, and take 10 ul samples for SDS-PAGE analysis.
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