Objectives and Requirements
- Learn the approaches and meaning of the expression of cloned genes.
- Learn the method of recombinant protein affinity chromatography, isolation and purification
Expression of gene clone in cells is of great significance in both theoretical research and experimental applications. We can explore and study gene function and expression mechanism expression with gene expression. What’s more, the encoded protein by cloned gene expression can be used in researching the structure and function.
E.coli (Escherichia coli ), whose exogenous expressing productivity is much higher than that of other gene expression systems, is currently the most widely used protein expression system. The number of target protein expressed may even exceed 80% of the total protein in bacteria. In this experiment, the plasmid carrying target protein overexpresses recombinant chloramphenicol acyltransferase protein with 6 consecutive histidine residues in E. coli BL21 under the induction of IPTG in 37 ℃. The protein can be purified with chromatographic media formed by immobilizing nickel ions (Ni2 +) with covalent coupling nitrilotriacetic acid (NTA), which is in fact Metal Chelate Affinity Chromatography (MCAC). The purity of the protein can be analyzed by polyacrylamide gel electrophoresis.
Reagents and apparatus
- LB fluid medium: Tryptone 10g, yeast extract 5g, NaCl 10g.Mix all together with distilled water to 1000mL.
- Ampicillin: 100mg / mL
- Sample: Buffer Solution. 100 mM NaH2PO4, 10 mMTris, 8M Urea, 10 mM2-ME, pH8.0
- Washing Buffer: 100 mM NaH2PO4, 10 mM Tris, 8 M Urea, pH6.3
- Elution Buffer: 100mM NaH2PO4, 10 mMTris, 8M Urea, 500 mM Imidazole, pH8.0
37°C shaking incubator, centrifuges, chromatography column (1’10 cm)
The induction of chloramphenicol acyltransferase recombinant protein
- Inoculate BL21 E. coli strain containing recombinant chloramphenicol acyltransferase protein in 5mL LB liquid medium (containing 100ug / mL ampicillin), Grow liquid cultures with agitation in shaking incubator overnight at 37 ℃.
- Transfer 1mL of this culture to 100mL LB liquid medium (containing 100 ug / mL ampicillin) and treat the mixture with shaking incubator until OD600 = 0.6 – 0.8. Take 10ul of the sample for SDS-PAGE analysis.
- Add IPTG to make a final concentration of 0.5 mmol / l and then continue to culture at 37 ℃ for 1-3h.
- Centrifuge for 10min at 12,000rpm. Discard the supernatant. Keep the insoluble material at -20 ℃ or -70 ℃.
Isolation and purification of chloramphenicol acyltransferase recombinant protein
- Preparation of NTA chromatography column: add 1mL NTA into the chromatography column media, wash with 8mL of deionized water and 8mL of buffer elution respectively.
- Denaturing of recombinant protein: thaw the insoluble material in ice bath, add 5mL buffer elution in it, resuspended with a straw, vortex the bacteria with ultrasonic. Mix gently the sample, centrifuge at 12000rpm for 60min at 4 ℃, and collect supernatant Take 10ul for SDS-PAGE analysis.
- Pour the supernatant onto the Ni2 + -NTA column at 10-15mL / h of flow rate. Collect the liquid flowed out and take 10ul for SDS-PAGE analysis.
- Elution of impure protein: wash the column at 10-15mL / h with Washing Buffer till OD280 = 0.01. Repeat elution two to three times and analyze each fraction by SDS-PAGE.
- Elution of the target protein: wash by adding Elution Buffer. Take 10ul of each sample respectively for SDS-PAGE analysis.