Protein expression, separation and purification - Database & Sql Blog Articles

Objectives (1) To understand the method and significance of cloned gene expression. (2) To understand the method of separation and purification of recombinant protein affinity chromatography. Experimental Principles The expression of cloned genes in cells is of great significance for both theoretical and experimental applications. By expressing the function of exploring and studying genes and the mechanism of gene expression regulation, the cloned genes express the encoded proteins for structural and functional studies. E. coli is the most widely used protein expression system, and its level of expression of foreign gene products is much higher than other gene expression systems, and the amount of protein expressed can exceed 80% of total bacterial protein. In this experiment, the plasmid carrying the target protein gene overexpresses the recombinant chloramphenicol acyltransferase protein carrying 6 consecutive histidine residues in E. coli BL21 under the induction of IPTG at 37 ° C. It can be purified by a chromatographic medium in which nickel ions (Ni2+) are solidified by covalently coupled sub-aminotriacetic acid (NTA), which is a metal chelating affinity chromatography (MCAC). The degree of purification of the protein can be analyzed by polyacrylamide gel electrophoresis. Reagents and equipment I. Reagents [1] LB liquid medium: Trytone 10g, yeast extract 5g, NaCl 10g, with distilled water to 1000mL. [2] Ampicillin: 100mg/mL [3] Loading buffer: 100 mM NaH2PO4 , 10 mMTris, 8M Urea, 10 mM2-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 II, equipment shaker, centrifuge, column (1'10 cm) method 1. Induction of chloramphenic acid acyltransferase recombinant protein 1. Inoculation contains recombination The chloramphenicol acyltransferase protein of Escherichia coli BL21 strain was cultured overnight in a 5 mL LB liquid medium (containing 100 ug/mL ampicillin) at 37 ° C with shaking. 2. Transfer 1 mL of the overnight culture to 100 mL (100 ug/mL ampicillin) in LB liquid medium and incubate at 37 °C until OD600 = 0.6 - 0.8. A 10 ul sample was taken for SDS-PAGE analysis. 3. Add IPTG to a final concentration of 0.5 mmol/l, continue to incubate at 37 °C for 1-3 h.4. 12,000 rpm for 10 min, discard the supernatant, and store the cells in a -20 ° C or -70 ° C freezer. 2. Isolation and purification of chloramphenic acid acyltransferase recombinant protein 1. Preparation of NTA column: 1 mL of NTA medium was added to the column, and washed with 8 mL of deionized water and 8 mL of loading buffer, respectively. 2. Denatured cleavage of recombinant protein: freeze-thaw the cell pellet in an ice bath, add 5 mL of the loading buffer, resuspend with a pipette, ultrasonically rupture the cells, and gently mix the sample with a shaker for 60 min, 4 ° C Centrifuge at 12000 rpm for 30 min, draw the supernatant into a clean container, and discard the pellet. 10 ul of the supernatant sample was taken for SDS-PAGE analysis. 3. The supernatant sample was subjected to a Ni2+-NTA column at a flow rate of 10-15 mL/h, and the effluent was collected, and 10 ul of the sample was taken for SDS-PAGE analysis. 4. Elution of the heteroprotein: Wash the column with Washing Buffer at a flow rate of 10-15 mL/h until OD280 = 0.01. The eluate was collected in steps, about 3-4 h, and 10 ul of the sample at the beginning of elution was used for SDS-PAGE. analysis. 5. Elution of target protein: Wash column with Elution Buffer, collect 1 mL fraction, and take 10 ul samples for SDS-PAGE analysis.