Purpose requirement
(1) Understanding the method and significance of cloning gene expression.
(2) Understand the method of separation and purification of recombinant protein affinity chromatography.
Experimental principle
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
First, the reagent
[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
Second, equipment
Shaker, centrifuge, column (1'10 cm)
Method of operation
I. Induction of recombinant protein of chloramphenic acid acyltransferase
1. Escherichia coli BL21 strain containing recombinant chloramphenic acid acyltransferase protein was inoculated in 5 mL of LB liquid medium (containing 100 ug/mL ampicillin), and cultured overnight at 37 ° C with shaking.
2. Transfer 1 mL of the overnight culture to 100 mL (containing 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, and continue to culture for 1-3 h at 37 °C.
4. Centrifuge at 12,000 rpm for 10 min, discard the supernatant, and store the pellet in a -20 ° C or -70 ° C freezer.
2. Isolation and purification of recombinant protein of chloramphenic acid acyltransferase
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 loading buffer, resuspend with a pipette, ultrasonically rupture the cells, gently mix the sample with a shaker, etc. 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 the target protein: Wash the column with Elution Buffer, collect 1 mL fraction, and take 10 ul samples for SDS-PAGE analysis.
Aobeki's Portable Pizza Maker crafted for ultimate speed, convenience and ease of use. Mini Pizza Maker portable design, easy bring to pizza or any outdoor party.Fully adjustable heat allows for cooking several styles of pizza.
We are professional Gas Grill , Steak Grill, Gas Griddle manufactures, welcome to visit our website.
Pizza Maker Machine,Pizza Making Machine,Mini Pizza Maker,Pizza Oven Machine Electric Pizza Maker,Pizzazz Pizza Maker,Home Pizza Maker,Portable Pizza Maker
GUANGDONG AOBEKI MANUFACTURING CO., LTD , https://www.aobeki.com