Protein expression, isolation and purification can: (1) explore and study the function of genes and the mechanism of gene expression regulation; (2) be used for the study of structure and function; (3) be used as catalysts and nutrients, etc.
Operation method
Genetic recombination - chromatography
Principle
Plasmids carrying the target protein gene were used to overexpress recombinant chloramphenicol acyltransferase protein carrying six consecutive histidine residues in Escherichia coli BL21 at 37°C, induced by IPTG, which could be purified by a chromatographic medium that solidifies nickel ions (Ni2+) through covalently coupled hypromellitic triacetic acid (NTA), which is in fact metal simmering affinity chromatography (MCAC). The purification of proteins can be accomplished through the use of a polymerization medium. The degree of protein purification can be analyzed by polyacrylamide gel electrophoresis.
Materials and Instruments
Escherichia coli BL21 Move I. Reagent preparation 1. LB liquid medium: Trytone 10 g, yeast extract 5 g, NaCl 10 g, prepared to 1000 mL with distilled water. 2. Ampicillin: 100 mg/mL. 3. Sampling 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, 8 M Urea, 500 mM Imidazole, pH 8.0. 6. IPTG: 100 mM IPTG (isopropylthio-β-D-galactopyranoside): 2.38 g IPTG dissolved in 100 ml ddH2O,0.22 um filter membrane extraction, -20 ℃ storage. 1. Inoculate E. coli BL21 strain containing recombinant chloramphenicol acyltransferase protein in 5 mL of LB liquid medium (containing 100 ug/mL ampicillin), and incubate overnight at 37℃ with shaking. 2. Dilute the overnight bacteria in the ratio of 1:50 or 1:100, generally transfer 1 mL of overnight culture in 100 mL (containing 100 ug/mL ampicillin) LB liquid medium, and incubate at 37℃ with shaking until the OD600 = 0.6 - 0.8 (preferably 0.6, and it takes about 3 h). A 10 ul sample was taken for SDS-PAGE analysis. 3. No inducer was added to the control group, and IPTG was added to the experimental group to a final concentration of 0.5 mmol/l, and the incubation was continued at 37℃ for 1-3h. 4. 12 000 rpm centrifugation for 10 min, discard the supernatant, and store the bacterial precipitate in the refrigerator at -20℃ or -70℃. VI. Isolation and purification of chloramphenicol acyltransferase recombinant protein 1. Preparation of NTA chromatography column: add 1 mL of NTA medium into the chromatography column and wash it with 8 mL of deionized water and 8 mL of sample buffer, respectively. 2. Denaturation and lysis of recombinant proteins: Freeze-thaw the bacterial precipitate in an ice bath, add 5 mL of sample buffer, resuspend by pipetting, rupture the bacterium by ultrasonic waves, mix the sample gently with an oscillator, etc. for 60 min, centrifuge the sample at 12000 rpm for 30 min at 4°C, aspirate the supernatant into a clean container, and discard the precipitate. The supernatant was aspirated into a clean container and the precipitate was discarded. 10 ul of the supernatant sample was used for SDS-PAGE analysis. 3. The supernatant sample was flowed at a rate of 10-15 mL/h onto a Ni2+-NTA column, the effluent was collected, and 10 ul of the sample was taken for SDS-PAGE analysis. 4. Elution of heteroprotein: Wash the column with Washing Buffer at a flow rate of 10-15 mL/h until OD280 = 0.01 and collect the eluate step by step for about 3-4 h. A 10 ul sample at the beginning of the elution was taken and used for SDS-PAGE analysis. 5. Elution of target proteins: Elute the column with Elution Buffer, collect each 1 mL step, and take 10 ul samples for SDS-PAGE analysis respectively. Caveat 1. The choice of expression vector should be based on the final application of the expressed protein. If it is for the convenience of purification, fusion expression can be chosen; if it is for obtaining natural protein, non-fusion expression can be chosen. 2. When selecting the exogenous DNA to connect with the vector molecule in fusion expression, it should not interfere with the reading of the codon structure in the process of transcription and translation. 3. the OD value of the bacterial solution should be less than 1, otherwise the cells will be too thick and too old, not easy to break, and the plasmid will be easy to be lost. 4. the time of induction should be made as long as possible. 4. The induction time should be a gradient, and the induction time of different proteins needs to be felt. 5. Induction temperature: 25℃, 30℃. 6. IPTG concentration: generally within 1 mM, can be appropriate to explore. 7. Ultrasonication conditions can be changed according to the actual situation, as long as the bacterial body can be sufficiently lysed, i.e., the bacterial liquid is clear and not sticky. Common Problems I. Prokaryotic expression 1. Introduction to prokaryotic expression For more product details, please visit Aladdin Scientific website.
LB liquid medium Ampicillin Washing Buffer Elution Buffer IPTG Distilled water Tryptone Yeast powder Sodium chloride
Shaker Centrifuge Chromatography Column Centrifuge Tube Pipette Gun Gun Case Beaker Glass Rods
II. Obtaining the target gene1. By PCR: take the cloned plasmid containing the target gene as template, design a pair of primers according to the gene sequence (introduce different enzymatic sites in the upstream and downstream primers respectively), and PCR cycle to obtain the required gene fragment.2. By RT-PCR: extract total RNA from cells or tissues by TRIzol method, use mRNA as template, reverse transcribe to form the first strand of cDNA, and then use the reverse transcription product as template for PCR cycle to obtain the product.Construction of recombinant expression vectors1. Vector digestion: the expression plasmid with restriction endonuclease (the same primer site) for double digestion, digestion product line agarose electrophoresis, gel recovery Kit or freeze-thawing method to recover large fragments of the carrier.2. PCR products were recovered after double digestion and ligated into the vector under the action of T4DNA ligase.Obtaining expression strains containing recombinant expression plasmids1. Transform E. coli DH5α with the ligation product, according to the mark of recombinant vector (anti-Amp or blue-white spot) for screening, pick a single spot, extract the plasmid by alkaline cleavage method in a small amount, and then initially identify it by double enzyme cleavage.2. Sequencing to verify that the insertion direction and reading frame of the target gene are correct, then proceed to the next step. Otherwise, more clones should be screened, and the subclones or subclones should be repeated to different cleavage sites.3. Transform the receptor cells of the host bacteria with this recombinant plasmid DNA. V. Induction of chloramphenicol acyltransferase recombinant protein
The method of inserting a cloned gene into a suitable vector and introducing it into E. coli for the expression of a large number of proteins is generally referred to as prokaryotic expression. This method has applications in protein purification, localization and functional analysis.
2. Characteristics of E. coli for expression of recombinant proteins
(2) Materials used for bacterial cultures are less expensive than those used in mammalian cell systems;
(3) A wide variety of E. coli strains and matching plasmids with various characteristics are available;
(4) Due to the lack of modification and post-translational processing such as glycosylation and phosphorylation, proteins expressed in E. coli often form inclusion bodies, which affect the biological activity and conformation of the expressed proteins.
Usually a plasmid, a typical expression vector should have the following components: