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IPTG(367-93-1) induced by step principle and method of protein expression

2014-07-29 来源:亚科官网

E.coli lactose operon (yuan) containing Z, Y and A three structural genes,respectively encoding - galactosidase, permease and acetyltransferase, in addition to a control sequence of O, a startup sequence P and a regulatory gene I. A repressor protein encoded by the I gene, which is combined with O sequences, the operon (yuan) repressed and is in a closed state. At the start ofthe sequence P upstream and a decomposition (metabolism) gene activator protein (CAP) binding sites. Constitute the regulatory region of lac operon by P sequences, O sequences and CAP binding sites, regulation of genes encodingenzymes from three of the same regulatory region, coordinated expression of gene products. In the absence of lactose exists, lac operon (yuan) in a repressed state. At this point, the I sequence in the PI promoter sequences under the control of expression of the combination of Lac repressor protein and O sequence, block RNA polymerase binding with P sequences, inhibiting transcription start. When lactose exists, lac operon (yuan) can be induced to.Lactose into the cell, the B - galactosidase catalysis, into galactose. The latter as an inducer molecule binding repressor protein, the protein conformational changes, resulting in repressor protein and O sequence, transcriptiondissociation. Isopropyl thiogalactoside (IPTG) is a strong inducer, no bacterialmetabolism and is very stable, so it is widely used in laboratory. Material Science1, induced the expression of materials

(1) LB (Luria - Bertani)) medium

Yeast extract (Yeast extract) 5g peptone (Peptone) 10g NaCl 10g agar (Agar) 1-2%

Yeast extract (Yeast extract) 5g peptone (Peptone) 10g NaCl 10g agar (Agar) 1-2%

Distilled water (Distilled water) 1000ml pH 7 scope of application: Escherichia coli

(2) the IPTG stock solution: 2 g IPTG dissolved in 10 mL distilled water, 0.22 μ m membrane filtration, packed into 1 mL / A, - 2℃ preservation

(3) l × gel electrophoresis sample buffer: 50 mmol / Tris -CI (pH 6.8) 50 mmol /DTT 2% SDS (electrophoresis grade) 0.1% bromophenol blue 10% glycerol

The purified material separation and protein 2, Escherichia coli inclusion body

1) enzyme digestion (1) lysis buffer:

50 mmol / L Tris-CI (pH 8) 1 mmol / L 100 mmol / LNaCI EDTA

(2) 50 mmol / L phenylmethylsulfonyl fluoride (PMSF). (3) 10 mg / mL lysozyme.(4) deoxycholic acid.

(5) 1 mg / mL DNase I. 2) ultrasonic crushing method (1) TE buffer.

(2) 2 * SDS -PAGE gel electrophoresis sample buffer: 100 mmol / L Tris-HCI (pH 8) 100 mmol / L DTT 4%SDS 0.2% bromophenol blue 20% glycerol experiment scheme

1, induced the expression of exogenous gene

(1) with restriction endonuclease digestion vector DNA and the appropriate target genes. (2) connected by steps. The target gene and vector, and transformed into host strain corresponding.

(3) selected transformed colonies containing recombinant plasmid DNA extraction, restriction endonuclease map, DNA sequencing, confirmed after the next.

(4) if the expression vector of prokaryotic promoter promoter PL, train 30 hoursat -32 ℃, the OD600 of the culture medium was 0.4-0.6, quickly make temperature rose to 42 ℃ cultured for 3 -5h; if the prokaryotic promoter for TACexpression vector, then cultured at 37 ℃ for several hours to bacteria the logarithmic growth phase after the addition of IPTG to a final concentration of 1 mmol / L. Cultured for 3 -5h.

(5) the culture fluid 1 mL, 1 min, 1000g centrifugation, precipitation, plus 100 μ L polyacrylamide gel electrophoresis sample buffer, SDS -PAGE detection

2, Escherichia coli inclusion body protein separation and purification of 1) lysis of bacteria

Commonly used methods are: ① high-temperature bead mill; the high-pressure homogenization; the ultrasonic method; the enzyme digestion method; chemicalpenetration. The first three methods of mechanical crushing method, and themethod, has been applied in industrial production, three methods are used in laboratory studies widely. The experimental steps enzyme solution andultrasonication.

(1) the enzyme digestion method. Lytic enzyme commonly used with lysozyme;-1,3 β - glucanase; -1,6 β - glucanase; protease; chitinase; glycosidase etc.. Lysozyme has effect on bacteria, and several other enzymes in yeast effect. The main steps are as follow

The 4 ℃, 5000rpm centrifugal, 15 min, bacteria culture fluid were collected to induce the expression of (100 mL). Kami Kiyo, about per gram of wet bacteriaplus 3 mL lysis buffer, suspended sediment.