Escherichia coli expression system of retinol binding protein and preparation method of protein thereof
A technology for binding proteins and Escherichia coli, applied in the field of microbial molecular biology, to achieve the effects of increased existence time, improved immunogenicity, simplified and accurate process methods
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Embodiment 1
[0032] Embodiment 1 Construction of retinol-binding protein genetically engineered bacteria expression system
[0033] 1.1 Design primers to clone retinol binding protein gene
[0034] RBPF: ATCG CCATGG ATGCTGCATCAGCCGACC;
[0035] RBPR: ATCG CTCGAGTTACTGCACTTTTTTAAACACC.
[0036] 2.2 Replicon amplification conditions
[0037] The reaction system includes 20mM (mmol / L, millimole per liter, the same below) Tris-HCl (trishydroxymethylaminomethane, pH 8.8), 10mM KCl (potassium chloride), 10mM (mol / L, mole per liter , the same below) (NH 4 ) 2 SO 4 (ammonium sulfate), 2mM MgSO 4 (Magnesium sulfate), 0.1% TritonX-100 (polyethylene glycol octyl phenyl ether), 50M dATP (adeoxynucleotide triphosphate), 50M dGTP (deoxynucleotide guanine triphosphate), 50μM ( μmol / L, micromole per liter, the same below) dTTP (thymidine triphosphate), 50 μM dCTP (cytosine triphosphate), 400nM primer SAMF (S-adenosylmethionine ), 400 nM primer SAMR, 1.5 U (unit) Pfu DNA polymerase (Promega, USA...
Embodiment 2
[0042] Embodiment 2 Fermentation preparation method of retinol binding protein
[0043] 2.1 Bacteria seed culture medium
[0044] The bacterial seed medium includes 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride. Sterilize at 121°C for 20 minutes before use, and add kanamycin sulfate after cooling to a final concentration of 50 mg / L.
[0045] 2.2 Fermentation initial medium
[0046] The fermentation initial medium includes 10g / L tryptone, 5g / L yeast extract, 6-12g / L dipotassium hydrogen phosphate, 3-6g / L sodium dihydrogen phosphate, 0.2-1g / L citric acid monohydrate, 0.2-2g / L magnesium sulfate, 1-10ml / L trace element mother solution, adjust the pH to 6.8-7.2, and sterilize at 121°C for 20min. Before inoculation, add sterilized glucose to a final concentration of 15-20 g / L, and use ammonia water to adjust the pH to 6.5-7.3. Add kanamycin sulfate to a final concentration of 50 mg / L.
[0047] Phosphate in the initial medium is calculated by volume ratio, dipo...
Embodiment 3
[0060] Example 3 Purification and renaturation of retinol binding protein
[0061] 3.1 Purification process
[0062] Adjust the pH of the fermentation broth to 7.0-8.0, and homogenate at 600-900 bar to release the target protein;
[0063] Add nuclease to the homogenate, stir well, and centrifuge at 5000g for 20min to remove most of the nucleic acid, cell debris, and some foreign proteins; then collect the precipitate;
[0064] Resuspend the pellet with 2M Urea and 50mM Tris, sonicate for 30min, centrifuge at 10000g for 30min to remove impurities, discard the supernatant and collect the pellet;
[0065] Add 1 / 5 to 1 times the original volume of 50Mm Tris and 1% Triton solution to the precipitate, stir for 30min, and centrifuge at 10000g for 30min;
[0066] The denaturing solution was 6M Urea, 50mM Tris and 2mM DTT solution, the denatured inclusion body was denatured with a denaturing solution with a volume ratio of 1:10, stirred for 2 hours, centrifuged at 10000g for 30 minut...
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