High-pressure refolding and combined chromatography preparation method of recombinant human interferon β-1b
A recombinant human interferon and combined layer technology, which is applied in the field of preparation of recombinant human interferon beta-1b, can solve the problems of difficult chromatography process, changes in protein structure, complicated steps, etc., and achieves cost reduction, easy operation, and improved production. The effect of efficiency
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Embodiment 1
[0053] Bacterial fermentation: ferment the genetically engineered bacteria with the target gene in conventional LB medium (1% peptone, 0.5% yeast extract, 1% NaCl), add a certain concentration of antibiotics, and use IPTG after the OD600 value reaches a certain level Induction is carried out for 3-5 hours, and the fermentation ends.
[0054] Bacteria destruction: suspend the bacteria with TE (50mmol / LTris-Cl, 5mmol / LEDTA) at a ratio of 1:10 (g / mL), place them in a high-pressure homogenizer for crushing, press 1000bar, and cycle 3 times. Centrifuge at 4°C and 8500rpm for 15 minutes after breaking the bacteria, discard the supernatant, resuspend the pellet in TE buffer, and repeat the above operation twice.
[0055] Inclusion bodies with a certain purity were obtained by washing the inclusion bodies. The specific steps were: (1) Wash the inclusion bodies with inclusion body washing solution 1 (20mM Tris-HCl, 1mM EDTA, 1% Triton-100, pH 8.0), washing solution 2, respectively. (2...
Embodiment 2
[0058] High-pressure renatured human interferon β-1b inclusion body: resuspend the obtained human interferon β-1b refined inclusion body with deionized water ultrasonic (power 225-300W, ultrasonic 3S, intermittent 6S, working time 30S), take Denature and dissolve a small amount of resuspension solution to measure the concentration, and use it as a reference value for the initial concentration of refolding.
[0059] The inclusion body suspension was added to different refolding buffer solutions at a certain initial concentration (1 mg / mL), mixed well and then subjected to 320MPa pressure for 16 hours to compare the refolding results.
[0060] From figure 2 It can be seen that the highest yield, close to 100%, was obtained when the renaturation buffer system was (20mM Tris-HCl, 0.5M Arg, pH 8.0, 0.5% Zwittergent3-14, GSH:GSSG=1.0mM:0.2mM).
Embodiment 3
[0062] SP FF purification: The sample obtained by high-pressure renaturation was diluted 10 times and then purified using SP FF column. The equilibrium buffer solution was 20mM PB, pH 7.0, and the elution buffer solution was 20mM PB, 1M NaCl, pH 7.0. After sample loading, rinse with 5% elution buffer, then elute at 5%-40% for 2-3 column volumes, and collect the elution peaks;
[0063] Butyl-S purification: add AS to the sample obtained by SP FF column purification to a final concentration of 0.4M, and then use Butyl-S column for purification, the equilibrium buffer solution is 20mM PB, 0.4M AS, pH 7.0, and the elution buffer solution is 20 mM PB, pH 7.0. After loading the sample, elute directly with 100% elution buffer solution, and collect the elution peak;
[0064] image 3 and Figure 4 They are SP FF purification chromatogram and Butyl-S purification chromatogram respectively.
[0065] pass image 3 It can be seen that most of the impurity proteins penetrated through ...
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