Preparation method and application of highly active recombinant human chymotrypsin
A chymotrypsin, high-activity technology, applied in the field of high-activity recombinant human chymotrypsin preparation, can solve problems such as waste of reagents, increased environmental purification cost, virus pollution and the like
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Embodiment 1
[0067] Embodiment 1, vector construction and expression identification
[0068] According to the human chymotrypsin amino acid sequence SEQ ID NO: 1, artificially synthesized gene sequence (Gene CT). GeneCT was integrated into the NdeI / EcoRI site of the expression vector pET24a (purchased from Invitrogen), and the recombinant plasmid was transformed into Escherichia coli BL21(DE3) to obtain transformants. Pick the monoclonal colony on the transformation plate, inoculate it into 30ml LB culture medium (containing 100μg / ml Kana), place it on a shaker at 37°C for overnight culture (10-12h), and transfer it to the secondary bottle with 2% inoculum , continue to cultivate. When the bacterial density reaches OD6000.5-0.6, add IPTG at a final concentration of 0.5mmo / L to induce for 4 hours, collect the bacterial cells by centrifugation at 10,000rpm, discard the supernatant, and ultrasonically disrupt the bacterial cells. After centrifugation, obtain the supernatant and precipitate, ...
Embodiment 2
[0072] Example 2, optimization of inclusion body renaturation conditions
[0073] Inclusion bodies collected by cell disruption can be denatured after being washed once with Triton X-100 and washed twice with pure water.
[0074] The denaturation conditions for inclusion bodies are as follows: 25 mg wet inclusion bodies are weighed, dissolved in 8M urea and 10 mM β-mercaptoethanol in denaturing solution, and denatured for 2 hours.
[0075] For the renaturation method, choose dilution renaturation.
[0076] Chymotrypsin activity assay method: Under the conditions of pH 7.8 and 25°C, hydrolysis of 1.0 μmol N-benzoyl-L-tyrosine ethyl ester (BTEE) per minute is defined as 1 enzyme activity unit (U).
[0077] 2.1 Refolding pH optimization
[0078]Each 1 mL of denaturing solution was slowly added to 10 mL of refolding solution (100 mM Tris-HCl, pH 8.0, 8.5, 9.0, 9.5). After refolding at 25°C for 24 hours, add recombinant trypsin to activate for 12 hours, see figure 2 .
[0079...
Embodiment 3
[0096] Embodiment 3, refolding liquid treatment
[0097] Filter 10,000 mL of the refolding solution with a 0.22 μm membrane filter, and concentrate the filtrate 10-100 times with a 10 kDa ultrafiltration membrane.
[0098] The concentrated solution was dialyzed against pure water (the temperature was controlled at 0-10° C.) for 3 days, and the water was changed every 12 hours.
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