Renaturation separation purification method of tissue plasminogen activator mutant
A technology of plasminogen and activator, which is applied in the field of separation and purification of recombinant protein in medical biotechnology, can solve the problems of high cost, achieve the effect of reducing loss, reducing renaturation volume, and promoting renaturation
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[0011] The preparation method of reteplase is as follows: the expressed reteplase engineered bacterial cells are collected, suspended in 20 mM Tris-Hcl pH8.0, ultrasonically broken and centrifuged at 12000 rpm at 4° C. for 15 minutes to collect inclusion bodies. Inclusion body solution I, its components are 6~8M urea, 10~100mM Tris-Hcl, 0.5M NaCl, 1~100mM imidazole, 0~5mM 2-mercaptoethanol pH8.0, stirred at room temperature, fully dissolved, 4 Centrifuge at 12000 rpm for 15 minutes, take the supernatant, and filter it with a 0.22um or 0.45um filter membrane to obtain a denatured reteplase solution.
[0012] The denatured reteplase was passed through the Ni solution equilibrated with solution I before 2+ -HiTrap column, after loading the sample, first use twice the column volume of solution I and solution II, the components of solution II are 6~8M urea, 10~100mM Tris-Hcl, 0.5M NaCl, 20~500mM imidazole, 0~ 100mM 2-mercaptoethanol PH7~10, wash once each, and then use six times c...
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