Font Size: a A A

Expression, Refolding, Purification And Pharmacology Of Douchi Fibrinolytic Enzyme (Subtilisin DFE)

Posted on:2006-04-30Degree:DoctorType:Dissertation
Country:ChinaCandidate:R H ZhangFull Text:PDF
GTID:1100360182972722Subject:Genetics
Abstract/Summary:PDF Full Text Request
The gene coding for a novel fibrinolytic enzyme, Douchi Fibrinolytic Enzyme (subtilisin DFE) from Bacillus amyliquefaciens, was previously reported and its intact coding region has also been identified. But its expression in Escherichia coli was not successful. In this study, four recombinant plasmids expressing pre-pro-DFE, Trx-pro-DFE, hisTrx-pro-DFE and Trx-DFE were first constructed and transformed into E. coli. Strong fibrinolytic activity was detected in the lysates of the induced Trx-pro-DFE and hisTrx-pro-DFE-transformants, but not in the lysates of other two transformants. Renaturation studies indicated that Trx-pro-DFE could refold easily from its inclusion bodies when denatured and dialyzed against an excess volume of 200 mM sodium potassium phosphate buffer or 0.5 mol/L (NH4)2SO4, 20 mmol/L Tris-HCl (pH 8.0). However, pre-pro-DFE could refold only with a low efficiency but Trx-DFE not at all. The recombinant subtilisin DFE was purified to homogeneity from the lysate of the induced cells expressing Trx-pro-subtilisin DFE by three-step chromatography. The results from fibrinolytic activity and Western-blotting analysis showed that the purified recombinant subtilisin DFE is the same as its native form.The inclusion body of fusion protein Trx-pro-DFE was purified and solubilized in 6 M guanidine hydrochloride. The denatured Trx-pro-subtilisin DFE was loaded on an immobilized metal affinity chromatography (IMAC). The subtilisin DFE was refolded, autoprocessed and partial purified by gradual removal of the guanidine hydrochloride and increase of imidazole concentration. The analysis of the chromatography behavior of Trx-pro-DFE on IMAC indicated that the refolding and processing of recombinant subtilisin DFE were occurred by its adsorption and desorption cycle on the matrix, and the surface of the matrix may have any effect on the refolding of the adsorpted proteins. By further purification of size exclusion chromatography, the recombinant subtilisin DFE was purified to be higher than 98% homogeneity with a 78% yield. The specific activity of the final purified enzyme was estimated to be 4611 urokinase units/mg protein. The purified recombinant subtilisin DFE has strong fibrinolytic activity with an apparent molecular weight of 27.4 kDa and can be recognized by the anti-subtilisin DFE antibody. SDS-PAGE electrophoresis and N-terminal sequencing indicated that the recombinant subtilisin DFE was processed accurately and same as its nature form.In order to test the function of pro-peptide and express the subtilisin DFE, the recombinant plasmids expressing pro-subtilisin DFE, and subtilisin DFE were first constructed in pPICZ a A and transformed into Pichia pastoris. Strong fibrinolytic activity was detected only in the culture supernatant of the methanol induced pro-subtilisin DFE-transformants, but not in that of the other kind of transformants. This result implies that the pro-peptide is important for the secretion of subtilisin DFE in P. pastoris, and after secretion, the pro-peptide is subsequently removed to produce active mature subtilisin DFE. The recombinant subtilisin DFE was purified to homogeneity from the culture supernatant of the induced P. pastoris transformant by three-step chromatography. The results from fibrinolytic activity, SDS-PAGE and Western-blotting analysis showed that the purified recombinant subtilisin DFE is same as its native form.Comparision of the production of subtilisin DFE from B. amyliquefaciens, E.coli, B. subtilis and P. pastoris, and the purification recovery from these strains,demonstrate that the best procedure to produce enough subtilisin DFE effectively is to express DFE gene in E.coli with Trx-pro-DFE form and to purify DFE from the inclusion bodies.Primary pharmacological reseach was done using subtilisin DFE purified from B. amyliquefaciens. When the mouse were orally administered or intravenously injected in a single maximal dose of 6,500 U/kg and 3x105 U/kg of subtilisin DFE, respectively, no acute toxicity was observed. The LD50 of subtilisin DFE in mouse by oral administration or intravenous injection was higher than 6,500 U/kg and 3><105 U/kg, respectively. When the rats were orally administered in a single dose of 60,000 U/kg/d subtilisin DFE for 5 d, or 30 min after the rats intravenously injected with a single dose of 1,300 U/kg subtilisin DFE, the value of PT, APTT, TT, CT, D-dimer was increased, and the thrombus formation decreased. These results indicate that the oral administration and intravenous injection of subtilisin DFE are safe and effective.
Keywords/Search Tags:Fibrinolytic enzyme, Subtilisin DFE, Escherichia coli, Pichia pastoris, Gene expression, Recombinant subtilisin DFE, Protein refolding, Pro-peptide, Purification, Immobilized metal affinity chromatography, Fibrinolytic activity, Pharmacology
PDF Full Text Request
Related items