Gene expression and characteristics of a novel fibrinolytic enzyme (subtilisin DFE) in Escherichia coli.
OBJECTIVE
The objective of this study is to actively express a novel fibrinolytic enzyme, subtilisin DFE (douchi fibrinolytic enzyme), in Escherichia coli.


RESULTS
The DNA fragments encoding pro-subtilisin DFE was amplified and cloned into the vector pET32a to obtain N-terminal Trx fusion expression plasmid. The recombinant subtilisin DFE was successfully expressed and processed in the soluble fraction of E. coli BL21(DE3) in a similar fashion as the endogenous one of Bacillus amyloliquefaciens DC-4, resulting in an active enzyme. Moreover, active enzyme can also be refolded from inclusion body.


CONCLUSIONS
Active subtilisin DFE can be expressed and processed in E. coli.


CONCLUSIONS
This study provides evidences that subtilisin DFE can be actively expressed in E. coli and the pro-peptide is essential for guiding the proper folding into the active conformation. As such, large quantities of recombinant subtilisin DFE can be produced for pharmacological and clinical research.
Zhang
R-H
RH
Sichuan Key Laboratory of Molecular Biology and Biotechnology, College of Life Science, Sichuan University, Chengdu, P.R. China.
Xiao
L
L
Peng
Y
Y
Wang
H-Y
HY
Bai
F
F
Zhang
Y-Z
YZ
D001407:Bacillus / Q000201:enzymology; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005342:Fibrinolysis; D005343:Fibrinolytic Agents / Q000494:pharmacology; D015870:Gene Expression; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D013381:Subtilisins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D005343:Fibrinolytic Agents; D011994:Recombinant Proteins; D013879:Thioredoxins; D013381:Subtilisins