Cloning And Expression Of Fibrinolytic Enzyme Genes

A strain, producing a fibrinolytic enzyme, was isolated from the environment and its morphorlogical, physiological and biochemical characteristics were studied. Results showed that most of these characteristics were very similar to those of Micrococcus luteus. The 16s rDNA sequence of the strain was also determined and compared with those in the GenBank.Its nucleotide sequence shares 99% identity with 16s rDNA of Micrococcus luteus. From the polyphasic taxonomical view, the strain belongs to Micrococcus Cohn Micrococcus luteus. named ML-909.According to the sequence edcoding Douchi fibrinolytic enzyme(DFE) gene, pro-preDFE(proDFE) and mature peptide of Bacillus amyloliquefaciens, three pairs of primers were synthesized and used to amplify the homologous DNA fragments from the total DNA of ML-909 strain. However, only the sequence coding for mature peptide was amplified and determined. The amplified fragment is 828bp long, encoding 275 amino acids residues. Its nucleotide sequence is 99% homologous to that of DFE, so named as Micrococcus luteus fibrinolytic enzyme(MLFE).In order to further study the heterogenous expression of genes, DFE and MLFE, two pairs of primers were synthesized based on the sequences coding for pre-peptide and mature-peptide of DFE. proDFE(1059bp) was amplified from the chromosomal DNA of B. amyloliquefaciens DC-4 islated from Douchi-a traditional Chinese fermented-soybean food, and matue-peptide-coding fragment (828bp) was amplifiedfrom the genomic DNA of Micrococcus luteus ML-909. The two fragments were cloned into pMAL-p2X, a highly expression vector of Escherichia coli, respectively. The two expression plasmids pMAL-pDFE and pMAL-MLFE were then transformed into E.coli. After 4 hours of induction by 1mM IPTG, the expressed products were analyzed through SDS-PAGE, and the fused proteins were found to exist in soluble form. DFE and MLFE were purified by Amylose affinity column and incised from the fused protein by Factor Xa. However, the purified fibrinolytic enzyme had no fibrinolytic activity.In order to highly express of DFE gene in B.subtilis, a fragment containing promoter and signal peptide-coding sequence of a -amylase gene was amplified from the chromosomal DNA of Bacillus amyloliquefaciens DC-4 by PCR and its sequence was determined.This sequence has 98% homology with that reported by Palva(GenBank accession No.J01542). Then, the fragment was ligated with pro-peptide-mature peptide-coding sequence of DFE gene and MLFE gene, respectively, resulted in two fusion genes called AmyDFE and AmyMLFE. These tow fused genes were cloned into a shuttle vector pSUGV4 and expression plasmids pSU-AmyDFE and pSU-AmyMLFE were constructed. After transformation with B. subtilis WB600, two transformant strains of WB600/pSU-AmyDFE and WB600/pSU-AmyMLFE were obtained. They produced clear hydrolyzed zones on fibrin plates. The fibrinolytic activity in supernants of 24h fermentation were tested and found to be 200IU/mL for WB600/pSU-AmyDFE and 238IU/mL for WB600/pSU-AmyMLFE. The results of SDS-PAGE analysis showed that there were indeed recombinant proteins in supernants. The Western blotting of the products of recombinant plasmid pSU-AmyDFE in E.coli and B. subtilis showed that the molecular weight of two proteins were same as expected. These results indicate that two genes, DFE and MLFE, are successfully expressed in B.subtilis.In order to further enhance production of recombinant Douchi fibrinolytic enzyme(rDFE),the optimal fermentation conditions of recombinant B. subtilis WB-DFE5 were determined. The compositions of fermentation medium are 2%soluble starch , 2% casein, 0.5% soybean peptone, 0.15% yeast extract, 0.25% K2HPO4, 0.05% KH2PO4, 0.02% CaCl2, 0.05% MgSO4, pH7.0.The optimal cultivation temperature is 37°C, 20mL of media are in 250mL flask and shaking is 200rpm for 96h.Under these conditions the fibrinolytic activity of the supernatant can reach 441U/mL. The recombinant DFE was purified with methods including ammonium sulfate fractional precipitation, SP-Sepharose FF, DEAE-Sepharose FF ion-exchang and Sephadex G-75 chromatography, and 12.5mg of purified products were obtained from one liter of culture. Its spectific activity is 5918.7U/mg, compared to that of original strain, B. amyloliquefaciens DC-4.To further improve the expression level of DFE, the sequence including promoter and signal sequence(sacR) of levansucrase gene was cloned from B. subtilis WB600, ligated to proDFE, then inserted into pSUGV4. The expression plasmid (pSU-SacDFE) was constructed and transformed into B.subtilis WB600. The resulted transformant was induced by sucrose, and DFE gene was highly expressed with 591IU/mL.