title: [Fusion expression of fibrinolytic enzyme gene PPFE-I from endophytic Paenibacillus polymyxa in Escherichia coli and activity analysis].
abstract: With the genomic DNA of strain EJS-3 as the template, we amplified the gene of fibrinolytic enzyme from Paenibacillus polymyxa (PPFE-I) by PCR. We purified the PCR product and ligated it into pMD19-T. After DNA sequencing, we cloned the PPFE-I gene into expression vector pET-DsbA and transformed it into Escherichia coli BL21(DE3). Upon induction of IPTG, we found that the activity of recombinant fibrinolytic enzyme fused with DsbA expressed in Escherichia coli was 228 IU/mL. SDS-PAGE analysis showed that the recombinant enzyme was soluble and accounted for about 18.4% of total cell protein. Western blotting demonstrated that the recombinant protein was DsbA-PPFE-I. We purified the recombinant enzyme by Ni affinity chromatography, thrombin digestion and sephadex G-100 gel-filtration, and identified the molecular weight of purified product to be 66.3 kDa with MALDI-TOF mass spectrometry. The purified enzyme exhibited distinct fibrinolytic activity on fibrin plate.
authors.affiliation: Laboratory of Enzyme Engineering, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Fengxia
authors.initials: F
authors.lastname: Lü
authors.forename: Zhaoxin
authors.initials: Z
authors.lastname: Lu
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Bie
authors.forename: Qian
authors.initials: Q
authors.lastname: Lin
authors.forename: Chong
authors.initials: C
authors.lastname: Zhang
authors.forename: Lin
authors.initials: L
authors.lastname: Cao
authors.forename: Yao
authors.initials: Y
authors.lastname: Guo
authors.forename: Yanchong
authors.initials: Y
authors.lastname: Tang
mesh_terms: D000933:Antifibrinolytic Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005343:Fibrinolytic Agents / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D056507:Paenibacillus / Q000737:chemistry* / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology
chemical_list: D000933:Antifibrinolytic Agents; D005343:Fibrinolytic Agents; D011993:Recombinant Fusion Proteins