Wheat peroxidases 1 (WP1) is the major cationic peroxidase of wheat (Triticum aestivum) grain, which is involved in the development of seeds and an important factor to affect the final processing quality of flour. We constructed a prokaryotic expression vector pET28a-WP1, and transformed it into E. coli host strain T7 Expression. His-tag fused WP1 existed as inclusion body, and the recombinant protein was purified by Ni-NTA resin affinity chromatography under denatured condition. The purity of target protein reached 98%. The recombinant WP1 was refolded by gradient urea dialysis, then used as antigen to immune rabbit to prepare polyclonal antibody. The result of ELISA showed that the titer of rabbit anti-WP1 antiserum was higher than 1:625 000. The result of Western blotting demonstrated that the prepared WP1 polyclonal antibody could be used to detect WP1 with high specificity.
Animals ; Antibodies ; immunology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Peroxidases ; biosynthesis ; genetics ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; immunology

To establish a prokaryotic expression and purification protocol for nuclease P1 (NP1), we first obtained a synthetic NP1 by splicing 22 oligonucleotides with overlapping PCR. We constructed and transformed a secretory expression vector pMAL-p4X-NP1 into Escherichia coli host strains T7 Express and Origami B (DE3) separately. Then, the recombinant NP1 was purified by amylose affinity chromatography, and its activity, thermo-stability and metal-ion dependence were investigated systematically. The results indicated that the expressed fusion proteins MBP-NP1 (Maltose binding protein-NP1) existed mainly in soluble form both in host strains T7 Express and Origami B (DE3), but the specific activity of recombinant protein from Origami B(DE3) strain was higher than T7 Express strain (75.48 U/mg : 51.50 U/mg). When the MBP-tag was cleaved by protease Factor Xa, the specific activity both increased up to 258.1 U/mg and 139.2 U/mg. The thermal inactivation experiments demonstrated that the recombinant NP1 was quite stable, and it retained more than 90% of original activity after incubated for 30 min at 80 degrees C. Zn2+ (2.0 mmol/L) could increase enzyme activity (to 119.1%), on the contrary, the enzyme activity was reduced by 2.0 mmol/L Cu2+ (to 63.12%). This research realized the functional expression of NP1 in the prokaryotic system for the first time, and provided an alternative pathway for NP1 preparation.
Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Fungal Proteins ; biosynthesis ; genetics ; metabolism ; Genes, Synthetic ; Genetic Vectors ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Single-Strand Specific DNA and RNA Endonucleases ; biosynthesis ; genetics ; metabolism

Wheat grain peroxidase 1 (WP1) belonged to class III plant peroxidase with cofactor heme, which not only has antifungal activity, but also influences the processing quality of flour. In order to enhance functional expression of WP1 in prokaryotic system by increasing endogenous heme synthesis, we constructed a recombinant plasmid pACYC-A-L containing hemA and hemL of Esherichia coli. Then, we co-transformed it into host strain T7 Express with secretive expression vector (pMAL-p4x-WP1) or non-secretive expression vector (pET21a-MBP-WP1), respectively. The MBP-WP1 fusion protein was further purified by amylose affinity chromatography and its peroxidase activity was assayed using 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) as substrate. At 12 h after induction at 28 degree, the extracellular 5-aminolevulinic acid (5-ALA) production of T7 Express/pACYC-A-L was up to 146.73 mg/L, simultaneously the extracellular porphrins also increased dramatically. The peroxidase activity of functional MBP-WP1 obtained from T7 Express/ (pACYC-A-L + pMAL-p4x-WP1) was 14.6-folds of that purified from T7 Express/ pET21a-MBP-WP1. This study not only successfully enhanced functional expression of wheat peroxidase 1 in Esherichia coli, but also provided beneficial references for other important proteins with cofactor heme.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Heme ; biosynthesis ; genetics ; Peroxidases ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transformation, Genetic

Objective To study the therapeutic effects of EUS-guided biliary drainage (EUS-BD) using a nitinol fully covered self-expandable metal stents in patients with malignant obstructive jaundice after failed ERCP.Methods From January 2016 to January 2018,all patients with malignant obstructive jaundice who failed ERCP underwent EUS-guided biliary drainage using nitinol fully covered self-expandable metal stent at Affiliated Hangzhou First People' s Hospital,Zhejiang University School of Medicine.The operation success rate,liver functional recovery time,complication rate,length of hospital stay and survival time were observed.Results Of 36 patients who underwent EUS-guided biliary drainage,34 were successfully performed,with 19 through the stomach,and 15 through the duodenum.The operation success rate was 94.4％ (34/36).The liver functional recovery time of the 34 patients were 25.8 ±.6.5 days.One patient developed hemobilia and one cholangitis,both improved after conservative treatment.The total complication rate was 5.6％ (2/36).The hospital stay and survival time were 21.5 ± 4.7 days and 220.5 ± 54.8 days,respectively.Conclusion EUS-BD using nitinol fully covered self-expandable metal stents was a feasible and effective treatment in patients with malignant biliary obstruction after failed ERCP.

Objective To evaluate the efficacy of endoscopic ultrasound guided biliary drainage (EUS-BD) in patients with biliary obstruction and surgically altered anatomies.Methods We collected data from 33 patients with biliary obstruction and surgically altered anatomies from January 2016 to January 2018 in Zhejiang University School of Medicine Affiliated Hangzhou First People's Hospital who underwent EUS-guided biliary drainage after unsuccessful ERCP.The operation success rate,clinical success rate,complication rate,hospital stay were studied.Results Of 33 patients,31 were successfully operated and stented using endoscopic ultrasound puncture:14 patients through the stomach,17 patients through the duodenum;8 patients by the rendezvous approach.The operation success rate was 93.9％.Of the 33 patients,28 had a significant decrease in jaundice,with a clinical success rate of 84.9％.Complications consisted of 2 patients with bleeding and 1 patient with cholangitis.These patients improved after conservative treatment.The complications rate was 9.1％.The hospital stay was (12.4±5.7) d.Conclusion EUS-BD can be the first choice for patients with biliary obstruction and surgically altered anatomy after failed endoscopic retrograde cholangiograohv in centers with exoertise in EUS-BD procedures.