Expression Of Pichia Pastoris And Its Biological Activity In GI - TLE1 Gene Of Agkistrodon Halys

Snake venom thrombin-like enzymes (SVTLEs) is widely present in the venoms of Viperidae crotalinae, it belongs to the venom serine protease family. It can directly act on fibrinogen in vitro, due to fibrin monomer is polymerized to form a clot, its function is very similar to thrombin enzyme, named as thrombin-like enzymes(TLEs). However, in vivo, since it did not activate the coagulation factor XIII, and just hydrolyzed fibrin clot without side chain cross-linking, this clot can easily be cleared by the body’s natural reticuloendothelial system or normal fibrinolysis, exhibiting anticoagulant and fibrinolytic effects. Since the isolation of the first thrombin-like enzyme from the venom of the Brazilian Bothrops in1936, and their potential application value, SVTLEs have been used in the clinical field for anti-thrombosis, bleeding, diagnostic reagents, etc. Gloydius intermedius belongs to Viperidae family, Crotalinae subfamily and Genus Gloydius, which distributed widely in the North and Northwest regions in China. Its toxicity is the strongest of the six species of pit-vipers in China. The thrombin-like enzymes were less studied. In the present study, thrombin-like enzyme1gene (GI-TLE1)from the venom gland cDNA library of Gloydius intermedius was chosen and expressed in Pichia pastrias and the biological activities were characterized.The experiment content:1. Firstly, the codons of GI-TLE1gene were optimized based on the codon usage bias of P. pastrias; Secondly, the codon optimized full gene was synthesized; Finally, the synthesized gene was amplified by PCR. Both the PCR fragments and pPIC9K plasmid were digested with SnaB I and Not I restrictive enzymes, later ligased. Recombinant plasmid was transformed into competent cells of E.coli TOP10by means of heat shock method. The recombinant plasmid pPIC9K/GI-TLE1was identified by PCR and sequencing.2. The recombinant pPIC9K/GI-TLE1plasmid and control (pPIC9K) were linearized by digestion with Sac I enzyme, then introduced into Pichia pastoris GS115by using electroporation (parameters:1500V,25μF,200Ω, discharge for4.7ms or4.5ms). Yeast clones with integrated foreign gene were screened through the YPD plate under different G418concentrations (0.5mg/ml-4mg/mI), and G418resistant recombinant transformant were obtained. The methanol utilization type of transformants were identified by using MM and MD palte. Yeast genome extraction kit was used to prepare genome DNA from the GS115/GI-TLE1yeast clones, genomic insert mode and phenotype were identified by PCR.3. The expression of GI-TLE1was induced in shaking flasks at different time (Oh,12h,24h,48h,72h,96h,120h). Target protein in the fermentation supernatants were detected by SDS-PAGE; The Amide hydrolase activity was detected with the simulation substrate BApNA.4. The expression of target protein rGI-TLE1was optimized under different fermentation conditions:different time, pH, different amount of methanol, temperature and the adding of different antioxidants. The optimal fermentation parameters were obtained by determining the amide hydrolase activities of the recombinant proteins in the fermentation supernatants.5. rGI-TLE1in the fermentation supernatant was purified by NI-NTA affinity method. The rGI-TLE1concentration was measured by Bradford method; The inhibiting effect of serine protease inhibitor PMSF on the amide hydrolase activity of purified rGI-TLE1was detected; The thrombin like activity was determined with bovine serum fibrinogen as substrate; fibrin plate assay was performed to detect the fibrinolytic activity.The experimental results:1. With the codon usage of Pichia pastoris as reference, synonymous mutation was performed to optimize the gene sequence of GI-TLE1. Results from PCR and DNA sequencing have shown that recombinant expression vector pPIC9K/GI-TLE1has been successfully constructed.2. Linearized expression vector pPIC9K/GI-TLE1and pPIC9K were introduced into the Pichia pastoris GS115. Screening under different G418concentrations,11high G418resistant transformants with genome integration were obtained, they can grow on the YPD plate. Screening with the MM and MD plates, the methanol utilization type was identified as Mut+. PCR with the yeast genome DNA of GS115-TLE1as template, the results showed that the target gene has been successfully integrated into the genome of GS115, this results were consistent with methanol rapid utilization type (Mut+). 3. SDS-PAGE analysis revealed the expression of recombinant protein, the bands with molecular weight of about35kDa increased from shallow to deep. The target protein could hydrolase substrate BApNA and showed amide hydrolase activity.4. By measuring the amide hydrolase activities of rGI-TLE1in the supernatants from different fermentation conditions, we got the optimal induction parameters:pH5, with0.75%methanol and the temperature of24℃. Experiments with the adding of different antioxidants in the fermentation system showed that only melatonin could improve the expression of target protein and biological activity, while the adding of GSH or Vc induced little expression of the target protein.5. Induction under the optimal condition, the expression of the target protein reached a level of242μg/ml. Compared with the initial (general) induction, the expression under optimized condition increased by48.7%, amide hydrolase activity also increased by29.2%. After purification by NI-NTA method, the concentration of the rGI-TLE1was59μg/ml. As to the characterization of the biological activities, the purified rGI-TLE1showed typical serine protease activity, and the activity can be inhibited by specific inhibitor PMSF, but EDTA did not. Thrombin-like enzyme activity assay revealed that when mixed with bovine serum fibrinogen solution, the recombinant protein did not cause the phenomenon of solidification. Fibrin plate assay with recombinant protein GI-TLE1showed the ability of fibrin degradation.Conclusion:Thrombin-like enzymel of Gloydius intermedius have been successfully expressed in Pichia pastoris. Induction under optimized condition(pH5,0.75%methanol and the temperature of24℃), the expression can reach a level of242μg/ml. The purified rGI-TLE1holds some biological activities. These works may provide information for the expression of other Gloydius intermedius serine proteases in Pichia pastoris and their exploitation.