Expression Of NK4, An HGF Variant In E.Coli And Detection Of Its Biological Activity

Objective1 . To express NK4 in Escherchia Coli 2. To detect the activity of NK4MethodNK.4 was produced in E.coli ,BL21. The DNA encoding the desired protein was generated as a BamH I-Sal I fragment using PCR. The template was PBV220-HGF- a strand that had been constructed in our lab. We cloned the interesting gene into a pGEX-4T- 1 expression vector, a kind of GST fusion vector. This vector is engineered with an internal lacI1 gene whose product is a represser protein that binds to the operator region of the tac promoter, preventing expression until induction by IPTG, thus maintaining tight control over expression of the insert. GST gene fusion system is based on inducible, high-level expression of genes or gene-fragments as fusions with Schistosoma japonicum (GST). GST can be cleavaged using a site-specific protease such as thrombin. We also compared the effects of different inducible time, inducible temperature and IPTG concentration. Recombinant protein was inclusion body that was expressed at 37 . After centrifugation at 8000rpm for 15 minutes, the bacterial pellets was subjected to ultrasonication in the suspension solution (50mmol/l, pH8.0 Tris-HCI,100mmol/L NaC1,0.5mmol/L EDTA,4mol/L urea,0.2%Triton X-100,0.2%DOC) at 300W, working 3s,intervul 3s for 240 times. Then, concentration at 8000rpm for 15 minutes at 4 . We used gradientdilution renaluration (Renaturation buffer:0.14mol/L Tris-HCl pH8.0.10mmol/LGSH,2mmol/LGSSG.5%glycerol,0.02mol/L L-Arg) and gel filter renaturation by Sephacral S200 to refold the inclusion body respectively. To get pure NK4, it was purified by GST affinity gromatochraphy. To detect the angiostatic activity of NK4 in vivo, it was tested using CAM.ResultBL21 with pGEX-4T-l-NK4 could correctly the targeting protein. The bacteria were cultured for 2h before being induced by IPTG. The high-level production was got by being induced at 37 for 2h. After washing inclusion body and GST affinity purification, GST-NK4 was cleavaged by thrombin. Antiangiogenic activity of NK4/GST-NK4 in vivo was detected. They inhibited new blood vessel formation.