Separation And Purification Of Gecko Anti-tumor Active Protein

Objective:Our preliminary study found there are anti-tumor protein macromolecules in gecko.The purpose of this subject is to isolate and purify the anti-tumor activity protein from gecko.Methods:1?Separation and purification methods of anti-tumor component from geckos(1)Gel filtration chromatography(Sephadex G-25):Sephadex G-25 was used for the purification of gecko coarse extraction liquid with high purity water.(2)Ion Exchange Chromatography:DEAE cellulose DE-52 weak anion exchange medium was used for the purification of Sephadex G-25 peak 1 with 0.1 M NaCl solution,0.2M NaCl solution,0.3M NaCl solution and 0.5 M NaOH solution.(3)Hydrophobic interaction chromatography:Phenyl agarose FF hydrophobic medium was used for the purification of 0.1 M NaCl component with 1.0 M(NH4)2SO4 mother solution,0.5 M(NH4)2SO4 mother solution,0M NaCl mother solution,high purity water and 0.5 M NaOH solution.(4)High Performance Size Exclusion Chromatography(HPSEC):The TSK-GEL G4000PWxl column(7.8*300 mm)was used for the purification of 0.5 M(NH4)2SO4 component at a flow rate of 1.0 mL/min and the mobile phase was high pure water.(5)Gel filtration chromatography(Sephadex G-200):Sephadex G-200 was used for the purification of 0.5 M(NH4)2SO4 component at a flow rate of 10 mL/min and the mobile phase was high pure water.(6)Native polyacrylamide gel electrophoresis(Native-PAGE):0.5 M(NH4)2SO4 component was further purified by Native-PAGE at the condition concentrated glue voltage 80 v,separation voltage 180 v.2?Methods for the examination of different components anti-tumor activities.(1)The proliferation inhibition of Bel-7402 cell was determined by MTT experiments.(2)Observe the Bel-7402 cells under a microscope.3?Methods for the determination of protein concentration(1)The BCA kit was used for the the determination of protein concentration.Results:1?Gel filtration chromatography Sephadex G-25 isolated 4 peaks from Gecko crude extract.Cell experiments showed that the peak 1 had the strongest anti-tumor activity 87.8%.The average yield rate of Sephadex G-25 peak 1 was 5.98%(n=3).2?DEAE cellulose DE-52 isolated 6 peaks from Sephadex G-25 peak 1.Cell experiments showed that the 20 mM pH8.0 Tris-HCl contained 0.1 M NaCl(hereinafter referred to as 0.1 M NaCl component)elution had the strongest anti-tumor activity 65.05%.The average yield rate of 0.1 M NaCl component was 0.91%(n=3).3?Phenyl agarose FF hydrophobic isolated five peaks from 0.1 M NaCl component.Cell experiments showed that the 20 mM pH8.0 Tris-HCl with 0.1%NaCl and 1 M(NH4)2SO4 as equilibrium liquid,20 mM pH 8.0 Tris-HCl with 0.1%NaCl and 0.5 M(NH4)2SO4 as eluent(hereinafter referred to as 0.5 M(NH4)2SO4 component)of elution had the strongest anti-tumor activity 73.57%.The average yield rate of 0.5 M(NH4)2SO4 component was 0.44%(n=3).4????samples were collected from the isolation of 0.5 M(NH4)2SO4 component by High gel filtration chromatography.Cell experiments showed that???samples had the anti-tumor activity 20.55%,43.43%,40.48%,43.43%,29.78%respectively.The?sample was further purified and got three sampes,The tumor inhibition rate of the 1?3 samples were 13.59%,724%and 13.59%respectively,and statistical analysis indicated tha there is no significant difference between the 1?3 samples.5?1?5 samples were collected from the isolation of 0.5 M(NH4)2SO4 component by Sephadex G-200.Cell experiments showed that 1?5 samples had the anti-tumor activity 74.12%,11.19%,18.10%,10.67%,30.26%respectively.Statistical analysis indicated that only sample 1 had significant difference with other samples.6?Three parts of the gel were cut down for the cell exprements.They were referred to as upper?middle?lower parts.Cell experiments showed that the tumor inhibition rate of the three parts were 93.52%,65.21%,64.21%respectively.The average yield rate of the three parts was 0.003%,0.035%,0.006%respectively.The single protein with anti-tumor activity may exist in the upper part of the gel.Conclusions:The gecko crude extract was effectively purified by gel filtration chromatography,ion exchange chromatography,hydrophobic interaction chromatography,Native PAGE gel electrophoresis.A comparatively single active protein was isolated and it's molecular weight was greater than 70 kD.