Study On The Separation And Purification Of High Molecular Weight Anti-tumor Protein From Geck

ObjectiveGecko has clear curative antineoplastic effect in clinic applications.However,gecko’s main active component is not clear.Based on the previous results,the purpose of this subject is to isolate and purify the gecko protein monomer from gecko with anti-tumor activity through protein separation and purification techniques.Methods1.Separation and purification methods of anti-tumor component from geckos(1)The preparation of crude gecko extract:the fresh gecko was homogenized and repeatedly frozen and thawed 5 times.The homogenate was filtered and centrifuged.The supernatant was passed through 0.45 μm filter to obtain a crude gecko extract.(2)Ammonium sulfate precipitation:0-40%ammonium sulfate precipitated fraction was obtained through ammonium sulfate and dialyzed to remove salts.(3)Ion exchange chromatography:DEAE cellulose DE-52 weak anion exchange medium was used for the purification of 0-40%ammonium sulfate precipitated fraction with 0.1M NaCl solution.(4)Hydrophobic interaction chroma tography:Phenyl agarose FF hydrophobic medium was used for the purification of 0.1 M NaCl component with 0M(NH4)2SO4 solution.(5)Ultrafiltration:0M(NH4)2SO4 component was further purified by Amicon Ultra-50 mL with molecular mass cut-off were 50 kDa and 100 kDa.2.Methods for screening the activities(1)In vivo:Select H22 tumor-bearing ICR mice for screening the vivo anti-tumor activities.(2)In vitro:The proliferation inhibition of Bel-7402 cell was determined by MTT experiments.And the morphological changes of Bel-7402 cells were observed directly using an inverted microscope.3.Methods for the determination of protein concentrationThe Bradford method was used for determining the concentration of protein.4.Methods for the component analysis of the gecko sampleChoose the SDS-PAGE electrophoresis and Native-PAGE electrophoresis to analysis the protein of sample.ResultsBase on the above methods,purification and activity screening test results are as follow:1.Methods exploration:(1)Sephadex G-100 gel filtrationSephadex G-100 molecular sieve was used to purify the SOURCE 15Q 0.1M NaCl elution component,the separation effect is not good.This method is abandoned.(2)Q-Sepharose FF strong anion exchange50-100 kDa component was prepared according to Liu Jianting’s method.The Q-Sepharose FF strong anionic medium was used to purify.The separation effect was unsatisfactory.This method is abandoned.(3)DEAE-Sepharose fast flow weak anion exchangeGecko crude extracts were directly purified by DEAE-Sepharose fast flow and the separation effect was poor.Ammonium sulfate precipitation was chosen as the primary purification step for pcko antitumor protein.2.The isolation and purification of gecko antitumor protein:(1)Ammonium sulfate precipitation:In vitro,20-30%,30-40%and 50-60%ammonium sulfate precipitated fractions showed a significant inhibitory effect on the growth of Bel-7402 cells,the inhibition rate was 6.99%,6.62%and 11.33%,respectively;in addition,0-20%,80-90%and 20-30%fractions showed an effect of promoting cell differentiation.In vivo,0-40%,40-60%and 60-90%saturation fraction were obtained with yields of 0.0534%,0.4317%and 0.3588%.The rates of tumor inhibition were 18.30%,23.00%,and 18.10%,respectively.(2)Ion exchange chromatography:The 0-40%saturation fraction was separated by DEAE-cellulose DE 52 ion medium,and the 0.1M NaCl fraction was collected.Its antitumor activity in vivo was verified and the rate of tumor inhibition was 53.33%.(3)Hydrophobic interaction chromatography:the 0.1M NaCl fraction were separated by Phenyl-Sepharose FF medium.The elution fractions of 0M(NH4)2SO4 and H2O were collected and the rate of tumor inhibition in vivo were 48.14%(P<0.05)and 31.28%(no significant difference),respectively.(4)Ultrafiltration:0M(NH4)2SO4 fraction was ultrafiltered and the>100 kDa and 50-100 kDa fractions were collected.The rate of tumor inhibition in vivo were 46.00%(P<0.01)and-7,87%,respectively.ConclusionThe gecko crude extract was effectively purified by ammonium sulfate precipitation,DEAE cellulose DE-52 weak anion exchange chromatography,hydrophobic interaction chromatography and ultrafiltration.Electrophoresis has less strips and a comparatively single active protein was isolated.Speculate the range of the anti-tumor protein molecular weight is 35 kDa-45 kDa.