Preparation Of CNBr-Stefin-Sepharose 4B Affinity Media And Purification Of Grass Carp Hepatopancreas Cathepsin L

Cathepsin L is a kind of Caspases with many types and wide distribution.It has been widely studied because of its various biological functions and has great potential value in food and pharmaceutical research.This experiment firstly induced the expression and purification of recombinant grass carp Stefin,and then prepared the affinity chromatography media CNBr-Stefin-Sepharose 4B with Stefin as ligand.The stability of affinity chromatography media was then determined.This experiment secondly isolated and purified cathepsin L from the hepatopancreatin of grass carp.Finally,the activity type and molecular structure characterization of purified cathepsin L was identified.This experiment provided experimental basis and scientific reference for the isolation and purification of cathepsin L,which was of high value in fish hepatopancreatin,and also provided new ideas and practical support for the preparation of affinity chromatography media.The results obtained in this experiment were as followed:First,the expression strain E.coli BL₂₁(DE₃)which was transferred into the grass carp Stefin-p ET-30a was induced to express.The recombinant grass carp Stefin was isolated and purified by freeze-thaw lysis,cell disruption,urea gradient washing and Ni⁺-NTA affinity chromatography.Ni⁺-NTA affinity chromatogram showed a single chromatographic peak,and SDS-PAGE electrophoresis showed that the molecular weight of recombinant grass carp Stefin was about 11.4 k D.Recombinant grass carp Stefin showed the inhibitory ability to Papain,but did not have inhibitory ability to Trypsin or Chymotrypsin.Its inhibitory activity of Papain increased with the increase of Stefin concentration.The stability detection of recombinant grass carp Stefin showed that Stefin has a wide range of acid-base and thermal stability.Stefin could maintain more than 64%of the residual inhibitory activity in the range of p H 4-11 and 4?-70?.Subsequently,the CNBr activation method was used to activate Sepharose 4B,and the affinity chromatography media CNBr-Stefin-Sepharose 4B was self-made with recombinant grass carp Stefin as ligand.Ultraviolet spectroscopy showed that the media was successfully activated and successfully coupled to Stefin.Using Stefin coupling capacity and Papain adsorption capacity as indicators,determined the optimal liquid-to-material ratio of CNBr-Sepharose 4B and Stefin.The result indicated that the optimal ratio was 1:8(w/v).In this condition,the Stefin coupling capacity was 11.22 mg/g,and the Papain adsorption capacity was 5.98 mg/g.The stability detection of the affinity chromatography media CNBr-Stefin-Sepharose 4B indicated that it showed good stability in the range of p H 2-10 and 4?-40?,and its optimal using condition was p H 7,4?.Next,the grass carp hepatopancreatin cathepsin L was extracted by crude extraction,acid treatment and ammonium sulfate precipitation,and then subjected to CNBr-Stefin-Sepharose 4B affinity chromatography.Grass carp hepatopancreatin cathepsin L was almost precipitated in 80%ammonium sulfate,at this time the purification factor was 1.91,and the recovery rate was 73.90%.SDS-PAGE and reversed-phase zymography analysis showed that two types of protease with molecular weights of 33 k D and 30 k D were obtained after affinity chromatography with CNBr-Stefin-Sepharose 4B.At this time the purification factor was 16.45,and the recovery rate was 46.81%.TSK gel G2000 SW_XLHPLC detection showed that the target protease showed its main activity peak at a retention time of 19.262 min(29.4 k D).At last,the molecular structure and activity type of target protease were characterized.Con A Sepharose 4B lectin affinity chromatography results indicated that there were two types of target protease,one was eluted when the concentration of?-methylmannosid was0%,proving that it had no carbohydrate side chains.The other one was eluted when the concentration of?-methylmannosides was among 0.15-0.3 mol/L,proving that it had carbohydrate side chains.The results of activity type identification indicated that the target protease would be activated by the thiol activators DTT,?-Me and L-Cys.Among them,DTT and?-Me reached the maximum activation effect at the concentration of 2 mmol/L.The activation effect of L-Cys increased with increasing concentration in the range of 0-20mmol/L.E-64 showed significant inhibitory effect on target protease,but PMSF did not inhibit it.