Isolation, Purification And Characterization Of Antifreeze Proteins From Ammopiptanthus Monglicus

Antifreeze proteins (AFPs) are a family of proteins which can interact directly with ice by adsorbing onto the surface of ice crystals and inhibiting the binding of water molecules to the ice crystals. Thermal hysteresis activity (THA) and ice crystal morphology are two important criteria to determine an antifreeze protein in aqueous solution.A modified assay for the morphology of ice crystal is described. Using this assay, two AFPs (both amAFP28 and amAFP37) are isolated from the leaves of cold-acclimated A. Mongolicus, an evergreen legume species surviving in the cold desert of northwest of China.The amAFP37 is isolated and purified using a combination of column chromatography and gel electrophoresis. After homogenization in buffer I (50mmol/L Tris-HCl,pH8.0, 10mmol/L EDTA,20mmol/L Vc), the soluble proteins were captured by DEAE-Cellulose 52 material. An elution with 0.1-0.3 MKC1 led to a crude active fraction. The crude fraction was further purified on a Superdex 75 prep grade column and finally a Poros 20HP2 column. A complex consisting of two proteins with relative molecular masses of 34.7kDa and 37.1kDa respectively in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was obtained by this protein purification protocol. The recovery of two proteins from the gel was carried out by electrophoresis. The purified protein with molecular mass of 37.1kDa showed thermal hysteresis activity (THA) and could modify the normal growth of ice crystals. The THA of this purified antifreeze protein is 0.24℃ at the concentration of 5 mg/ml.The amAFP28 is a heat-stable antifreeze protein. The crude extract from the leaves of A. Mongolicus is obtained with buffer II (25mmol/L Tris-HCl, pH7.4, 0.1mmol/L EDTA, 1mmol/L PMSF, 0.1mol/L KCl) .Purification is achieved by using, a procedure consisting of a heat treatment step followed by consecutive chromatography including ion-exchange chromatography on DEAE-Cellulose A52, molecular exclusion chromatography on Sephacryl S300, hydrophobic chromatography on Poros20HP2, and followed by ion-exchange chromatography on Source 15Q. The purified amAFP28 shows a single protein band on SDS-PAGE and IEF; Its molecular mass as estimated by SDS-PAGE is 28kDa and its isoelectric point (pI) is 6.68 by IEF analysis. The purified amAFP28 can modify the morphology of ice crystals and its THA is 0.15℃ at the concentration of 10 mg/mL . The antifreeze activity in amAFP28 does not disappear after treatment with 0.1mol/Ldithiothreitol, which means that it is not necessary for the antifreeze activity in amAFP28 to maintain intramolecular disulflde bonds. The maximum absorption of the purified amAFP28 is at 278 nm and the minimum absorption 250 nm. amAFP28 does not contain carbohydrate. The result of amino acid composition analysis showed that the AFP is relatively enriched in the hydrophilic amino acid residues of Asp , Thr , Glu, and amino acids residues of low molecular weight including Ser , Ala and Val. The reduction of the disulflde bonds does not influence the secondary structure of amAFP28. A well-defined secondary structure of 17.2% a -helix, 4.1% parallel -pleated sheet and -rum, 39.7% antiparallel -pleated sheet, 34.4% random coil and Y -turn, and 4.6% disulphide and aromatic amino acid is deduced from circular dichroism(CD) of AFP spectral data from 190-250nm. The N-terminus of the AFP is blocked and its part sequence is GKSINHLPGQND. Antifreeze proteins appear to be localized within the apoplast.