Characterization And Functional Study Of Antifreeze Proteins From The Desert Beetle Anatolica Polita

Antifreeze genes Apafp752 and Apafp914 isolated from Xinjiang desert insect Anatolica polita in previous work were constructed with different expressive plasmids and the corresponding mature peptide has been expressed in E. coli respectively. The thermal hysteresis activity (THA) of ApAFPs was measured by micro-liter osmometer or differentiated scanning calorimeter (DSC), and the modification of the ApAFPs to ice crystals was observed. The antifreeze activity of the ApAFPs was further confirmed by assay of the cryopreservation of the proteins on the bacteria in vitro. The study focused on the heat stability, pH stability and the hydrophilicity of the ApAFPs was performed. And the enhancing effects of some salts, polyhydroxyl and polycarboxyl compounds on recombinant ApAFP752 was assayed. The secondary structure of the ApAFPs was derived from the data of circular dichroism (CD), and 3-dimentional structure was predicted based on the homologous protein from Tenebrio molita. Bioinformatic methods were employed to analyze the antifreeze protein genes Apafp752 and Apafp914 from Anotolica polita and their deduced proteins ApAFP752 and ApAFP914 in terms of amino acid composition, physical and chemical properties, evolution relationship and the relationship between structure and function. In this study, the cryoprotective effects of the recombinant ApAFPs on mammalian red blood cells and lactic dehydrogenase were determined and the cold resistance of the E.coli expressing Apafp914 under different low temperature and its growth curve were examined. The main results are as follows:(1) An antifreeze protein gene Apafp752 was expressed in a high level in Escherichia coli strain BL21 (DE3). An approximately 30 kDa fusion protein Trx-ApAFP752 was purified through Ni-NTA affinity chromatography, ion-exchange and gel filtration chromatography. The activity of the purified fusion protein Trx-ApAFP752 was analysed by thermal hysteresis activity (THA) and in vitro antifreeze activity. The results suggested that Trx-ApAFP752 conferred cold resistance on bacteria in a concentration- and time-dependent manner and the cryoprotective effect was increased under alkaline conditions. Circular Dichroism (CD) spectrum analysis showed that the recombinant protein of ApAFP752 possessingβ-sheet as the main structure was stable under a wide range of pH from 2.0 to 11.0 and thermal stability below 50℃.(2) Thermal hysteresis activity (THA) of ApAFP752 from the desert beetle Anatolica polita was measured with differential scanning calorimetry (DSC). When the ice fraction was less than 25.3%, a delay in the onset temperature of refreezing was observed, indicating that the ApAFP752 solution has thermal hysteresis effect. When the amount of ice in the solution was less than 5.1 %, THA of the ApAFP752 reached as high as 0.76℃. THA of ApAFP752 was concentration-dependent. Hydrophilic ability of ApAFP752 was evaluated by Thermal gravimetry (TG). The results of TG showed that ApAFP752 has strong hydrophilicity.(3) In this study, the salts involved in NaCl, KCl, CaCl2, MgCl2 can enhance colligatively the thermal hysteresis activity of antifreeze protein ApAFP752 from Anatolica polita and the bivalent ions were more effective than monovalent ions at low concentration. By comparing the enhancing effect of 4 kinds of polycarboxyl compounds on ApAFP752, the sequence is EDTA > sodium citrate > sodium acetic acid≥sodium carbonic acid. The enhancement of the polycarboxyl compounds was higher than that of the neutral salts, which was determined by the number of carboxyl group. Glycerol and trehalose, the polyhydroxyl compounds studied, just had obvious enhancement on ApAFP752 under high concentration.(4) An antifreeze protein gene from the desert beetle Anatolica polita (named as Apafp914) constructed on pET28a and pET28a-egfp expressed in the Escherichia coli BL21 (DE3) in the form of inclusion body and soluble form, respectively. An approximately 20 kDa recombinant antifreeze protein His-ApAFP914 dissolved in 8M urea was purified through Ni-NTA affinity chromatography and then refolded. The activity of the two recombinant ApAFP914s were analysed by thermal hysteresis activity (THA) and in vitro antifreeze activity. The results suggested that the two recombinant ApAFP914s conferred cold resistance on E. coli in a concentration- and time-dependent manner. The recombinant proteins ApAFP914s had limited pH stability and were comparatively heat stable, THA of which was decreased to a great degree under acidic condition, while alkaline condition has little effect on it. The recombinant ApAFP914 with EGFP tag had higher heat stable level than that of recombinant ApAFP914 with only His-tag. Circular Dichroism (CD) spectrum analysis showed thatβ-sheet was main secondary-structure component of His-ApAFP914, which was a little unstable under acidic condition.(5) ApAFPs have higher sequence similarity with the other insect AFPs from other Coleoptera beetles than the AFPs from Lepidoptera insect. ApAFP914 has closer relationship with MpAFP from the beetle Microdera punctipennis, while ApAFP752 has greater divergence according to the homology tree constructed with 15 homologous AFPs from insects. As the main composition of the secondary structure, ApAFP914 has moreβ-sheet than ApAFP752. Their tertiary structure has high similarity with TmAFP from Tenebrio molita. The number of loops of the ApAFP914 is more than that of ApAFP752 in their right-handβ-helix. The bigger size and higher hydrophilicity of the ApAFP914 may be the dominant factor to its higher thermal hysteresis activity and heat stability.(6) The recombinant antifreeze protein rApAFP914 can decrease the hemolysis of murine red blood cell under -3℃, and increase the resistance of the cell to the salt osmotic stress. Simultaneously, the hemolysis of the red blood cell was decrease at 4℃with the presence of rApAFP914. The cryopreservation effect was also concentration-dependent. It was deduced that cryoprotective effect of ApAFP914 was based on the binding to the ice nucleaor, and the binding to the membrane of the red blood cell to stabilize the membrane.(7) The survival rate of the host expressing ApAFP914 was significantly higher than that of the other bacteria expressing control protein under different low temperature. The in vivo cryoprotective effect was increasing with the accumulation of the ApAFP914 resulted from the concentration of the inducer and the duration of the induction. The research makes the basis to the further elucidation how the AFP functions in the nature body.(8) The recombinant ApAFPs can significantly improve the remained activity of the LDH after freeze-thaw cycles in liquid nitrogen in a manner of concentration-dependency. The recombinant ApAFP752 has higher protective effect than recombinant ApAFP914.