Effect Of Structure Of Antifreeze Protein ApAFP914 From Anatolica Polita On The Antifreeze Activity

Antifreeze gene 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. Based on the desert insect antifreeze protein gene ApAFP914 cloned previously, using site-directed mutagenesis PCR reaction by overlapping and three mutants site on the primers, to obtain ApAFP914-A19 T, ApAFP914-T33 F, ApAFP914-T33&45F, mutants genes.The first structure of the ApAFPs and the data 3-dimentional structure was predicted based on the homologous protein from Tenebrio molita. In addition, ExPAsy Swiss Model prediction bioinformatics software such as the wild type and mutant protein in high-level structure.The antifreeze protein gene ApAFP914 and its mutants were expressed in a high level in Escherichia coli strain BL21(DE3). An approximately 30 kDa fusion protein Trx-ApAFP914 was purified through Ni-NTA affinity chromatography, be confirmed by SDS-PAGE and WesternBlot.Thermal hysteresis activity(THA) of ApAFP914 and its mutants was measured with differential scanning calorimetry(DSC). When the ice fraction was less than 25%, a delay in the onset temperature of refreezing was observed, indicating that the ApAFP914 s solution had thermal hysteresis effect. When the amount of ice in the solution was less than 5%, THA of the ApAFP914 reached as high as 0.49℃, but ApAFP914-A19 T was 0.55℃, ApAFP914-T33 F was 0.38℃, ApAFP914-T33&45F was 0.31℃. THA of ApAFP914 s was concentration-dependent. By comparing the thermal hysteresis activity(THA) of 3 kinds of mutants and ApAFP914, the sequence is ApAFP914-A19T> Ap AFP914 > ApAFP914-T33F≥ApAFP914-T33&45F.The antifreeze activity of the ApAFPs was further confirmed by assay of the cryopreservation of the proteins on the bacteria, yeast or red blood cells in vitro. The results suggested that the recombinant Ap AFP914 s and its mutants conferred cold resistance on E. coli and yeast in a concentration- and time-dependent manner. By comparing the antifreeze effect level of 3 kinds of mutants and ApAFP914, The TrxAApAFP914 or TrxA-Ap AFP914-A19 T had higher antifreeze effect level than that of TrxA-ApAFP914-T33 F or TrxA-ApAFP914-T33&45F.The hemolysis of the red blood cell was decrease at 4℃、-4℃ or-7℃ with the presence of rAp AFP914. 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.The enhancing effects of some salts NaCl、LiCl、CaCl2、MgCl2, polyhydroxyl and polycarboxyl compounds on recombinant ApAFP914 was measured with differential scanning calorimetry(DSC). And the salts can enhance colligatively the thermal hysteresis activity of antifreeze protein ApAFP914 from Anatolica polita and the bivalent ions were more effective than monovalent ions at low concentration. Sorbitol and trehalose, the polyhydroxyl compounds studied, just had obvious enhancement on ApAFP914 under high concentration.In order to explore the role of insect antifreeze proteins in the vitrification of mammalian germ cells, the survival rate of apafp914 vitrification of mouse oocytes and the mouse oocytes were observed by morphological observation and FDA and PI double staining technique. The results showed that the survival rate of the apafp914 group was significantly higher than that of the control group, while the insect antifreeze protein apafp914 could be used as the oocytes.