Gene Cloning And Prokaryotic Expression Of Antifreeze Protein From Tenebrodae Insects

Antifreeze proteins (AFPs) are proteins inhibiting the growth of ice crystals and decreasing the freezing point of water by a non-colligative mechanism without depressing the melting point, therefore producing a difference between the freezing and melting points termed thermal hysteresis which is widely used as an indicator of AFP activity. AFPs have three main founctional characteristics:①thermal hysteresis activity ②changing the shape of the ice crystal ③suppressing the recrystallization of ice.AFPs have been identified from many orgnisims such as fish, plants, insects, fungi and bacteria.Comparing with these species , the insect AFP has the highest thermal hysteresis activity of 3-6℃, while the fish of 0.7～1.5℃, and the plant AFP has the lowest of 0.2～0.5℃. Thus the insect AFP has great potential in practical application. The AFPs from different sources vary greatly in terms of the composition of base pairs, the composition of the amino acids, and the second structure of proteins.So it is difficult to classify these protein,only the fish AFPs can be divided into four types. Besides, there are different isoforms within the same species due to the length change of the genes and positions changes of a few amino acids. To date, the insect AFPs reported are different from fish AFPs and plant AFPs. Homology analysis showed that the identity of the Tenebrio moliter AFP sequences with the Dendroides Canadensis AFP sequences is over 50%. Both these two types of AFPs have 12-13 amino-acid repeat units with more pairs of disulfide-bonds which play important role in sustaining the stability of the second structure. Because of these diversities, AFPs do not show conservativeness in evolution. Only the typeII AFP in fish was found homologous to the carbohydrate-binding domain of C-type lectins. Although the structures of AFPs change greatly, they accord with the same fundmental rule –the ice adsorption–inhibition mechanism, a widely accepted theory for explaining the AFPs function. With the advances in the research of AFPs, the application study for AFPs made great progress and brought prosperous potential. For example, AFPs could be used as food additive to improve food quality in food industy. It could be used as antifreezer in preserving tissues, organs, sperm and cellular materials stored at lower temperature. Other application like AFP gene transferring strategy is employed to provide plants cold resistance, and expand the living range of animals and plants. The main purpose of this study was to clone AFP genes from different insects and detect the characteristics of these AFPs. The research including: cloning of antifreeze protein genes from Tenebrio molitor larvas, beetle Pterocoma loczyi and beetleBlaps kashgarensis bates; making constructs of pGEX4T-1-tafp-416, pGEX4T-1-tafp -430, pGEX4T-1-plafp -743; and expressing these proteins in prokaryotic system. After purified the fusion protein GST-TAFP-416, GST-TAFP-430, GST-PLAFP-743 by Glutathione Sepharose 4B, the AFP activities of GST-TAFP-416 and GST-PLAFP-743 were tested by the protactive effect of AFP on bacteria at low temperature. Meanwile, the antiserum of tafp-430 produced by immunizing mice with plasmid pcDNA3-tafp-430 was prepared for the detection of AFP. Our work were started at cloning the full length fragment of AFP from Tenebrio molitor .At first, the primers for amplifying the full length fragment were designed according to the conservative region of Tenebrio moliter AFP published in GenBank. Six pairs of primers were applied for RT-PCR. The primers for AFP core and full length fragments were designed according to conservative region from different insect AFPs published in GenBank and those from beetle Microderapunctipenis dzunarica previously cloned in our laboratory. The core and full length fragments were cloned separately from beetle Pterocoma loczyi and beetle Blaps kashgarensis bates by 3'RACE. All the PCR products were constructed to pMD18-T vector for sequencing. Sequence analysis revealed the obtained genes as following: ten of full length fragments from Tenebrio moliter, three core fragments and one full fragment from Pterocoma loczyi and three core fragments from Blaps kashgarensis bates. These fragments were of very similar with Tenebrio moliter AFP genes. They were composed of 12-or 13-amino-acid repeat units: C1T2X3X4X5X6C7X8X9X10X11X12.The size of the AFP genes from Tenebrio moliter were between 300bp-444bp in length and had seven repeat units in common, and the largest protein TAFP-433 had ten repeat units and the shortest protein TAFP-408 had five repeat units. Only the gene TAFP-433 was completely identical with the published sequence (AF160497), while the other sequences had more or less difference in non-conservative region. The fragments from Pterocoma loczyi and Blaps kashgarensis bates had very high identity with published Tenebrio moliter AFP sequences and also with 12-or 13-amino-acid repeat units: C1T2X3X4X5X6C7X8X9X10X11X12. Considering that Tenebrio moliter, Pterocoma loczyi and Blaps kashgarensis bates all belong to the Tenebrionidae family, it is acceptable that the obtained beetle AFP genes were conservative in the family. In order to detect the biological activity of AFPs, the cDNA fragment of tafp-430, tafp-416 and plafp-743 were constructed into the express vector pGEX4T-1 respectively. Positive clones were transformed into E.coli BL21(DE3)and induced by IPTG for the expression. The SDS-PAGE analysis showed these three proteins were expressed correctly in a fusion way. Then these fusion proteins were induced at large scale and further purified by Glutathione Sepharose 4B. The Western blot analysis with antisera of Dendroides Canadensis prepared in our laboratorypreviously showed the purified protein GST-TAFP-416, GST-TAFP-430 and GST-PLAFP-743 could react specifically with the DAFP antibody. Besides, the specific antibody was prepared successfully by immunizing mice with plasmid pcDNA3-tafp-430. In addition, the biological activity of antifreeze protein GST-TAFP-416 and GST-PLAFP-743 were detected by AFP protection experiment on bacteria at low temperature. The results indicated that the bacteria could be protected from the low temperature damage at least at the concentrations of GST-TAFP-416 40μg/ml and GST-PLAFP-743 20μg/ml. In this experiment,the water, elution buffer, and GST(70μg/ml)were set as the control. In addition,to detect the heat steability of AFPs, the protein GST-TAFP-416 and GST-PLAFP-743 were boiled in 100℃for 10 minutes , the biological activity were lost.