Expression And Activity Comparsion Of Insect Antifreeze Proteins With Different Structures

Antifreeze proteins (AFPs) are kinds of functional biological micromolecules that can noncolligatively lower the freezing point of body fluids by a without significantly affecting the melting point. As a result, a gap is generated between the freezing and melting temperature, which is known as thermal hysteresis activity (THA). AFPs interact with ice surfaces or ice nuclei and inhibit ice growth. These proteins have been identified in a variety of organisms including plants,bacteria, fungi ,fish and insects. Insect AFPs have antifreeze activities 10–100 times greater than those of fish AFPs .The recombinant plasmid containing Blaps kashgarensis AFP gene Bkafp972 was transformed into E. coli, and induced by IPTG. The soluble fusion protein MBP-BkAFP972 was obtained. The cryoprotective activity of the fusion protein was checked on bacterial and mouse muscle. The results showed that the fusion protein made with genetic engineering technique had cryoprotective activity. Thus, the insect AFP from the prokaryotic expression system possessed biological function, and could a way to provide the source for the far-flung application of AFP.Based on the successful expression of insect antifreeze protein in E. coli, the biological activity and thermal hysteresis activity of the recombinant AFPs from two different insects, Microdera punctipennis and Blaps kashgarensis, fused with both MBP and GST tags were compared. The results showed that the MpAFP5, from Microdera punctipennis had higher biological activity and thermal hysteresis activity than the BkAFP972, the protein from Blaps kashgarensis. The structures of the two AFPs from two kinds of insects are similar in that they both contain several of the typical 12-amino acid repeat units TCTxSxxCxxAx except that MpAFP5 has one more repeat unit than BkAFP972. So it was speculated that the number of the coils of antifreeze protein might play an important role in the cryoprotective activity of the proteins. In addition, it was found that the activity of the fusion protein with MBP tag was higher than that with GST tag, and the purified MBP-proteins had more clear solutions.In order to remove the influence of the tag on the activity of the AFPs, GST-MpAFP5 and GST-BkAFP972 were digested with thrombin. It was found that both MpAFP5 and BkAFP972 had higher activities than their recombinant proteins, and the activity of MpAFP5 was still higher than that of BkAFP972.To further understand the effect of coils number on the activity of insect AFPs, the fifth coil in the tertiary of the Mpafp5 was deleted by PCR. The mutant gene (named: sMpafp5) was then constructed into two prokaryotic expression vectors. The two recombinant plasmids, pGEX4T-1-sMpafp5and pMAL-p2x-sMpafp5, were transformed into E.coli and expressed with the induction of IPTG. The two mutant fusion proteins GST-sMpAFP5and MBP-sMpAFP5 were purified respectively. It proved out that the activity of the eight coils MpAFP5 was higher than that of the seven coils sMpAFP5. This result demonstrated that that the number of the coils played an important role in the cryoprotective activity of insect AFPs.