›› 2010, Vol. 53 ›› Issue (11): 1207-1212.

• RESEARCH PAPERS • Previous Articles     Next Articles

Highly efficient expression and activity detection of Tenebrio molitor antifreeze protein AFP84a in Escherichia coli

YAN Qing-Hua, YANG Li, SHAO Qiang   

  • Online:2010-12-29 Published:2010-11-20

Abstract:

For the expression and purification of Tenebrio molitor antifreeze proteins, RT-PCR method was adopted to obtain the cDNA of antifreeze protein gene afp84a which was then cloned into prokaryotic plasmid pMAL-p2X and expressed in Escherichia coli TBI strain. The recombination protein was purified through an amylose affinity column and assayed for antifreeze activity by observing bacterial survival rate in the presence of purified antifreeze protein following incubation for various intervals at 0. The results showed that the content of fusion protein was 40% of total dissolved proteins. SDS-PAGE analysis indicated that fusion protein could be released from cells treated with MgSO4 or sonicate. A single band of target protein was acquired after the fusion protein was purified through amylose affinity column and incised by Factor Xa. Furthermore, the antifreeze protein AFP84a could increase the low temperature resistance of bacteria as shown in the biological activity analysis. The cloning and prokaryotic expression of afp84a from T. molitor could provide useful experimental materials for the further study of the nature and function of antifreeze protein AFP84a.

Key words: Tenebrio molitor, Escherichia coli, antifreeze protein, fusion expression, affinity purification, antifreeze activity