Expression,purification and activity determination of the beetle tenebrio molitor antifreeze protein AFP84c in Escherichia coli

A cdna encoding antifreeze protein (afp84c) was cloned by rt-pcr from the larva of the yellow mealworm tenebrio molitor. the coding fragment of 252 bp encodes a protein of 84 amino acid residues and was fused to the expression vectors pmal-c2x and pmal-p2x. the expression plasmids pmal-c2x-afp84c and pmal-p2x-afp84c were constructed and transformed into escherischia coli strains tbi,respectively. strategy of optimization of induction conditions were used for expression of the highly disulfide-bonded β-helix-contained protein with the activity of antifreeze in pmaltm expression system. the target fusion protein was released from the cytoplasm and periplasm by sonication and cold osmotic shock procedure respectively. recombinant afp84c was purified by amylose affinity column. the purified target protein displayed a single band in sds-page. expressed afp84c exhibits to increase low temperature resistance of bacteria.