EXPRESSION AND PURIFICATION OF CRY8DA RECOMBINANT PROTEIN AGAINST COLEOPTERAN INSECTS OF BACILLUS THURINGIENSIS IN E. COLI

Abstract

Bacillus thuringiensis (Bt) is widely used in agriculture for pest control against Lepidoptera, Diptera and Coleoptera orders. It is very difficult to control of Coleoptera pests such as larvae of Scarabaeidae family, which damagethe roots of tusf grass and other horticultural and agricultural plants, because theyare living in soil where sprayable insecticidal formulation is hard to reach the target insects. The Cry8-type proteins specified to larvae of scarab beetles. The Cry8-type proteins are insecticidal to larvae of scarab beetles. Bacillus thuringiensis Cry8D (Cry8Da, Cry8Db) insecticidal proteins are unique among Cry8 family protein in terms of its insecticidal activity against larvae and adult Scarab beetles, such as Japanese beetle (Anomala cuprea, Popillia japonica). In this study, cry8Dagene encoding nature protein Cry8Da, which were isolated in Vietnam, was expressed in  E. coli. The 2031 bp of  cry8D agene is digested from recombinant plasmid pGEM-cry8Dawith  BamHI và  sacI and ligated into pET32a(+) vector treated with the sameenzymes. The recombinant plasmids pET32a(+)-cry8Da were expressed in BL21(DE3)LysS strain under induction of IPTG. The highest level for expression of Cry8Da recombinant E. coli has been obtained after induction by 1 mM IPTG at 30 ⁰C for 4 hrs. Recombinant Cry8Da protein has molecular mass about 96 kDa and purified by Ni⁽²⁺⁾ affinity chromatography column, concentration 0,45mg/ml  after purification. The expressed recombinant Cry8Da protein was confirmed by Western blot using anti-Cry8Da antibody.