Thermo-Inducible Expression of δ Endotoxin Gene of Bacillus thuringiensis HD1 Derived under Lambda PL Promoter in Escherichia coli

Abstract

The insecticidal crystal protein gene cryIA(a) from Bacillus thuringiensis HD1 has been cloned as a single 3.765 kb Ndel fragment on the expression vector pRE1. The pBR322 based clone pES1 was digested with restriction endonuclease Ndel and the 3.765 kb fragment carrying the intact gene was eluted and cloned on pUC18 to confirm its functional integrity. This Ndel fragment was then cloned on the vector pRE1 carrying strong promoter PL of lambda upstream to the cloning site. The recombinant construct pUSR14.1 carried crystal protein (CP) gene under PL and was temperature inducible at 42°C in MZ1 host strain of Escherichia coli because of temperature sensitive CI857 gene carried by it as lysogen. Dilution based insect bioassays showed hyper-expression of toxin in these constructs. SDS-PAGE analysis indicated that polypeptides corresponding to 132 kD and 66 kD bands of HD1 endotoxin constituted 20.1% of the total soluble protein in this recombinant strain to be delta-endotoxin.