A dye elution method for the quantification of insecticidal crystal proteins from Bacillus thuringiensis and its compatibility with the presence of agro-industrial raw materials and waste products

Abstract

The insecticidal crystal proteins produced by Bacillus thuringiensis during sporulation are active ingredients against lepidopteran, dipteran, andcoleopteran insects. Several methods have been reported for their quantification, such as crystal counting, ELISA, and SDS-PAGE/densitometry.One of the major tasks in industrial processes is the analysis of raw material dependency and costs. Thus, the crystal protein quantificationmethod is expected to be compatible with the presence of complex and inexpensive culture medium components. This work presents a reval-idated elution-based method for the quantification of insecticidal crystal proteins produced by the native strain B. thuringiensis RT. To quantifyproteins, a calibration curve was generated by varying the amount of BSA loaded into SDS-PAGE gels. First, SDS-PAGE was performed for qual-ity control of the bioinsecticide. Then, the stained protein band was excised from 10% polyacrylamide gel and the protein-associated dye waseluted with an alcoholic solution of SDS (3% SDS in 50% isopropanol) during 45 min at 95◦C. This protocol was a sensitive procedure to quantifyproteins in the range of 2.0–10.0 μg. As proof of concept, proteins of samples obtained from a complex fermented broth were separated bySDS-PAGE. Then, Cry1 and Cry2 proteins were properly quantified.