IN GEL DETECTION OF A HIS-TAGGED TRANSGENE FOLLOWING THE SEPARATION OF CRUDE PLANT PROTEIN EXTRACTS WITH SDS PAGE

Abstract

In the present study, we visualised sundew His6-tagged chitinase protein in crude protein extracts of transgenic tobacco plants following protein separation with sodium dodecyl sulfate polyacrylamide gel electrophoresis by detecting chitinase activity as well as the His-tag. A short sequence encoding six histidines was fused downstream of the DNA sequence encoding the last amino acid of the mature protein. Following binary vector construction and plant transformation, a set of 10 transgenic plants was analysed for transgene expression. Except for one, all transgenic plants exhibited the presence of sundew chitinase protein of ~52 kDa, which was different from the calculated molecular weight of ~32 kDa. Clear identification of DrChitHis protein was performed with a Ni2+:NTA complex conjugated to a fluorescent dye and visualized using light laser-based scanner. A subsequent endochitinolytic activity assay using a N-fluorescein-labelled chitin substrate confirmed that the two transgenic lines with the strongest expression of DrChitHis protein had endochitinolytic activity 6.4 and 6.7 times higher than non-transgenic control.