Prokaryotic expression, purification, and sub-cellular localization of a novel alcohol acyltransferase from apple

The coding region of the alcohol acyltransferase gene (MdAAT2) from apple was sub-cloned into expression vectors, pET30a and pET32a, and introduced into E. coli for expression. The purified pET30a/MdAAT2 fusion proteins were used to immunize rabbits following standard protocols. The partially soluble fusion proteins had alcohol acyltransferase activity and were detected only in the pET32a/Origami B(DE3) expression system. Immunolocalization analysis indicated that MdAAT2 is mainly in the cytoplasm, in agreement with the prediction of sub-cellular localization obtained by the LOCSVMpsi program. Western blot analysis indicated that ester biosynthesis in different apple cultivars was related positively to the accumulation of MdAAT2.