Recombinant expression and characterization of Solanum tuberosum aspartic protease

Abstract

Unique to aspartic proteinases from plants are the presence of approximately 100 amino acid regions, which are usually excised during activation of the zymogen. These sequences are termed ‘Plant-Specific Inserts’ and are implicated in membrane interactions of their parent enzymes, including vacuolar targeting and host defense. The need to further characterize the structure-function rol (s) of plant-specific inserts stimulated the current study of the characterization of Solanum tuberosum AP (StAP). Recombinant expression of wild-type StAP resulted in the 54 kDa protein being visualized by SDS-PAGE analysis with a protein yield of 0.03%. A protein purification factor could not be established since activation of the protein at pH 2.2, 3.0, 3.7 and 5.5 was not achieved as evidenced by a lack of change in band patterns on SDS-PAGE as well as acidification and hemoglobin degradation assays. To potentially improve enzyme folding and activation ability, two mutants, (1) lacking the pre-signal sequence and (2) lacking both the signal sequence and the prosegment, were designed, sub-cloned, and expressed. Both products proved to be insoluble and inactive. New constructs were designed for the expression of StAP inclusion bodies for insoluble expression and subsequent re-folding of the protein. Additionally, CysAla mutations for each PSI Cys residue were made to investigate the rol (s) of plant-specific insert disulfide bonds in plant aspartic proteinase enzyme folding and structure. All PSI cysteine mutations (eight mutants) were successfully created using QuickChange MutagenesisTM