THE ENGINEERING OF ACTINIDIN PROTEIN AND ITS EXPRESSION IN THEYEAST SACCHAROMYCES CEREVISIAE
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Actinidin is an intracellular cysteine protease found in the fruit of Chinese gooseberry (Actinidia chinensis) or Kiwi fruit. A variant of actinidin-encoding DNA sequence has been constructed by amplifying a specific region of the full-length actinidin cDNA using PCR method. Amplification was targeted to construct an actinidin-encoding DNA sequence consisting of mature actinidin-encoding DNA plus part of the N-terminal extension (carrying the QRTN motif) and part of the C-terminal extension without QR sequence motif. The amplified fragment was then cloned in an expression-secretion vector (pYSV9) and expressed in Saccharomyces cerevisiae. The results of this study demonstrated that the cloned fragment had a smaller size as compared to the PCR amplification product, suggesting that a rearrangement event occured during the cloning process. Yeast culture supernatant was analysed for expression of actinidin by using anti-actinidin polyclonal antibody raised in chicken. The expression of the cloned fragment in S. cerevisiae, however, did not result in any detectable expression product on the immunoblot.