| Summary: | An E. coli yeast shuttle vector for the anchoring of heterologous protein to the yeast host's cell wall was constructed. The vector PYDSM01 includes a DNA sequence constructed from the signal sequence from the yeast sucrose isomerase gene, a multiple cloning site and a DNA fragment encoding the carboxyl-terminal of the yeast cell wall protein 2 (CWP2). This construct was then inserted into the HindIII site on pGAD424, replacing the GAL4 fusion tag and the original MCS sequence. DNA sequencing confirmed the correct insertion of both signal and anchor proteins in the vector. To test for proper expression and functional anchoring to the cell wall, the coding sequence for a bacterial alpha-amylase enzyme was cloned into the vector and transformed into a yeast host. A total of 22 yeast transformant were recovered which were able to degrade starch, indicating successful expression and function of the bacterial alpha-amylase gene. Enzyme assay of the washed cell pellet and supernatant fractions indicate that the both activity and anchoring efficiency are variable. © 2012 Academic Journals Inc.
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