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Mutagenesis of AbPPO4

MP Matthias Pretzler
AB Aleksandar Bijelic
AR Annette Rompel
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As the enzyme isolated from the natural source did not contain the last 46 amino acids that are encoded by its gene27, the cloned gene was adjusted accordingly. Towards that end the base triplets encoding the respective amino acids were deleted from the gene using the Q5® Site-Directed Mutagenesis Kit (NEB). Removal of the sequence corresponding to amino acids A566 to F611 was accomplished with the two primers lAbPPO4_fwd and lAbPPO4_rev, while E384 to F611 was removed using lAbPPO4_fwd and aAbPPO4_rev resulting in the bases encoding just the main domain of the tyrosinase27, 33.

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