COMBINING HETEROLOGOUS BACTERIAL EXPRESSION SYSTEM
WITH AFFINITY CHROMATOGRAPHY PURIFICATION TO OBTAIN
NATIVE MOUSE TYROSINASE
GABRIELA N. CHIRIȚOIU1*#, CRISTIAN V.A. MUNTEANU1#, NICULINA MITREA2
1Institute of Biochemistry of the Romanian Academy
2Department of Biochemistry, Faculty of Pharmacy, “Carol Davila” University of Medicine and Pharmacy
*corresponding author: email@example.com
#These authors contributed equally to this work.
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Tyrosinase is a type 1 transmembrane protein, with a pivotal role in melanin synthesis within melanocytes. Tyrosinase is also
upregulated in melanoma cells and for this reason it is used as a conventional biomarker in melanoma diagnostic. Moreover,
many scientific reports present tyrosinase as a well-known autoantigen capable to elicit the immune system and as a
consequence, one of the main goals in melanoma therapy is to develop methods to increase tyrosinase immunogenicity.
Among the experimental approaches in achieving this goal is the manipulation of the protein in vitro, followed by studies on
animal models. Here we report expression in a bacterial system of a tagged tyrosinase from which the protein is further
purified. It is known that foreign transmembrane proteins often aggregate and form inclusion bodies when they are expressed
in bacteria because of their highly hydrophobic aminoacids that span the membrane bilayer. Protein extraction from these
structures is usually done using denaturant agents. We were able to express mouse tyrosinase without the signal sequence and
to co-purify in non-denaturant conditions this protein. Sequence confirmation of the purified product was obtained using
mass-spectrometry and Western blotting.