Using Dictyostelium discoideum to Express Recombinant Squid Myosin Protein (2012)

Undergraduates: Carlos de Castro, Justin Shaffer

Faculty Advisor: William Kier
Department: Biology

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Previous research has determined that the squid Doryteuthis pealeii express multiple isoforms of the contractile protein myosin in their muscular tissues. Because it is difficult to isolate each myosin isoform from squid tissues to study each isoforms’ biochemical function, we have decided to make the proteins recombinantly instead. The amoeba Dictyostelium discoideum was chosen as a host to express the specific recombinant squid myosin isoform. D. discoideum was used because of its ability to manufacture eukaryotic proteins and because it is very easy to grow and maintain in a laboratory setting. An axenic strain of D. discoideum (AX3-ORF+) was ordered and grown in the lab in 10 mL cultures of HL-5 media. A culture of D. discoideum was transformed with an expression vector (pDXA-3H) containing the DNA sequence of squid muscle myosin controlled by the actin promoter. The amoeba will then express the squid myosin protein from this vector. Once expressed, we will then purify the squid myosin protein using column chromatography and further study its function using biochemical assays, specifically measuring its ATPase activity. If successful, these same methods could then be used to express other myosin isoforms for future studies.