Solubilization, purification and characterization of ME31B, a putative RNA helicase
Lewis, Suzanne Scott
Master of Arts
ME31B, a putative RNA helicase belongs to the DEAD box family. A subclass of the DEAD box family is growing around ME31B. The members are DHH1 (S. cerevisiae), ste 13 (S. pombe), and rck/p54 (human and murine). In a conserved region of approximately 390 amino acids, these proteins all share greater than 70% identity with ME31B. The construct pOTS-Nco12-ME31B was used to produce an easily discernible quantity of insoluble ME31B. Following treatment with 8 M urea, ME31B was allowed to renature. Approximately 66% of the ME31B protein remained soluble during these manipulations. This soluble protein was purified on an HPLC hydrophobic column with a purification yield from total protein loaded of 35% soluble ME31B. With this soluble pure protein, I was able to investigate ME31B's ability to hydrolyze ATP. In an ATPase assay, ME31B hydrolyzed ATP in the presence and absence of an RNA substrate.