HIGH-YIELD EXPRESSION AND PURIFICATION OF ENTAMOEBA HISTOLYTICA ALCOHOL DEHYDROGENASE 2 (EHADH2) Layla Ferland, Steven Mcdonough, Andrew Mitchell, Avelina Espinos, Department of Biology, Roger Williams University, Bristol, RI; Dana Lord, Rebecca Page, Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI RI-INBRE Summer Undergraduate Research Fellowship Program The bifunctional alcohol/aldehyde dehydrogenase enzyme in E. histolytica (EhADH2) belongs to the ADHE iron dependent family of enzymes and is essential for trophozoite survival. Using bioinformatics and sequence comparisons with distant structural homologs, we have identified the optimal domain boundaries for the isolated ADH and ALDH domains of EhADH2. We are fully confident that we will obtain protein yields suitable for structure determination using x-ray crystallography. Five constructs, EhADH2 full length, EhADH2 truncated, ALDH, ADH, ADH truncated, have been successfully cloned into pRP1B-NheI vector and sequence verified. The Nterminal ALDH domain expresses to high levels, has been purified to homogeneity and is a monomer, as determined using size exclusion chromatography. The protein is also stable, can be concentrated to more than 39 mg/ml (40x higher than the 1.5 mg/ml of the previous protocol). This domain has produced small crysta ls that are being optimized. The pRP1B-Nhe_EhADH2 five constructs have been transformed in an E. coli adhe deficient strain to test for activity. We have obtained enzymatic activities for EhADH2 full length and ALDH domain that are twice more active than the proteins expressed with the original expression vector. Once diffraction quality crystals of these constructs are obtained, data will be collected and phased by molecular replacement using structures 3K9D and 1RRM as search models.
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