This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Human Fhit and mutated Fhit-H96G enzymes were isolated from E. coli strain SG100 sells transformed from pSGA02-Fhit according the published method (b). An overnight cell culture was diluted by 100 folds to 16 litters of LB medium containing kanamycin and ampicillin. The cell culture was grown at 37 C untill OD600 reaches 0.6. After addition of isopropyl thio-b-D-galactoside (IPTG) to 1 mM and induction at 37 C for 6 hours, cells were harvested (~ 17 g), suspended in 100 ml buffer A (50 mM HEPES, pH 6.8, 10% vol/vol glycerol) containing 0.5 mM phenylmethylsulfonyl fluoride (PMSF). The cells were sonicated 5 minutes and centrifuged at 100,000 g for 15 minutes to collect the supernatant. The supernatant was diluted to 150 ml and subjected to a 400 ml DEAE-Sephacel column as described (b). The fractions containing Ap3A hydrolase activity from the DEAE-Sephacel column were combined and concentrated by PM10 memberane to 40 ml, which was diluted to 100ml and subjected to a 200 ml Q-Sepharose column eluted with 1000 ml gradient of buffer A to 0.2 M NaCl in buffer A at a flow rate of 2 ml/min. Two peaks showing Fhit activity were collected and checked for purity for SDS-PAGE gel electrophoresis. The subunit concentration of Fhit was determined by use of e280 = 8310 M-1cm-1 calculated from the amino acid sequence of Fhit according to a published method ( c).
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