Expression and simple, one-step purification of fragile histidine triad (Fhit) tumor suppressor mutant forms in Escherichia coli and their interaction with protoporphyrin IX.
The product of human fragile histidine triad (FHIT) gene is a tumor suppressor protein of still largely unknown cellular background. We have shown previously that it binds protoporphyrin IX (a photosensitizer) which alters its enzymatic activity in vitro. Fhit, diadenosine triphosphate (Ap3A) hydrolase, possesses the active site with histidine triad His-phi-His-phi-His-phiphi. So-called histidine Fhit mutants (His94Asn, His96Asn and His98Asn) exhibit highly reduced activity in vitro, however, their antitumor function has not been fully described yet. In this work we have cloned the cDNAs of histidine mutants into pPROEX-1 vector allowing the production of His6-fusion proteins. The mutated proteins: Fhit-H94N, Fhit-H96N and Fhit-H98N, were expressed in Escherichia coli BL21(DE3) and purified (up to 95%) by an improved, one-step affinity chromatography on Ni-nitrilotriacetate resin. The final yield was 2 mg homogenous proteins from 1 g bacteria (wet wt). The activity of purified proteins was assessed by previously described assay. The same purification procedure yielded 0.8 mg/ml and highly active wild-type Fhit protein (Km value for Ap3A of 5.7 microM). Importantly, purified mutant forms of Fhit also interact with a photosensitizer, protoporphyrin IX in vitro.
Zawacka-Pankau
Joanna
J
Division of Molecular Diagnostics, Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822, Gdansk, Poland. jzawacka@biotech.ug.gla.pl
Podhajska
Anna J
AJ
D017766:Acid Anhydride Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D015226:Dinucleoside Phosphates / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011485:Protein Binding; D011524:Protoporphyrins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D000099170:Bis(5'-adenosyl)triphosphatase
D017766:Acid Anhydride Hydrolases; D015226:Dinucleoside Phosphates; D009363:Neoplasm Proteins; D011524:Protoporphyrins; D011994:Recombinant Proteins; D000099170:Bis(5'-adenosyl)triphosphatase; C011440:diadenosine triphosphate; C028025:protoporphyrin IX