Title page for ETD etd-08142006-110059


Type of Document Dissertation
Author Leng, Jie
URN etd-08142006-110059
Title Purification and characterization of a protein phosphatase (PP1-Arch) from the archaebacterium Sulfolobus solfataricus, isolation and expression of its gene
Degree PhD
Department Biochemistry and Anaerobic Microbiology
Advisory Committee
Advisor Name Title
No Advisors Found
Keywords
  • Phosphoprotein phosphateses Purification
  • Archaebacteria
Date of Defense 1994-11-05
Availability restricted
Abstract

PP1-Arch was verified as a protein phosphatase by both acid molybdate extraction and thin layer electrophoresis. Soluble fraction was prepared from Sulfolobus solfataricus, from which PP1-Arch was purified over 1OOO-fold by DE-52 ion-exchange, hydroxyapatite, gel filtration (G- 100), and Mono Q FPLC chromatography. PP1-Arch was identified from the fmal purified sample by renaturation on an SDS-polyacrylamide gel. The molecular size of PP1-Arch was determined by both gel filtration chromatography and SDS-PAGE as 28 kDa and 33 kDa, respectively, which suggests that PP1-Arch is a monomer. PP1-Arch was found stable at temperatures as high as 90°C. Activation constants for the divalent metal ions Mn2+ and Ni2+, and the Km for phosphocasein were determined. Myosin light chain was found to be a substrate for PP1-Arch in vitro. EDTA, Cu2+, Zn2+, Pi' and PPi were shown to be inhibitors of PP1-Arch, while many compounds known to affect eukaryotic protein phosphatase activities were found to be without noticeable effect.

N-terminal and an internal peptide sequence of the enzyme were obtained. The gene for PP1-Arch was cloned by a combination of "touchdown" PCR and conventional cloning techniques. The PP1-Arch gene was sequenced on both strands, and the sequence was compared with ones from eukaryotes and bacteriophage λ. The sequence homology between PP1-Arch and PP1/PP2A/PP2B suggests that they belongs to the same genetic family.

A recombinant plasmid which was derived from pT7-7 was constructed for expression of PP1-Arch. The PP1-Arch gene was expressed in E. coli and the activity of the expressed enzyme was tested and shown to be divalent metal ion-dependent. Formation of inclusion bodies of expressed PP1-Arch was demonstrated.

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