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Purification and characterization of a protein phosphatase (PP1-Arch) from the archaebacterium Sulfolobus solfataricus, isolation and expression of its gene

PP1-Arch was verified as a protein phosphatase by both acid molybdate extraction and thin layer electrophoresis. Soluble fraction was prepared from <i>Sulfolobus solfataricus</i>, 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 final 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 K<sub>m</sub> for phosphocasein were determined. Myosin light chain was found to be a substrate for PP1-Arch <i>in vitro</i>. EDTA, Cu²⁺, Zn²⁺, P<sub>i</sub>' and PP<sub>i</sub> 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 <i>E. coli</i> 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. / Ph. D.

Identiferoai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/39141
Date14 August 2006
CreatorsLeng, Jie
ContributorsBiochemistry and Anaerobic Microbiology, Kennelly, Peter J., Bender, Patrick K., Bunce, George E., Sitz, Thomas O., Lee, John C.
PublisherVirginia Tech
Source SetsVirginia Tech Theses and Dissertation
LanguageEnglish
Detected LanguageEnglish
TypeDissertation, Text
Formatx, 117 leaves, BTD, application/pdf, application/pdf
RightsIn Copyright, http://rightsstatements.org/vocab/InC/1.0/
RelationOCLC# 32749882, LD5655.V856_1994.L464.pdf

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