Molecular cloning and expression of protein phosphatase-1

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Biochemistry and Molecular Biology

First Committee Member

Ernest Y. C. Lee, Committee Chair


A cDNA coding for the catalytic subunit of protein phosphatase-1 (ppase-1) was cloned from a rabbit muscle cDNA library by screening with oligonucleotide probes. A total of 10 clones were analyzed. Systems for the expression of ppase-1 in E. coli were studied. Phosphatase-1 was expressed in the pET3a vector as an insoluble aggregate. The insoluble enzyme could be partially renatured ($<$5%) by high dilutions of the urea-solubilized protein in buffers containing dithiothreitol, Mn$\sp{++}$ and high NaCl concentrations. A system was found for the expression of the enzyme as a soluble protein in the pTACTAC vector at levels of 3-4% of the soluble E. coli protein. The recombinant enzyme displayed similar sensitivities to inhibition by inhibitor-2, okadaic acid and microcystin-LR as for the protein isolated from rabbit muscle. Unlike the enzyme purified from rabbit muscle, the recombinant enzyme is dependent on Mn$\sp{++}$ for activity.Four rat ppase-1 isoforms were expressed in E. coli using the pTACTAC vector. The four recombinant isoforms were purified to near homogeneity and their properties were examined in terms of substrate specificity and sensitivity to okadaic acid and inhibitor-2. All four of the isoforms displayed similar dependencies of Mn$\sp{++}$ as that for recombinant rabbit muscle ppase-1.One N-terminal and five C-terminal deletion mutants of ppase-1 were constructed and expressed in pTACTAC vectors. The N-terminal deletion mutant, N-310, leads to a complete loss of the enzyme activity. Six cysteine mutants were constructed and expressed in the pTACTAC vector. The C62S, C171S and C202S mutations led to loss of more than 80% of activity in the E. coli crude lysates, while the other three cysteine mutants give a similar phosphorylase phosphatase activity to that of wild type. All of the six cysteine mutants were purified near to homogeneity using affinity chromatography and confirmed to be catalytically active. These results suggest that cysteine residues are not involved in catalytic mechanism.The coding region of the cDNA of protein phosphatase inhibitor-2 was isolated by polymerase chain reaction amplification. The deduced amino-acid sequence was identical with that of the sequence reported by chemical sequencing methods. Inhibitor-2 was expressed in E. coli using the pET3a vector. The purified recombinant inhibitor-2 exhibited similar properties to native rabbit muscle inhibitor-2. (Abstract shortened by UMI.)


Biology, Molecular

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