Expression, Purification, and Characterization of the Protein Repair l-Isoaspartyl Methyltransferase from Arabidopsis thaliana
| Autor: | Nitika Thapar, Steven Clarke |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Methyltransferase
Molecular Sequence Data Arabidopsis medicine.disease_cause Substrate Specificity Evolution Molecular Complementary DNA Protein D-Aspartate-L-Isoaspartate Methyltransferase Escherichia coli medicine Arabidopsis thaliana Amino Acid Sequence Protein Methyltransferases Cloning Molecular Promoter Regions Genetic Peptide sequence Plant Proteins Sequence Deletion chemistry.chemical_classification Chromatography Base Sequence biology Temperature Hydrogen-Ion Concentration biology.organism_classification Molecular biology Recombinant Proteins Kinetics Enzyme chemistry Biochemistry L-isoaspartyl methyltransferase Protein repair Electrophoresis Polyacrylamide Gel Plasmids Biotechnology |
| Zdroj: | Protein Expression and Purification. 20:237-251 |
| ISSN: | 1046-5928 |
| DOI: | 10.1006/prep.2000.1311 |
| Popis: | Protein l-isoaspartate (d-aspartate) O-methyltransferase (EC 2.1.1. 77) is a repair enzyme that methylates abnormal l-isoaspartate residues in proteins which arise spontaneously as a result of aging. This enzyme initiates their conversion back into the normal l-aspartate form by a methyl esterification reaction. Previously, partial cDNAs of this enzyme were isolated from the higher plant Arabidopsis thaliana. In this study, we report the cloning and expression of a full-length cDNA of l-isoaspartyl methyltransferase from A. thaliana into Escherichia coli under the P(BAD) promoter, which offers a high level of expression under a tight regulatory control. The enzyme is found largely in the soluble fraction. We purified this recombinant enzyme to homogeneity using a series of steps involving DEAE-cellulose, gel filtration, and hydrophobic interaction chromatographies. The homogeneous enzyme was found to have maximum activity at 45 degrees C and a pH optimum from 7 to 8. The enzyme was found to have a wide range of affinities for l-isoaspartate-containing peptides and displayed relatively poor reactivity toward protein substrates. The best methyl-accepting substrates were KASA-l-isoAsp-LAKY (K(m) = 80 microM) and VYP-l-isoAsp-HA (K(m) = 310 microM). We also expressed the full-length form and a truncated version of this enzyme (lacking the N-terminal 26 amino acid residues) in E. coli under the T7 promoter. Both the full-length and the truncated forms were active, though overexpression of the truncated enzyme led to a complete loss of activity. |
| Databáze: | OpenAIRE |
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