Chaperone-assisted expression and purification of putrescine monooxygenase from Shewanella putrefaciens-95
| Autor: | Anil H. Shyam Mohan, D. Srividya, Narsing Rao Saroja, K. Supreetha |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
Shewanella putrefaciens Erwinia Hydroxylation medicine.disease_cause 01 natural sciences Streptomyces Mixed Function Oxygenases 03 medical and health sciences chemistry.chemical_compound Bacterial Proteins 010608 biotechnology Escherichia coli Putrescine medicine Amino Acid Sequence Cloning Molecular 030304 developmental biology Shewanella baltica 0303 health sciences Expression vector biology biology.organism_classification Recombinant Proteins Biochemistry chemistry Flavin-Adenine Dinucleotide bacteria Heterologous expression Sequence Alignment NADP Molecular Chaperones Biotechnology |
| Zdroj: | Protein Expression and Purification. 157:9-16 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2019.01.006 |
| Popis: | A putrescine monooxygenase from Shewanella putrefaciens 95 (SpPMO) is the initial enzyme catalyzing the hydroxylation of putrescine to N-hydroxyl putrescine, the precursor for the synthesis of a siderophore putrebactin was identified. This PMO clustered together with known characterized NMOs from Shewanella baltica, Bordetella pertussis, Erwinia amylovora, Streptomyces sp. Gordonia rubripertincta, Pseudomonas aeruginosa and outgrouped from Escherichia coli, Nocardia farcinica, and Rhodococcus erythropolis. The deduced SpPMO protein showed 53% and 36% sequence identity with other characterized bacterial NMOs from Erwinia amylovora and Gordonia rubripertincta respectively. In this investigation, we have cloned the complete 1518bp coding sequence of pubA from Shewanella putrefaciens 95 encoding the corresponding protein SpPMO. It comprises 505 amino acid residues in length and has approximately a molecular weight of 54 kDa. Chaperone-assisted heterologous expression of SpPMO in pET151Topo expression vector under the control of bacteriophage T7 promoter permitted a stringent IPTG dependent expression. It has been successfully cloned, overexpressed and purified as a soluble His6 –tagged enzyme using E. coli as a cloning and expression host. The expression of recombinant SpPMO was confirmed by Western blotting using anti-His6 antibody. The purified protein showed FAD and NADPH dependent N-hydroxylation activity. This study has paved a way to understand the hydroxylation step of putrebactin synthesis which can be further investigated by studying its kinetic mechanism and physiological role. |
| Databáze: | OpenAIRE |
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