High-Level Production of DNA-Specific Endonuclease AsEndI with Synonymous Codon and its Potential Utilization for Removing DNA Contamination.
Appl Biochem Biotechnol
; 185(3): 641-654, 2018 Jul.
Article
en En
| MEDLINE
| ID: mdl-29250754
ABSTRACT
Endonuclease I is a widely distributed periplasmic or extracellular enzyme. A method for the high-level production of recombinant AsEndI (endonuclease I from Aliivibrio salmonicida) in Escherichia coli with secretion expression is investigated. The coding sequence of AsEndI gene was assembled according to the E. coli codon usage bias, and AsEndI was expressed in the periplasm of E. coli TOP10 with a C-terminal 6× His-tagged fusion. The recombinant AsEndI (His-AsEndI) was purified by Ni-NTA resin with a yield of 1.29 × 107 U from 1-L LB medium. His-AsEndI could be classified into Ca2+/Mg2+-dependent nucleases and showed highest nuclease activity to dsDNA at pH 8.0 and 37 °C. His-AsEndI is highly active in a broad range of salt concentration range up to 1.0 M with optimal NaCl concentration at 0.4 M. His-AsEndI can effectively remove DNA contamination in RNA sample or in PCR reagents to the level that cannot be detected by highly sensitive nested PCR and without adverse effects on the subsequent PCR reaction. His-AsEndI can remove DNA contamination at high salt conditions, especially for the DNA that may be shielded by DNA-binding protein at low salt conditions.
Palabras clave
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Codón
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ADN de Cadena Simple
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Aliivibrio salmonicida
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Desoxirribonucleasa I
Idioma:
En
Año:
2018
Tipo del documento:
Article