Codon optimization of a gene encoding DNA polymerase from Pyrococcus furiosus and its expression in Escherichia coli.
Codon optimization
Polymerase activity
Purification
Recombinant enzyme
Journal
Journal, genetic engineering & biotechnology
ISSN: 2090-5920
Titre abrégé: J Genet Eng Biotechnol
Pays: Netherlands
ID NLM: 101317150
Informations de publication
Date de publication:
21 Nov 2023
21 Nov 2023
Historique:
received:
03
05
2023
accepted:
14
11
2023
medline:
21
11
2023
pubmed:
21
11
2023
entrez:
21
11
2023
Statut:
epublish
Résumé
DNA polymerase is an essential component in PCR assay for DNA synthesis. Improving DNA polymerase with characteristics indispensable for a powerful assay is crucial because it can be used in wide-range applications. Derived from Pyrococcus furiosus, Pfu DNA polymerase (Pfu pol) is one of the excellent polymerases due to its high fidelity. Therefore, we aimed to develop Pfu pol from a synthetic gene with codon optimization to increase its protein yield in Escherichia coli. Recombinant Pfu pol was successfully expressed and purified with a two-step purification process using nickel affinity chromatography, followed by anion exchange chromatography. Subsequently, the purified Pfu pol was confirmed by Western blot analysis, resulting in a molecular weight of approximately 90 kDa. In the final purification process, we successfully obtained a large amount of purified enzyme (26.8 mg/L). Furthermore, the purified Pfu pol showed its functionality and efficiency when tested for DNA amplification using the standard PCR. Overall, a high-level expression of recombinant Pfu pol was achieved by employing our approach in the present study. In the future, our findings will be useful for studies on synthesizing recombinant DNA polymerase in E. coli expression system.
Sections du résumé
BACKGROUND
BACKGROUND
DNA polymerase is an essential component in PCR assay for DNA synthesis. Improving DNA polymerase with characteristics indispensable for a powerful assay is crucial because it can be used in wide-range applications. Derived from Pyrococcus furiosus, Pfu DNA polymerase (Pfu pol) is one of the excellent polymerases due to its high fidelity. Therefore, we aimed to develop Pfu pol from a synthetic gene with codon optimization to increase its protein yield in Escherichia coli.
RESULTS
RESULTS
Recombinant Pfu pol was successfully expressed and purified with a two-step purification process using nickel affinity chromatography, followed by anion exchange chromatography. Subsequently, the purified Pfu pol was confirmed by Western blot analysis, resulting in a molecular weight of approximately 90 kDa. In the final purification process, we successfully obtained a large amount of purified enzyme (26.8 mg/L). Furthermore, the purified Pfu pol showed its functionality and efficiency when tested for DNA amplification using the standard PCR.
CONCLUSIONS
CONCLUSIONS
Overall, a high-level expression of recombinant Pfu pol was achieved by employing our approach in the present study. In the future, our findings will be useful for studies on synthesizing recombinant DNA polymerase in E. coli expression system.
Identifiants
pubmed: 37987973
doi: 10.1186/s43141-023-00605-7
pii: 10.1186/s43141-023-00605-7
pmc: PMC10663413
doi:
Types de publication
Journal Article
Langues
eng
Pagination
129Subventions
Organisme : Kementerian Riset dan Teknologi /Badan Riset dan Inovasi Nasional
ID : 45/A/DH/2021
Informations de copyright
© 2023. The Author(s).
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