Expression of thermostable MMLV reverse transcriptase in Escherichia coli by directed mutation.
Murine Moloney Leukemia Virus (MMLV)
Recombinant protein expression
Reverse transcriptase
Sequence optimization
Site-directed mutagenesis
Thermostability
Journal
AMB Express
ISSN: 2191-0855
Titre abrégé: AMB Express
Pays: Germany
ID NLM: 101561785
Informations de publication
Date de publication:
03 Oct 2024
03 Oct 2024
Historique:
received:
21
08
2024
accepted:
19
09
2024
medline:
4
10
2024
pubmed:
4
10
2024
entrez:
3
10
2024
Statut:
epublish
Résumé
The functionality of Moloney murine leukemia virus reverse transcriptase (MMLV RT) will increase with the improvement of its solubility and thermal stability. Introduce directed mutation at specific positions of the MMLV RT sequence and codon optimization is needed to achieve these properties. The two RT coding sequences with (rRT-K) and without directed mutations (rRT-L) were versatility optimized and expressed to analyze the ribonuclease H (RNase H) inactivity and thermostable polymerase activity. For this purpose, the five-point mutations (438-442aa) and three-point mutations (530, 568, and 659 aa) were done at the RT connection domain and RNase H active site, respectively. High expression levels of rRT-L and rRT-K were obtained in E. coli BL21(DE3) and BL21(shuffle) strains, 0.5 mM IPTG concentration at 37 °C, and 8 hours' post-induction condition. Then, recombinant enzymes were purified and verified by Ni-NTA resin and western blotting. Insilico analysis (IUpred 3.0) showed that the directed mutation in the RNase H domain caused the formation of disorder regions or instability in the RNase H domain of rRT-K compared to rRT-L. The modified RT-PCR and the RT-LAMP reactions proved the RNase H inactivity of rRT-K. In addition, increasing of thermostability of rRT-K compared to rRT-L and commercial RT was evaluated by the RT-PCR and RT-LAMP reactions. The results showed that rRT-K could successfully tolerate 60 ºC in the two methods. This study revealed that the directed mutations and the versatile sequence optimization can promise to produce thermostable commercial enzymes to decrease non-specific one-step RT-PCR and RT-LAMP products.
Identifiants
pubmed: 39363125
doi: 10.1186/s13568-024-01773-6
pii: 10.1186/s13568-024-01773-6
doi:
Types de publication
Journal Article
Langues
eng
Pagination
113Subventions
Organisme : Golestan University of Medical Sciences
ID : Golestan University of Medical Sciences
Informations de copyright
© 2024. The Author(s).
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