Optimization of Conditions for Production of Soluble <i>E. coli</i> Poly(A)-Polymerase for Biotechnological Applications
Poly(A) polymerase (PAP 1) from <i>Escherichia coli</i> is the primary enzyme responsible for synthesizing poly(A) tails on RNA molecules, signaling RNA degradation in bacterial cells. In vitro, PAP 1 is used to prepare libraries for RNAseq and to produce mRNA vaccines. However, <i>...
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2025-01-01
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| author | Igor P. Oscorbin Maria S. Kunova Maxim L. Filipenko |
| author_facet | Igor P. Oscorbin Maria S. Kunova Maxim L. Filipenko |
| author_sort | Igor P. Oscorbin |
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| description | Poly(A) polymerase (PAP 1) from <i>Escherichia coli</i> is the primary enzyme responsible for synthesizing poly(A) tails on RNA molecules, signaling RNA degradation in bacterial cells. In vitro, PAP 1 is used to prepare libraries for RNAseq and to produce mRNA vaccines. However, <i>E. coli</i> PAP 1’s toxicity and instability in low-salt buffers complicate its expression and purification. Here, we optimized the conditions for the production of recombinant PAP 1. For that, <i>E. coli</i> PAP 1 was expressed in seven <i>E. coli</i> strains with different origins and genetic backgrounds, followed by assessment of the overall protein yield, solubility, and enzymatic activity. Among the tested strains, BL21 (DE3) pLysS achieved the best balance of cell density, total PAP 1 yield, solubility, and specific activity. Rosetta 2 (DE3) and Rosetta Blue (DE3) hosting the pRARE plasmid exhibited the lowest solubility, likely due to excessive translation efficiency. Higher induction temperatures (>18 °C) exacerbated PAP 1’s insolubility. Interestingly, PAP 1 accumulation correlated with an increase in the plasmid copy number encoding the enzyme, indicating its potential utility as a surrogate marker of PAP 1 activity. These findings provide insights into optimizing <i>E. coli</i> PAP 1 production for biotechnological applications. |
| format | Article |
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| institution | Kabale University |
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| language | English |
| publishDate | 2025-01-01 |
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| spelling | doaj-art-2768f0e15e354744947182284d314ccc2025-01-24T13:23:25ZengMDPI AGBiology2079-77372025-01-011414810.3390/biology14010048Optimization of Conditions for Production of Soluble <i>E. coli</i> Poly(A)-Polymerase for Biotechnological ApplicationsIgor P. Oscorbin0Maria S. Kunova1Maxim L. Filipenko2Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (ICBFM SB RAS), 8, Lavrentiev Avenue, Novosibirsk 630090, RussiaInstitute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (ICBFM SB RAS), 8, Lavrentiev Avenue, Novosibirsk 630090, RussiaInstitute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (ICBFM SB RAS), 8, Lavrentiev Avenue, Novosibirsk 630090, RussiaPoly(A) polymerase (PAP 1) from <i>Escherichia coli</i> is the primary enzyme responsible for synthesizing poly(A) tails on RNA molecules, signaling RNA degradation in bacterial cells. In vitro, PAP 1 is used to prepare libraries for RNAseq and to produce mRNA vaccines. However, <i>E. coli</i> PAP 1’s toxicity and instability in low-salt buffers complicate its expression and purification. Here, we optimized the conditions for the production of recombinant PAP 1. For that, <i>E. coli</i> PAP 1 was expressed in seven <i>E. coli</i> strains with different origins and genetic backgrounds, followed by assessment of the overall protein yield, solubility, and enzymatic activity. Among the tested strains, BL21 (DE3) pLysS achieved the best balance of cell density, total PAP 1 yield, solubility, and specific activity. Rosetta 2 (DE3) and Rosetta Blue (DE3) hosting the pRARE plasmid exhibited the lowest solubility, likely due to excessive translation efficiency. Higher induction temperatures (>18 °C) exacerbated PAP 1’s insolubility. Interestingly, PAP 1 accumulation correlated with an increase in the plasmid copy number encoding the enzyme, indicating its potential utility as a surrogate marker of PAP 1 activity. These findings provide insights into optimizing <i>E. coli</i> PAP 1 production for biotechnological applications.https://www.mdpi.com/2079-7737/14/1/48<i>pcnB</i>poly(A) polymerasePAP 1polyadenylation<i>E. coli</i>recombinant protein |
| spellingShingle | Igor P. Oscorbin Maria S. Kunova Maxim L. Filipenko Optimization of Conditions for Production of Soluble <i>E. coli</i> Poly(A)-Polymerase for Biotechnological Applications Biology <i>pcnB</i> poly(A) polymerase PAP 1 polyadenylation <i>E. coli</i> recombinant protein |
| title | Optimization of Conditions for Production of Soluble <i>E. coli</i> Poly(A)-Polymerase for Biotechnological Applications |
| title_full | Optimization of Conditions for Production of Soluble <i>E. coli</i> Poly(A)-Polymerase for Biotechnological Applications |
| title_fullStr | Optimization of Conditions for Production of Soluble <i>E. coli</i> Poly(A)-Polymerase for Biotechnological Applications |
| title_full_unstemmed | Optimization of Conditions for Production of Soluble <i>E. coli</i> Poly(A)-Polymerase for Biotechnological Applications |
| title_short | Optimization of Conditions for Production of Soluble <i>E. coli</i> Poly(A)-Polymerase for Biotechnological Applications |
| title_sort | optimization of conditions for production of soluble i e coli i poly a polymerase for biotechnological applications |
| topic | <i>pcnB</i> poly(A) polymerase PAP 1 polyadenylation <i>E. coli</i> recombinant protein |
| url | https://www.mdpi.com/2079-7737/14/1/48 |
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