23
DOI: https://doi.org/10.1186/s43141-023-00605-7
English
eng
Nuryana, Isa
Contributor
<p><ul id="issue-navigation" class="issue-navigation u-margin-s-bottom u-bg-grey1" style="box-sizing: border-box; margin-top: 0px; margin-right: 0px; margin-bottom: 16px !important; margin-left: 0px; padding: 0px; list-style: none; background-color: rgb(245, 245, 245) !important; overflow: hidden; font-size: 16px; line-height: 24px; color: rgb(31, 31, 31); font-family: ElsevierSans, Arial, Helvetica, Roboto, "Lucida Sans Unicode", "Microsoft Sans Serif", "Segoe UI Symbol", STIXGeneral, "Cambria Math", "Arial Unicode MS", sans-serif; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; orphans: 2; text-align: start; text-indent: 0px; text-transform: none; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; white-space: normal; text-decoration-thickness: initial; text-decoration-style: initial; text-decoration-color: initial;"><li class="previous move-left u-padding-s-ver u-padding-s-left" style="box-sizing: border-box; margin: 0px; padding-top: 16px !important; padding-right: 0px; padding-bottom: 16px !important; padding-left: 16px !important; float: left; left: 0px;"><a class="button-alternative button-alternative-tertiary u-display-flex button-alternative-icon-left" href="https://www.sciencedirect.com/science/article/pii/S1687157X23009927" style="box-sizing: border-box; margin: 0px; padding: 0px; background-color: rgba(0, 0, 0, 0); word-break: break-word; --sd-ui-button-alt-border-bottom-colour: transparent; --sd-ui-button-alt-border-bottom-colour-hover: #eb6500; --sd-ui-button-alt-border-bottom-width: 2px; --sd-ui-button-alt-colour: #1f1f1f; --sd-ui-button-alt-colour-focus: #eb6500; --sd-ui-button-alt-cursor: pointer; --sd-ui-button-alt-font-size: 1em; --sd-ui-button-alt-gap: calc(0.5em - 2px); --sd-ui-button-alt-min-width: 5.5rem; --sd-ui-button-alt-icon-background-colour: transparent; --sd-ui-button-alt-icon-background-colour-hover: transparent; --sd-ui-button-alt-icon-border-colour: #1f1f1f; --sd-ui-button-alt-icon-border-colour-hover: #eb6500; --sd-ui-button-alt-icon-fill: #1f1f1f; --sd-ui-button-alt-icon-fill-hover: #1f1f1f; --sd-ui-button-alt-icon-padding: 0.5rem; --sd-ui-button-alt-icon-size: 2.5rem; --sd-ui-button-alt-icon-external-link-size: 0.625rem; -webkit-box-align: baseline; align-items: baseline; border-width: medium; border-style: none; border-color: currentcolor; border-image: initial; color: rgb(31, 31, 31); cursor: pointer; display: flex !important; font-family: inherit; font-size: 16px; gap: 6px; line-height: 24px; min-width: 88px; text-decoration: none; user-select: text;"><svg focusable="false" viewBox="0 0 54 128" height="20" class="icon icon-navigate-left"><path d="M1 61l45-45 7 7-38 38 38 38-7 7z"></path></svg></a></li></ul></p><div class="Abstracts u-font-serif" id="abstracts" style="box-sizing: border-box; margin: 0px; padding: 0px; --sd-ui-line-height: calc(1em + 10px); line-height: 26px; font-family: ElsevierGulliver, Georgia, "Times New Roman", Times, STIXGeneral, "Cambria Math", "Lucida Sans Unicode", "Microsoft Sans Serif", "Segoe UI Symbol", "Arial Unicode MS", serif, sans-serif !important; color: rgb(31, 31, 31); font-size: 16px; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; orphans: 2; text-align: start; text-indent: 0px; text-transform: none; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; white-space: normal; text-decoration-thickness: initial; text-decoration-style: initial; text-decoration-color: initial;"><div class="abstract author" id="ab0005" style="box-sizing: border-box; margin: 0px 0px 8px; padding: 0px;"><div id="abs0005" style="box-sizing: border-box; margin: 0px; padding: 0px;"><h3 class="u-h4 u-margin-m-top u-margin-xs-bottom" id="sect0010" style="box-sizing: border-box; margin-top: 24px !important; margin-right: 0px; margin-bottom: 8px !important; margin-left: 0px; padding: 0px; font-weight: normal !important; font-size: 20px !important; line-height: 30px !important;">Background</h3><div class="u-margin-s-bottom" id="sp0050" style="box-sizing: border-box; margin-top: 0px; margin-right: 0px; margin-bottom: 16px !important; margin-left: 0px; padding: 0px;"><span style="box-sizing: border-box; margin: 0px; padding: 0px;"><span style="box-sizing: border-box; margin: 0px; padding: 0px;"><a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/dna-polymerase" class="topic-link" style="box-sizing: border-box; margin: 0px; padding: 0px; background-color: rgba(0, 0, 0, 0); word-break: break-word; text-decoration: underline 1px rgb(31, 31, 31); color: rgb(31, 31, 31); text-underline-offset: 1px;">DNA polymerase</a><span style="box-sizing: border-box; margin: 0px; padding: 0px;"><span> </span>is an essential component in<span> </span><a href="https://www.sciencedirect.com/topics/immunology-and-microbiology/polymerase-chain-reaction" class="topic-link" style="box-sizing: border-box; margin: 0px; padding: 0px; background-color: rgba(0, 0, 0, 0); word-break: break-word; text-decoration: underline 1px rgb(31, 31, 31); color: rgb(31, 31, 31); text-underline-offset: 1px;">PCR</a><span> </span>assay for<span> </span></span></span><a href="https://www.sciencedirect.com/topics/immunology-and-microbiology/dna-synthesis" class="topic-link" style="box-sizing: border-box; margin: 0px; padding: 0px; background-color: rgba(0, 0, 0, 0); word-break: break-word; text-decoration: underline 1px rgb(31, 31, 31); color: rgb(31, 31, 31); text-underline-offset: 1px;">DNA synthesis</a><span style="box-sizing: border-box; margin: 0px; padding: 0px;">. Improving<span> </span><a href="https://www.sciencedirect.com/topics/immunology-and-microbiology/dna-polymerase" class="topic-link" style="box-sizing: border-box; margin: 0px; padding: 0px; background-color: rgba(0, 0, 0, 0); word-break: break-word; text-decoration: underline 1px rgb(31, 31, 31); color: rgb(31, 31, 31); text-underline-offset: 1px;">DNA polymerase</a><span> </span>with characteristics indispensable for a powerful assay is crucial because it can be used in wide-range applications. Derived from<span> </span></span></span><span style="box-sizing: border-box; margin: 0px; padding: 0px;"><span style="box-sizing: border-box; margin: 0px; padding: 0px;"><a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/pyrococcus-furiosus" class="topic-link" style="box-sizing: border-box; margin: 0px; padding: 0px; background-color: rgba(0, 0, 0, 0); word-break: break-word; text-decoration: underline 1px rgb(31, 31, 31); color: rgb(31, 31, 31); text-underline-offset: 1px;">Pyrococcus furiosus</a></span><span style="box-sizing: border-box; margin: 0px; padding: 0px;"><em style="box-sizing: border-box; margin: 0px; padding: 0px;">,<span> </span></em><em style="box-sizing: border-box; margin: 0px; padding: 0px;"><a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/phenformin" title="Learn more about Pfu from ScienceDirect's AI-generated Topic Pages" class="topic-link" style="box-sizing: border-box; margin: 0px; padding: 0px; background-color: rgba(0, 0, 0, 0); word-break: break-word; text-decoration: underline 1px rgb(31, 31, 31); color: rgb(31, 31, 31); text-underline-offset: 1px;">Pfu</a></em></span></span><span style="box-sizing: border-box; margin: 0px; padding: 0px;"><span> </span><a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/dna-polymerase" class="topic-link" style="box-sizing: border-box; margin: 0px; padding: 0px; background-color: rgba(0, 0, 0, 0); word-break: break-word; text-decoration: underline 1px rgb(31, 31, 31); color: rgb(31, 31, 31); text-underline-offset: 1px;">DNA polymerase</a><span> </span>(</span><em style="box-sizing: border-box; margin: 0px; padding: 0px;">Pfu</em><span style="box-sizing: border-box; margin: 0px; padding: 0px;"><span> </span>pol) is one of the excellent<span> </span><a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/polymerase" class="topic-link" style="box-sizing: border-box; margin: 0px; padding: 0px; background-color: rgba(0, 0, 0, 0); word-break: break-word; text-decoration: underline 1px rgb(31, 31, 31); color: rgb(31, 31, 31); text-underline-offset: 1px;">polymerases</a><span> </span>due to its high fidelity. Therefore, we aimed to develop<span> </span></span><em style="box-sizing: border-box; margin: 0px; padding: 0px;">Pfu</em><span style="box-sizing: border-box; margin: 0px; padding: 0px;"><span> </span>pol from a<span> </span><a href="https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/synthetic-dna" class="topic-link" style="box-sizing: border-box; margin: 0px; padding: 0px; background-color: rgba(0, 0, 0, 0); word-break: break-word; text-decoration: underline 1px rgb(31, 31, 31); color: rgb(31, 31, 31); text-underline-offset: 1px;">synthetic gene</a><span> </span>with codon optimization to increase its protein yield in<span> </span></span><em style="box-sizing: border-box; margin: 0px; padding: 0px;">Escherichia coli</em>.</div></div><div id="abs0010" style="box-sizing: border-box; margin: 0px; padding: 0px;"><h3 class="u-h4 u-margin-m-top u-margin-xs-bottom" id="sect0015" style="box-sizing: border-box; margin-top: 24px !important; margin-right: 0px; margin-bottom: 8px !important; margin-left: 0px; padding: 0px; font-weight: normal !important; font-size: 20px !important; line-height: 30px !important;">Results</h3><div class="u-margin-s-bottom" id="sp0055" style="box-sizing: border-box; margin-top: 0px; margin-right: 0px; margin-bottom: 16px !important; margin-left: 0px; padding: 0px;">Recombinant<span> </span><em style="box-sizing: border-box; margin: 0px; padding: 0px;">Pfu</em><span style="box-sizing: border-box; margin: 0px; padding: 0px;"><span> </span>pol was successfully expressed and purified with a two-step<span> </span><a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/copurification" class="topic-link" style="box-sizing: border-box; margin: 0px; padding: 0px; background-color: rgba(0, 0, 0, 0); word-break: break-word; text-decoration: underline 1px rgb(31, 31, 31); color: rgb(31, 31, 31); text-underline-offset: 1px;">purification</a><span style="box-sizing: border-box; margin: 0px; padding: 0px;"><span style="box-sizing: border-box; margin: 0px; padding: 0px;"><span> </span>process using nickel<span> </span><a href="https://www.sciencedirect.com/topics/immunology-and-microbiology/affinity-chromatography" class="topic-link" style="box-sizing: border-box; margin: 0px; padding: 0px; background-color: rgba(0, 0, 0, 0); word-break: break-word; text-decoration: underline 1px rgb(31, 31, 31); color: rgb(31, 31, 31); text-underline-offset: 1px;">affinity chromatography</a>, followed by<span> </span></span><a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/anion-exchange-chromatography" class="topic-link" style="box-sizing: border-box; margin: 0px; padding: 0px; background-color: rgba(0, 0, 0, 0); word-break: break-word; text-decoration: underline 1px rgb(31, 31, 31); color: rgb(31, 31, 31); text-underline-offset: 1px;">anion exchange chromatography</a>. Subsequently, the purified<span> </span></span></span><em style="box-sizing: border-box; margin: 0px; padding: 0px;">Pfu</em><span style="box-sizing: border-box; margin: 0px; padding: 0px;"><span> </span>pol was confirmed by<span> </span><a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/western-blot" class="topic-link" style="box-sizing: border-box; margin: 0px; padding: 0px; background-color: rgba(0, 0, 0, 0); word-break: break-word; text-decoration: underline 1px rgb(31, 31, 31); color: rgb(31, 31, 31); text-underline-offset: 1px;">Western blot analysis</a><span style="box-sizing: border-box; margin: 0px; padding: 0px;">, resulting in a molecular weight of approximately 90 kDa. In the final purification process, we successfully obtained a large amount of purified<span> </span><a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/enzyme" class="topic-link" style="box-sizing: border-box; margin: 0px; padding: 0px; background-color: rgba(0, 0, 0, 0); word-break: break-word; text-decoration: underline 1px rgb(31, 31, 31); color: rgb(31, 31, 31); text-underline-offset: 1px;">enzyme</a><span> </span>(26.8 mg/L). Furthermore, the purified<span> </span></span></span><em style="box-sizing: border-box; margin: 0px; padding: 0px;">Pfu</em><span style="box-sizing: border-box; margin: 0px; padding: 0px;"><span> </span>pol showed its functionality and efficiency when tested for<span> </span><a href="https://www.sciencedirect.com/topics/immunology-and-microbiology/gene-amplification" class="topic-link" style="box-sizing: border-box; margin: 0px; padding: 0px; background-color: rgba(0, 0, 0, 0); word-break: break-word; text-decoration: underline 1px rgb(31, 31, 31); color: rgb(31, 31, 31); text-underline-offset: 1px;">DNA amplification</a><span style="box-sizing: border-box; margin: 0px; padding: 0px;"><span> </span>using the standard<span> </span><a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/polymerase-chain-reaction" class="topic-link" style="box-sizing: border-box; margin: 0px; padding: 0px; background-color: rgba(0, 0, 0, 0); word-break: break-word; text-decoration: underline 1px rgb(31, 31, 31); color: rgb(31, 31, 31); text-underline-offset: 1px;">PCR</a>.</span></span></div></div><div id="abs0015" style="box-sizing: border-box; margin: 0px; padding: 0px;"><h3 class="u-h4 u-margin-m-top u-margin-xs-bottom" id="sect0020" style="box-sizing: border-box; margin-top: 24px !important; margin-right: 0px; margin-bottom: 8px !important; margin-left: 0px; padding: 0px; font-weight: normal !important; font-size: 20px !important; line-height: 30px !important;">Conclusions</h3><div class="u-margin-s-bottom" id="sp0060" style="box-sizing: border-box; margin-top: 0px; margin-right: 0px; margin-bottom: 16px !important; margin-left: 0px; padding: 0px;">Overall, a high-level expression of recombinant<span> </span><em style="box-sizing: border-box; margin: 0px; padding: 0px;">Pfu</em><span style="box-sizing: border-box; margin: 0px; padding: 0px;"><span> </span>pol was achieved by employing our approach in the present study. In the future, our findings will be useful for studies on synthesizing<span> </span><a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/recombinant-dna" class="topic-link" style="box-sizing: border-box; margin: 0px; padding: 0px; background-color: rgba(0, 0, 0, 0); word-break: break-word; text-decoration: underline 1px rgb(31, 31, 31); color: rgb(31, 31, 31); text-underline-offset: 1px;">recombinant DNA</a><span> </span>polymerase in<span> </span></span><em style="box-sizing: border-box; margin: 0px; padding: 0px;">E. coli</em><span style="box-sizing: border-box; margin: 0px; padding: 0px;"><span> </span><a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/gene-expression-system" class="topic-link" style="box-sizing: border-box; margin: 0px; padding: 0px; background-color: rgba(0, 0, 0, 0); word-break: break-word; text-decoration: underline 1px rgb(31, 31, 31); color: rgb(31, 31, 31); text-underline-offset: 1px;">expression system</a>.</span></div></div></div></div>
Elsevier
December 2023
pdf
Journal of Genetic Engineering and Biotechnology
Codon optimization of a gene encoding DNA polymerase from Pyrococcus furiosus and its expression in Escherichia coli
mixed material
Elsevier