Enhanced Production of a Thermostable Carbonic Anhydrase in <i>Escherichia coli</i> by Using a Modified NEXT Tag
Carbonic anhydrase (CA) is an ultrafast enzyme that catalyzes the reversible conversion of carbon dioxide (CO<sub>2</sub>) to bicarbonate. CA is considered to be a green catalyst for enzyme-based CO<sub>2</sub> capture and utilization. In particular, the CA of <i>Thermo...
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| Format: | Article |
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MDPI AG
2021-09-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/26/19/5830 |
| _version_ | 1797515945013411840 |
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| author | In Seong Hwang Joo Hyeon Kim Byung Hoon Jo |
| author_facet | In Seong Hwang Joo Hyeon Kim Byung Hoon Jo |
| author_sort | In Seong Hwang |
| collection | DOAJ |
| description | Carbonic anhydrase (CA) is an ultrafast enzyme that catalyzes the reversible conversion of carbon dioxide (CO<sub>2</sub>) to bicarbonate. CA is considered to be a green catalyst for enzyme-based CO<sub>2</sub> capture and utilization. In particular, the CA of <i>Thermovibrio ammonificans</i> (taCA) has attracted increasing attention as a highly stable enzyme. However, the poor solubility and the low expression level in <i>Escherichia coli</i> have hampered further utilization of taCA. In a recent study, these limitations were partly resolved by using a small solubility-enhancing fusion tag named NEXT, which originates from the N-terminal extension of <i>Hydrogenovibrio marinus</i> CA. In this study, the NEXT tag was engineered by adding small peptides to the N terminus to further increase the production yield of NEXT-tagged taCA. The addition of ng3 peptide (His-Gly-Asn) originating from the N-terminal sequence of <i>Neisseria gonorrhoeae</i> CA improved the expression of NEXT-taCA, while the previously developed translation-enhancing element (TEE) and Ser-Lys-Ile-Lys (SKIK) tag were not effective. The expression test with all 16 codon combinations for the ng3 sequence revealed that the change in translation initiation rate brought about by the change in nucleotide sequence was not the primary determinant for the change in expression level. The modified ng3-NEXT tag may be applied to increase the production yields of various recombinant proteins. |
| first_indexed | 2024-03-10T06:55:23Z |
| format | Article |
| id | doaj.art-c6f0173786dc44e1bb2d72b17f504841 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-10T06:55:23Z |
| publishDate | 2021-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-c6f0173786dc44e1bb2d72b17f5048412023-11-22T16:33:18ZengMDPI AGMolecules1420-30492021-09-012619583010.3390/molecules26195830Enhanced Production of a Thermostable Carbonic Anhydrase in <i>Escherichia coli</i> by Using a Modified NEXT TagIn Seong Hwang0Joo Hyeon Kim1Byung Hoon Jo2Division of Applied Life Science, Gyeongsang National University, Jinju 52828, KoreaDivision of Life Science, Gyeongsang National University, Jinju 52828, KoreaDivision of Life Science and Research Institute of Life Science, Gyeongsang National University, Jinju 52828, KoreaCarbonic anhydrase (CA) is an ultrafast enzyme that catalyzes the reversible conversion of carbon dioxide (CO<sub>2</sub>) to bicarbonate. CA is considered to be a green catalyst for enzyme-based CO<sub>2</sub> capture and utilization. In particular, the CA of <i>Thermovibrio ammonificans</i> (taCA) has attracted increasing attention as a highly stable enzyme. However, the poor solubility and the low expression level in <i>Escherichia coli</i> have hampered further utilization of taCA. In a recent study, these limitations were partly resolved by using a small solubility-enhancing fusion tag named NEXT, which originates from the N-terminal extension of <i>Hydrogenovibrio marinus</i> CA. In this study, the NEXT tag was engineered by adding small peptides to the N terminus to further increase the production yield of NEXT-tagged taCA. The addition of ng3 peptide (His-Gly-Asn) originating from the N-terminal sequence of <i>Neisseria gonorrhoeae</i> CA improved the expression of NEXT-taCA, while the previously developed translation-enhancing element (TEE) and Ser-Lys-Ile-Lys (SKIK) tag were not effective. The expression test with all 16 codon combinations for the ng3 sequence revealed that the change in translation initiation rate brought about by the change in nucleotide sequence was not the primary determinant for the change in expression level. The modified ng3-NEXT tag may be applied to increase the production yields of various recombinant proteins.https://www.mdpi.com/1420-3049/26/19/5830recombinant proteinsolubility enhancerNEXT tagcarbonic anhydrase<i>Thermovibrio ammonificans</i>CO<sub>2</sub> capture |
| spellingShingle | In Seong Hwang Joo Hyeon Kim Byung Hoon Jo Enhanced Production of a Thermostable Carbonic Anhydrase in <i>Escherichia coli</i> by Using a Modified NEXT Tag Molecules recombinant protein solubility enhancer NEXT tag carbonic anhydrase <i>Thermovibrio ammonificans</i> CO<sub>2</sub> capture |
| title | Enhanced Production of a Thermostable Carbonic Anhydrase in <i>Escherichia coli</i> by Using a Modified NEXT Tag |
| title_full | Enhanced Production of a Thermostable Carbonic Anhydrase in <i>Escherichia coli</i> by Using a Modified NEXT Tag |
| title_fullStr | Enhanced Production of a Thermostable Carbonic Anhydrase in <i>Escherichia coli</i> by Using a Modified NEXT Tag |
| title_full_unstemmed | Enhanced Production of a Thermostable Carbonic Anhydrase in <i>Escherichia coli</i> by Using a Modified NEXT Tag |
| title_short | Enhanced Production of a Thermostable Carbonic Anhydrase in <i>Escherichia coli</i> by Using a Modified NEXT Tag |
| title_sort | enhanced production of a thermostable carbonic anhydrase in i escherichia coli i by using a modified next tag |
| topic | recombinant protein solubility enhancer NEXT tag carbonic anhydrase <i>Thermovibrio ammonificans</i> CO<sub>2</sub> capture |
| url | https://www.mdpi.com/1420-3049/26/19/5830 |
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