Extraction of ADP-Heptose and Kdo<sub>2</sub>-Lipid A from <i>E. coli</i> Deficient in the Heptosyltransferase I Gene
The enzymes involved in lipopolysaccharide (LPS) biosynthesis, including Heptosyltransferase I (HepI), are critical for maintaining the integrity of the bacterial cell wall, and therefore these LPS biosynthetic enzymes are validated targets for drug discovery to treat Gram-negative bacterial infecti...
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MDPI AG
2021-09-01
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| Series: | Applied Sciences |
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| Online Access: | https://www.mdpi.com/2076-3417/11/18/8314 |
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| author | Jozafina Milicaj Colleen D. Castro Nadiya Jaunbocus Erika A. Taylor |
| author_facet | Jozafina Milicaj Colleen D. Castro Nadiya Jaunbocus Erika A. Taylor |
| author_sort | Jozafina Milicaj |
| collection | DOAJ |
| description | The enzymes involved in lipopolysaccharide (LPS) biosynthesis, including Heptosyltransferase I (HepI), are critical for maintaining the integrity of the bacterial cell wall, and therefore these LPS biosynthetic enzymes are validated targets for drug discovery to treat Gram-negative bacterial infections. Enzymes involved in the biosynthesis of lipopolysaccharides (LPSs) utilize substrates that are synthetically complex, with numerous stereocenters and site-specific glycosylation patterns. Due to the relatively complex substrate structures, characterization of these enzymes has necessitated strategies to generate bacterial cells with gene disruptions to enable the extraction of these substrates from large scale bacterial growths. Like many LPS biosynthetic enzymes, Heptosyltransferase I binds two substrates: the sugar acceptor substrate, Kdo<sub>2</sub>-Lipid A, and the sugar donor substrate, ADP-<span style="font-variant: small-caps;">l</span>-<i>glycero</i>-<span style="font-variant: small-caps;">d</span>-<i>manno</i>-heptose (ADPH). HepI characterization experiments require copious amounts of Kdo<sub>2</sub>-Lipid A and ADPH, and unsuccessful extractions of these two substrates can lead to serious delays in collection of data. While there are papers and theses with protocols for extraction of these substrates, they are often missing small details essential to the success of the extraction. Herein detailed protocols are given for extraction of ADPH and Kdo<sub>2</sub>-Lipid A (KLA) from <i>E. coli</i>, which have had proven success in the Taylor lab. Key steps in the extraction of ADPH are clearing the extract through ultracentrifugation and keeping all water that touches anything in the extraction, including filters, at a pH of 8.0. Key steps in the extraction of KLA are properly lysing the dried down cells before starting the extraction, maximizing yield by allowing precipitate to form overnight, appropriately washing the pellet with phenol and dissolving the KLA in 1% TEA using visual cues, rather than a specific volume. These protocols led to increased yield and a higher success rate of extractions thereby enabling the characterization of HepI. |
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| issn | 2076-3417 |
| language | English |
| last_indexed | 2024-03-10T07:56:15Z |
| publishDate | 2021-09-01 |
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| spelling | doaj.art-94665a32d82e41588a5f921d7ee1dae12023-11-22T11:50:45ZengMDPI AGApplied Sciences2076-34172021-09-011118831410.3390/app11188314Extraction of ADP-Heptose and Kdo<sub>2</sub>-Lipid A from <i>E. coli</i> Deficient in the Heptosyltransferase I GeneJozafina Milicaj0Colleen D. Castro1Nadiya Jaunbocus2Erika A. Taylor3Department of Chemistry, Wesleyan University, Middletown, CT 06459, USADepartment of Chemistry, Wesleyan University, Middletown, CT 06459, USADepartment of Chemistry, Wesleyan University, Middletown, CT 06459, USADepartment of Chemistry, Wesleyan University, Middletown, CT 06459, USAThe enzymes involved in lipopolysaccharide (LPS) biosynthesis, including Heptosyltransferase I (HepI), are critical for maintaining the integrity of the bacterial cell wall, and therefore these LPS biosynthetic enzymes are validated targets for drug discovery to treat Gram-negative bacterial infections. Enzymes involved in the biosynthesis of lipopolysaccharides (LPSs) utilize substrates that are synthetically complex, with numerous stereocenters and site-specific glycosylation patterns. Due to the relatively complex substrate structures, characterization of these enzymes has necessitated strategies to generate bacterial cells with gene disruptions to enable the extraction of these substrates from large scale bacterial growths. Like many LPS biosynthetic enzymes, Heptosyltransferase I binds two substrates: the sugar acceptor substrate, Kdo<sub>2</sub>-Lipid A, and the sugar donor substrate, ADP-<span style="font-variant: small-caps;">l</span>-<i>glycero</i>-<span style="font-variant: small-caps;">d</span>-<i>manno</i>-heptose (ADPH). HepI characterization experiments require copious amounts of Kdo<sub>2</sub>-Lipid A and ADPH, and unsuccessful extractions of these two substrates can lead to serious delays in collection of data. While there are papers and theses with protocols for extraction of these substrates, they are often missing small details essential to the success of the extraction. Herein detailed protocols are given for extraction of ADPH and Kdo<sub>2</sub>-Lipid A (KLA) from <i>E. coli</i>, which have had proven success in the Taylor lab. Key steps in the extraction of ADPH are clearing the extract through ultracentrifugation and keeping all water that touches anything in the extraction, including filters, at a pH of 8.0. Key steps in the extraction of KLA are properly lysing the dried down cells before starting the extraction, maximizing yield by allowing precipitate to form overnight, appropriately washing the pellet with phenol and dissolving the KLA in 1% TEA using visual cues, rather than a specific volume. These protocols led to increased yield and a higher success rate of extractions thereby enabling the characterization of HepI.https://www.mdpi.com/2076-3417/11/18/8314Heptosyltransferase Ilipopolysaccharide biosynthesisADP-HeptoseKdo<sub>2</sub>-Lipid A |
| spellingShingle | Jozafina Milicaj Colleen D. Castro Nadiya Jaunbocus Erika A. Taylor Extraction of ADP-Heptose and Kdo<sub>2</sub>-Lipid A from <i>E. coli</i> Deficient in the Heptosyltransferase I Gene Applied Sciences Heptosyltransferase I lipopolysaccharide biosynthesis ADP-Heptose Kdo<sub>2</sub>-Lipid A |
| title | Extraction of ADP-Heptose and Kdo<sub>2</sub>-Lipid A from <i>E. coli</i> Deficient in the Heptosyltransferase I Gene |
| title_full | Extraction of ADP-Heptose and Kdo<sub>2</sub>-Lipid A from <i>E. coli</i> Deficient in the Heptosyltransferase I Gene |
| title_fullStr | Extraction of ADP-Heptose and Kdo<sub>2</sub>-Lipid A from <i>E. coli</i> Deficient in the Heptosyltransferase I Gene |
| title_full_unstemmed | Extraction of ADP-Heptose and Kdo<sub>2</sub>-Lipid A from <i>E. coli</i> Deficient in the Heptosyltransferase I Gene |
| title_short | Extraction of ADP-Heptose and Kdo<sub>2</sub>-Lipid A from <i>E. coli</i> Deficient in the Heptosyltransferase I Gene |
| title_sort | extraction of adp heptose and kdo sub 2 sub lipid a from i e coli i deficient in the heptosyltransferase i gene |
| topic | Heptosyltransferase I lipopolysaccharide biosynthesis ADP-Heptose Kdo<sub>2</sub>-Lipid A |
| url | https://www.mdpi.com/2076-3417/11/18/8314 |
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