Transfer of a Rational Crystal Contact Engineering Strategy between Diverse Alcohol Dehydrogenases
Protein crystallization can serve as a purification step in biotechnological processes but is often limited by the non-crystallizability of proteins. Enabling or improving crystallization is mostly achieved by high-throughput screening of crystallization conditions and, more recently, by rational cr...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-08-01
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| Series: | Crystals |
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| Online Access: | https://www.mdpi.com/2073-4352/11/8/975 |
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| author | Brigitte Walla Daniel Bischoff Robert Janowski Nikolas von den Eichen Dierk Niessing Dirk Weuster-Botz |
| author_facet | Brigitte Walla Daniel Bischoff Robert Janowski Nikolas von den Eichen Dierk Niessing Dirk Weuster-Botz |
| author_sort | Brigitte Walla |
| collection | DOAJ |
| description | Protein crystallization can serve as a purification step in biotechnological processes but is often limited by the non-crystallizability of proteins. Enabling or improving crystallization is mostly achieved by high-throughput screening of crystallization conditions and, more recently, by rational crystal contact engineering. Two selected rational crystal contact mutations, Q126K and T102E, were transferred from the alcohol dehydrogenases of <i>Lactobacillus brevis</i> (<i>Lb</i>ADH) to <i>Lactobacillus kefir</i> (<i>Lk</i>ADH). Proteins were expressed in <i>E. coli</i> and batch protein crystallization was performed in stirred crystallizers. Highly similar crystal packing of <i>Lk</i>ADH wild type compared to <i>Lb</i>ADH, which is necessary for the transfer of crystal contact engineering strategies, was achieved by aligning purification tag and crystallization conditions, as shown by X-ray diffraction. After comparing the crystal sizes after crystallization of <i>Lk</i>ADH mutants with the wild type, the mean protein crystal size of <i>Lk</i>ADH mutants was reduced by 40–70% in length with a concomitant increase in the total amount of crystals (higher number of nucleation events). Applying this measure to the <i>Lk</i>ADH variants studied results in an order of crystallizability T102E > Q126K > <i>Lk</i>ADH wild type, which corresponds to the results with <i>Lb</i>ADH mutants and shows, for the first time, the successful transfer of crystal contact engineering strategies. |
| first_indexed | 2024-03-10T08:53:39Z |
| format | Article |
| id | doaj.art-770d49777e2147e592b97a912e3fb421 |
| institution | Directory Open Access Journal |
| issn | 2073-4352 |
| language | English |
| last_indexed | 2024-03-10T08:53:39Z |
| publishDate | 2021-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Crystals |
| spelling | doaj.art-770d49777e2147e592b97a912e3fb4212023-11-22T07:17:38ZengMDPI AGCrystals2073-43522021-08-0111897510.3390/cryst11080975Transfer of a Rational Crystal Contact Engineering Strategy between Diverse Alcohol DehydrogenasesBrigitte Walla0Daniel Bischoff1Robert Janowski2Nikolas von den Eichen3Dierk Niessing4Dirk Weuster-Botz5Institute of Biochemical Engineering, Technical University of Munich, Boltzmannstraße 15, 85748 Garching, GermanyInstitute of Biochemical Engineering, Technical University of Munich, Boltzmannstraße 15, 85748 Garching, GermanyInstitute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764 Neuherberg, GermanyInstitute of Biochemical Engineering, Technical University of Munich, Boltzmannstraße 15, 85748 Garching, GermanyInstitute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764 Neuherberg, GermanyInstitute of Biochemical Engineering, Technical University of Munich, Boltzmannstraße 15, 85748 Garching, GermanyProtein crystallization can serve as a purification step in biotechnological processes but is often limited by the non-crystallizability of proteins. Enabling or improving crystallization is mostly achieved by high-throughput screening of crystallization conditions and, more recently, by rational crystal contact engineering. Two selected rational crystal contact mutations, Q126K and T102E, were transferred from the alcohol dehydrogenases of <i>Lactobacillus brevis</i> (<i>Lb</i>ADH) to <i>Lactobacillus kefir</i> (<i>Lk</i>ADH). Proteins were expressed in <i>E. coli</i> and batch protein crystallization was performed in stirred crystallizers. Highly similar crystal packing of <i>Lk</i>ADH wild type compared to <i>Lb</i>ADH, which is necessary for the transfer of crystal contact engineering strategies, was achieved by aligning purification tag and crystallization conditions, as shown by X-ray diffraction. After comparing the crystal sizes after crystallization of <i>Lk</i>ADH mutants with the wild type, the mean protein crystal size of <i>Lk</i>ADH mutants was reduced by 40–70% in length with a concomitant increase in the total amount of crystals (higher number of nucleation events). Applying this measure to the <i>Lk</i>ADH variants studied results in an order of crystallizability T102E > Q126K > <i>Lk</i>ADH wild type, which corresponds to the results with <i>Lb</i>ADH mutants and shows, for the first time, the successful transfer of crystal contact engineering strategies.https://www.mdpi.com/2073-4352/11/8/975technical protein crystallization from impure sourcesrational crystal contact engineeringdownstream processingcrystal image analysis |
| spellingShingle | Brigitte Walla Daniel Bischoff Robert Janowski Nikolas von den Eichen Dierk Niessing Dirk Weuster-Botz Transfer of a Rational Crystal Contact Engineering Strategy between Diverse Alcohol Dehydrogenases Crystals technical protein crystallization from impure sources rational crystal contact engineering downstream processing crystal image analysis |
| title | Transfer of a Rational Crystal Contact Engineering Strategy between Diverse Alcohol Dehydrogenases |
| title_full | Transfer of a Rational Crystal Contact Engineering Strategy between Diverse Alcohol Dehydrogenases |
| title_fullStr | Transfer of a Rational Crystal Contact Engineering Strategy between Diverse Alcohol Dehydrogenases |
| title_full_unstemmed | Transfer of a Rational Crystal Contact Engineering Strategy between Diverse Alcohol Dehydrogenases |
| title_short | Transfer of a Rational Crystal Contact Engineering Strategy between Diverse Alcohol Dehydrogenases |
| title_sort | transfer of a rational crystal contact engineering strategy between diverse alcohol dehydrogenases |
| topic | technical protein crystallization from impure sources rational crystal contact engineering downstream processing crystal image analysis |
| url | https://www.mdpi.com/2073-4352/11/8/975 |
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