DOI: 10.1111/j.1432-1033.1993.tb18148.x. Large‐scale preparation of fully deuterated cell components

Large‐scale preparation of fully deuterated cell components

10.1111/j.1432-1033.1993.tb18148.x

Crossref journal-article

Wiley

European Journal of Biochemistry (311)

Abstract

Some applications of NMR and of neutron scattering require fully deuterated biological material which should be highly active and available in large quantities. These requirements are hardly compatible since full deuteration is achieved easily only if cells are grown in minimal media. This condition used in standard batch fermentation results in both low yields and reduced activities of the biological mass. Here we report a method which combines the apparently incompatible requirements taking advantage of a recent observation according to which the appearance of growth inhibiting extracellular products could be prevented. The method was applied for growing Escherichia coli cells, strain MRE600rif (resistance against high doses of rifampicin is used as selection marker) on partially deuterated media (76% and 84% D2O) with glucose as carbon source and on deuterated acetate and succinate with 100% D2O when full deuteration was to be achieved. The essential point for preserving the log‐phase character of the cells is that the cultivation is carried out at substrate limiting conditions thus keeping the growth rate at low levels (for glucose the growth rate, μ≤ 0.35 h−1, for acetate/succinate μ≤ 0.1 h−1) which avoids the accumulation of the substrate or of by‐products in the medium. Our data suggest that acetate is a main extracellular component for accompanying or triggering the transition from logarithmic growth to stationary phase of E. coli cells cultivated on glucose as carbon source.The cells were first grown in fed‐batch to high cell densities (above 50 g wet cells/1) under conditions of substrate limitations. A steady‐flow fermentation followed keeping the growth rate at about μ of 0.1 h−1. Cells were harvested in kg quantities, the extracted ribosomes showed a normal complement of proteins, contained intact rRNA and were fully active. The ribosomal protein and rRNA fractions could be efficiently reconstituted to highly active particles. In the case of full deuteration a matching point of 120% (tentative D2O scale) was achieved. The reported method facilitates the preparation of deuterated biological material for applications in NMR and neutron scattering analysis.

Bibliography

VANATALU, K., PAALME, T., VILU, R., BURKHARDT, N., JÃNEMANN, R., MAY, R., RÃHL, M., WADZACK, J., & NIERHAUS, K. H. (1993). Largeâscale preparation of fully deuterated cell components. European Journal of Biochemistry, 216(1), 315â321. Portico.

Authors 9

Kalju VANATALU (first)
Toomas PAALME (additional)
Raivo VILU (additional)
Nils BURKHARDT (additional)
Ralf JÜNEMANN (additional)
Roland MAY (additional)
Martin RÜHL (additional)
Jörg WADZACK (additional)
Knud H. NIERHAUS (additional)

References 14 Referenced 31

{'key': 'e_1_2_2_2_1', 'volume-title': 'Small angle scattering of X‐rays', 'author': 'Guinier A.', 'year': '1955'} / Small angle scattering of X‐rays by Guinier A. (1955)
10.1016/0022-2836(75)90131-X
10.1099/00221287-103-2-345
10.1099/00221287-103-2-353
10.1016/0921-4526(92)90524-V
10.1111/j.1365-2621.1981.tb14529.x
10.1002/j.1460-2075.1992.tb05060.x
10.1016/0076-6879(79)59119-8
10.1016/0022-2836(75)90374-5
{'key': 'e_1_2_2_11_1', 'first-page': '161', 'volume-title': 'Ribosomes and protein synthesis', 'author': 'Nierhaus K. H.', 'year': '1990'} / Ribosomes and protein synthesis by Nierhaus K. H. (1990)
10.1016/S0076-6879(88)64039-0
10.1002/bit.260350313
10.1016/S0076-6879(88)64076-6
10.1111/j.1432-1033.1979.tb02038.x

Dates

Type	When
Created	20 years, 5 months ago (March 4, 2005, 3:50 a.m.)
Deposited	1 year, 9 months ago (Nov. 22, 2023, 10:15 p.m.)
Indexed	1 month, 4 weeks ago (July 2, 2025, 2:46 p.m.)
Issued	32 years, 1 month ago (Aug. 1, 1993)
Published	32 years, 1 month ago (Aug. 1, 1993)
Published Online	20 years, 5 months ago (March 3, 2005)
Published Print	32 years, 1 month ago (Aug. 1, 1993)

Funders 0

None

BibTeX

@article{VANATALU_1993, title={Large‐scale preparation of fully deuterated cell components: Ribosomes from Escherichia coli with high biological activity}, volume={216}, ISSN={1432-1033}, url={http://dx.doi.org/10.1111/j.1432-1033.1993.tb18148.x}, DOI={10.1111/j.1432-1033.1993.tb18148.x}, number={1}, journal={European Journal of Biochemistry}, publisher={Wiley}, author={VANATALU, Kalju and PAALME, Toomas and VILU, Raivo and BURKHARDT, Nils and JÜNEMANN, Ralf and MAY, Roland and RÜHL, Martin and WADZACK, Jörg and NIERHAUS, Knud H.}, year={1993}, month=aug, pages={315–321} }

JSON

{
  "indexed": {
    "date-parts": [
      [
        2025,
        7,
        2
      ]
    ],
    "date-time": "2025-07-02T18:46:08Z",
    "timestamp": 1751481968031
  },
  "reference-count": 14,
  "publisher": "Wiley",
  "issue": "1",
  "license": [
    {
      "start": {
        "date-parts": [
          [
            2005,
            3,
            3
          ]
        ],
        "date-time": "2005-03-03T00:00:00Z",
        "timestamp": 1109808000000
      },
      "content-version": "vor",
      "delay-in-days": 4232,
      "URL": "http://onlinelibrary.wiley.com/termsAndConditions#vor"
    }
  ],
  "content-domain": {
    "domain": [],
    "crossmark-restriction": false
  },
  "published-print": {
    "date-parts": [
      [
        1993,
        8
      ]
    ]
  },
  "abstract": "<jats:p>Some applications of NMR and of neutron scattering require fully deuterated biological material which should be highly active and available in large quantities. These requirements are hardly compatible since full deuteration is achieved easily only if cells are grown in minimal media. This condition used in standard batch fermentation results in both low yields and reduced activities of the biological mass. Here we report a method which combines the apparently incompatible requirements taking advantage of a recent observation according to which the appearance of growth inhibiting extracellular products could be prevented. The method was applied for growing <jats:italic>Escherichia coli</jats:italic> cells, strain MRE600rif (resistance against high doses of rifampicin is used as selection marker) on partially deuterated media (76% and 84% D<jats:sub>2</jats:sub>O) with glucose as carbon source and on deuterated acetate and succinate with 100% D<jats:sub>2</jats:sub>O when full deuteration was to be achieved. The essential point for preserving the log\u2010phase character of the cells is that the cultivation is carried out at substrate limiting conditions thus keeping the growth rate at low levels (for glucose the growth rate, \u03bc\u2264 0.35 h<jats:sup>\u22121</jats:sup>, for acetate/succinate \u03bc\u2264 0.1 h<jats:sup>\u22121</jats:sup>) which avoids the accumulation of the substrate or of by\u2010products in the medium. Our data suggest that acetate is a main extracellular component for accompanying or triggering the transition from logarithmic growth to stationary phase of <jats:italic>E. coli</jats:italic> cells cultivated on glucose as carbon source.</jats:p><jats:p>The cells were first grown in fed\u2010batch to high cell densities (above 50 g wet cells/1) under conditions of substrate limitations. A steady\u2010flow fermentation followed keeping the growth rate at about \u03bc of 0.1 h<jats:sup>\u22121</jats:sup>. Cells were harvested in kg quantities, the extracted ribosomes showed a normal complement of proteins, contained intact rRNA and were fully active. The ribosomal protein and rRNA fractions could be efficiently reconstituted to highly active particles. In the case of full deuteration a matching point of 120% (tentative D<jats:sub>2</jats:sub>O scale) was achieved. The reported method facilitates the preparation of deuterated biological material for applications in NMR and neutron scattering analysis.</jats:p>",
  "DOI": "10.1111/j.1432-1033.1993.tb18148.x",
  "type": "journal-article",
  "created": {
    "date-parts": [
      [
        2005,
        3,
        4
      ]
    ],
    "date-time": "2005-03-04T08:50:21Z",
    "timestamp": 1109926221000
  },
  "page": "315-321",
  "source": "Crossref",
  "is-referenced-by-count": 31,
  "title": "Large\u2010scale preparation of fully deuterated cell components",
  "prefix": "10.1111",
  "volume": "216",
  "author": [
    {
      "given": "Kalju",
      "family": "VANATALU",
      "sequence": "first",
      "affiliation": []
    },
    {
      "given": "Toomas",
      "family": "PAALME",
      "sequence": "additional",
      "affiliation": []
    },
    {
      "given": "Raivo",
      "family": "VILU",
      "sequence": "additional",
      "affiliation": []
    },
    {
      "given": "Nils",
      "family": "BURKHARDT",
      "sequence": "additional",
      "affiliation": []
    },
    {
      "given": "Ralf",
      "family": "J\u00dcNEMANN",
      "sequence": "additional",
      "affiliation": []
    },
    {
      "given": "Roland",
      "family": "MAY",
      "sequence": "additional",
      "affiliation": []
    },
    {
      "given": "Martin",
      "family": "R\u00dcHL",
      "sequence": "additional",
      "affiliation": []
    },
    {
      "given": "J\u00f6rg",
      "family": "WADZACK",
      "sequence": "additional",
      "affiliation": []
    },
    {
      "given": "Knud H.",
      "family": "NIERHAUS",
      "sequence": "additional",
      "affiliation": []
    }
  ],
  "member": "311",
  "published-online": {
    "date-parts": [
      [
        2005,
        3,
        3
      ]
    ]
  },
  "reference": [
    {
      "key": "e_1_2_2_2_1",
      "volume-title": "Small angle scattering of X\u2010rays",
      "author": "Guinier A.",
      "year": "1955"
    },
    {
      "key": "e_1_2_2_3_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0022-2836(75)90131-X"
    },
    {
      "key": "e_1_2_2_4_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1099/00221287-103-2-345"
    },
    {
      "key": "e_1_2_2_5_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1099/00221287-103-2-353"
    },
    {
      "key": "e_1_2_2_6_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0921-4526(92)90524-V"
    },
    {
      "key": "e_1_2_2_7_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1365-2621.1981.tb14529.x"
    },
    {
      "key": "e_1_2_2_8_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/j.1460-2075.1992.tb05060.x"
    },
    {
      "key": "e_1_2_2_9_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0076-6879(79)59119-8"
    },
    {
      "key": "e_1_2_2_10_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0022-2836(75)90374-5"
    },
    {
      "key": "e_1_2_2_11_1",
      "first-page": "161",
      "volume-title": "Ribosomes and protein synthesis",
      "author": "Nierhaus K. H.",
      "year": "1990"
    },
    {
      "key": "e_1_2_2_12_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0076-6879(88)64039-0"
    },
    {
      "key": "e_1_2_2_13_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/bit.260350313"
    },
    {
      "key": "e_1_2_2_14_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0076-6879(88)64076-6"
    },
    {
      "key": "e_1_2_2_15_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1432-1033.1979.tb02038.x"
    }
  ],
  "container-title": "European Journal of Biochemistry",
  "original-title": [],
  "language": "en",
  "link": [
    {
      "URL": "https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1111%2Fj.1432-1033.1993.tb18148.x",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "text-mining"
    },
    {
      "URL": "https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1993.tb18148.x",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "similarity-checking"
    }
  ],
  "deposited": {
    "date-parts": [
      [
        2023,
        11,
        23
      ]
    ],
    "date-time": "2023-11-23T03:15:09Z",
    "timestamp": 1700709309000
  },
  "score": 1,
  "resource": {
    "primary": {
      "URL": "https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1993.tb18148.x"
    }
  },
  "subtitle": [
    "Ribosomes from <i>Escherichia coli</i> with high biological activity"
  ],
  "short-title": [],
  "issued": {
    "date-parts": [
      [
        1993,
        8
      ]
    ]
  },
  "references-count": 14,
  "journal-issue": {
    "issue": "1",
    "published-print": {
      "date-parts": [
        [
          1993,
          8
        ]
      ]
    }
  },
  "alternative-id": [
    "10.1111/j.1432-1033.1993.tb18148.x"
  ],
  "URL": "http://dx.doi.org/10.1111/j.1432-1033.1993.tb18148.x",
  "relation": {},
  "ISSN": [
    "0014-2956",
    "1432-1033"
  ],
  "subject": [],
  "container-title-short": "European Journal of Biochemistry",
  "published": {
    "date-parts": [
      [
        1993,
        8
      ]
    ]
  }
}