EP1325027A2 - Fragment peptidique de caseine presentant une activite influant sur la croissance de cultures cellulaires - Google Patents

Fragment peptidique de caseine presentant une activite influant sur la croissance de cultures cellulaires

Info

Publication number
EP1325027A2
EP1325027A2 EP01986693A EP01986693A EP1325027A2 EP 1325027 A2 EP1325027 A2 EP 1325027A2 EP 01986693 A EP01986693 A EP 01986693A EP 01986693 A EP01986693 A EP 01986693A EP 1325027 A2 EP1325027 A2 EP 1325027A2
Authority
EP
European Patent Office
Prior art keywords
peptide
amino acid
peptide according
growth
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01986693A
Other languages
German (de)
English (en)
Inventor
Juan Claus
Marcelo Comini
Georgina Tonarelli
Juan Carlos Perin
Jorge Luis Salvetti
Ronald Frank
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Original Assignee
Universidad Nacional del Litoral
Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universidad Nacional del Litoral, Helmholtz Zentrum fuer Infektionsforschung HZI GmbH filed Critical Universidad Nacional del Litoral
Publication of EP1325027A2 publication Critical patent/EP1325027A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4732—Casein

Definitions

  • Eukaryotic cell cultures are increasingly being used for the biotechnological production of valuable recombined proteins.
  • Most culture media for such cells are complex and many require the additional addition of fetal calf serum (FCS, "fetal calf serum”). Since the use of “serum in culture media causes many problems and causes high costs, the replacement of the serum component with cheap and defined additives would result in a significant improvement of biotechnological processes based on cell cultures.
  • FCS fetal calf serum
  • insect cells BHK (“baby hamster kidney”) cells, CHO (“Chinese hamster ovary”) cells, HEK ("human epithelial kidney”) cells, etc. are used.
  • Protein hydrolysates and natural protein extracts are often added to the culture media of eukaryotic cells as growth-promoting additives.
  • knowledge of their activity is very limited, the active components are unknown and their role in influencing growth is unknown.
  • IPLB-Sf21 Spodoptera frugiperda
  • the object of the invention is to identify the peptide components of the casein hydrolyzate which are responsible for the growth-promoting action of the satin hydrolyzate and to provide them as chemically defined additives for culture media of, for example, insect cell cultures.
  • the invention thus relates, according to patent claim 1, to a peptide which promotes the growth of eukaryotic cell cultures and is selected from
  • peptides in which one or more amino acid residue (s) in the sequence according to a) is / are replaced by amino acid residues for example the degree of homology can be 30%, 40%, 50%, 60%, 70%, 80%, 85% is 90% or 95%,
  • amino acid residues in which one or more amino acid residue (s) in the sequence according to a) or b) is / are chemically modified, for example the amino acid residue at the N-terminus may have an acyl group such as an acetyl group,
  • a fusion protein that contains a peptide according to a), b) or d) without the additional amino acid residues impairing the growth-promoting effect or these being easily cleavable.
  • the peptide according to the invention according to d) can be the peptide HQP, which can be chemically modified.
  • the amino acid residue at the N-terminus can have an acyl group such as an acetyl group.
  • the peptides according to the invention can be isolated from natural casein sources (for example from bovine casein), chemically synthesized or produced by genetic engineering.
  • the invention further relates to a DNA sequence which encodes a fusion protein according to the invention according to e), a recombined expression vector which contains such a DNA sequence and prokaryotic or eukaryotic cells which are linked to a DNA sequence according to the invention or an expression vector according to the invention are transformed or transfected.
  • the invention relates to a method for producing a peptide according to a), b) or d), in which cells according to the invention are grown in a suitable culture medium, the fusion protein is obtained from the cells or the culture medium and the additional amino acid residues are cleaved off.
  • the invention relates to a method for culturing eukaryotic cell cultures using a peptide according to the invention.
  • the peptide according to the invention can advantageously be used in a concentration in the range from 0.1 ⁇ g / l to 10 ⁇ g / ml culture medium.
  • cells from the insect cell line IPLB-Sf-21 are suitable as cells.
  • IPLB-Sf-21 cells (ACC1 19, DSMZ in Braunschweig, Germany) were developed by Dr. Victor Romanowski (Instituto de Bioquimica y
  • the starting material was obtained from Sigma-Aldrich Co. under the product name N-Z Case Plus (order number N4642). This hydrolyzate was first separated by gel filtration chromatography in a manner known per se into fractions with different molecular sizes, lyophilized and stored at ⁇ 20 ° C. These fractions were checked for their biological activity. The active fraction was then, in a manner known per se, by reverse phase high-pressure liquid chromatography (“reversed phase HPLC”) with a column with dimensions of 4 ⁇ 250 mm, filled with Cl ⁇ -modified silica gel (pore size 300 ⁇ ) with a grain size of 7 ⁇ m, and further fractionated a solvent gradient from water to acetonitrile with 0.1% trifluoroacetic acid.
  • reverse phase HPLC reverse phase high-pressure liquid chromatography
  • the column eluate was passed through a UV absorption detector and UV absorbing compounds were collected in separate fractions.
  • the fractions were concentrated in vacuo, the residue was taken up in 500 ⁇ l of water with 0.1% trifluoroacetic acid and stored at -20 ° C.
  • the peptide fragment, which was biologically active, and partial segments from the casein hydrolyzate were prepared chemically and synthetically manually according to the Fmoc / tBu method (Fields and Noble, Int. J. Peptide Protein Res. 35, 1992, 161-214).
  • PEG polystyrene resin (Rapp Polymer, Tübingen) with a Rink linker served as the carrier material.
  • the peptides were deprotected with a mixture of trifluoroacetic acid / water / triethylsilane (90/5/5, v / v / v) and split off from the support, precipitated in cold diethyl ether, centrifuged off, dissolved in water and freeze-dried. These crude products were then, in a manner known per se, by reverse phase high-pressure liquid chromatography (“reversed phase HPLC”) with a column of 10 ⁇ 250 mm, filled with C18-modified silica gel with a particle size of 7 ⁇ m, and a solvent gradient from water to acetonitrile 0.1% trifluoroacetic acid purified. The analytical HPLC showed a purity of more than 90%.
  • the tripeptide was also analyzed by nuclear magnetic resonance spectroscopy (NMR).
  • IPLB-Sf-21 cells were seeded in an initial density of 5 ⁇ 10 4 cells in 1 ml of culture medium (TC-100 + 10% FCS) in the respective wells of 24-well culture plates. The plates were incubated at 27 ° C in a humidified chamber for two days. During the following two days, the cultures were weaned from the serum addition by exchanging the medium several times for pure TClOO medium and then provided with the peptides or peptide fractions in different concentrations. Control cultures were cultivated in pure TClOO medium.
  • the number of cells per culture bowl was determined by counting in a Neubauer chamber with an inverted microscope.
  • the Neubauer Chamber had two areas for counting. Each sample was counted three times in both areas (six values per sample).
  • the main component of the biologically active fraction of the casein was identified as the peptide of the sequence HQPHQPLPPT. Synthetic peptides
  • AC-HQP-OH tripeptide
  • Ac-HQPHQPLPPT-OH decapeptide
  • Table 1 shows the cell yields after treatment with various concentrations of the decapeptide Ac-HQPHQPLPPT-OH in comparison with the cell yields in cultures without peptide and without FCS.
  • the maximum growth-promoting effect was achieved at a peptide concentration of 1 ⁇ g / ml. Higher concentrations led to growth inhibition.
  • p standard error deviation calculated with the ANOVA (Analysis of Variance) option of the Microcal Origin program.
  • the growth-influencing effect of the tripeptide AC-HQP-OH which is contained twice in the casein sequence, is shown in Table 2. Again, the cell yields are compared for cell yield in cultures without peptide and without FCS. The maximum growth-promoting effect was already achieved here at a peptide concentration of 0.1 ⁇ g / ml. Very high concentrations (> 10 ⁇ g / ml) also led to growth inhibition.
  • Tripeptide concentration ( ⁇ g / ml) 100 10 1 0.1 0.01

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Selon l'invention, un décapeptide a été isolé à partir d'un hydrolysat enzymatique de caséine bovine, décapeptide dont la séquence a pu être, sans équivoque, identifiée dans la séquence primaire de la caséine. Ce décapeptide, qui a également été réalisé de façon chimiosynthétique, influe sur la croissance de la lignée cellulaire d'insectes IPLB-Sf-21 en fonction de la concentration. Un tripeptide qui est contenu deux fois dans la séquence du décapeptide ou de la caséine présente un effet encore plus fort que ledit décapeptide, cet effet étant également fonction de la concentration. Ces peptides peuvent donc, de façon avantageuse, être utilisés comme additifs pour des milieux de culture cellulaire exempts de sérum et chimiquement définis.
EP01986693A 2000-10-09 2001-10-09 Fragment peptidique de caseine presentant une activite influant sur la croissance de cultures cellulaires Withdrawn EP1325027A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10050091 2000-10-09
DE10050091A DE10050091A1 (de) 2000-10-09 2000-10-09 Casein-Peptidfragmente mit wachstumsbeeinflußender Aktivität auf Zellkulturen
PCT/DE2001/003849 WO2002030958A2 (fr) 2000-10-09 2001-10-09 Fragment peptidique de caseine presentant une activite influant sur la croissance de cultures cellulaires

Publications (1)

Publication Number Publication Date
EP1325027A2 true EP1325027A2 (fr) 2003-07-09

Family

ID=7659237

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01986693A Withdrawn EP1325027A2 (fr) 2000-10-09 2001-10-09 Fragment peptidique de caseine presentant une activite influant sur la croissance de cultures cellulaires

Country Status (5)

Country Link
US (1) US20040086965A1 (fr)
EP (1) EP1325027A2 (fr)
JP (1) JP2004535152A (fr)
DE (1) DE10050091A1 (fr)
WO (1) WO2002030958A2 (fr)

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2522358B1 (fr) * 2007-06-27 2016-11-09 Laboratorios Ordesa, S.l. Peptides contres les infections par le rotavirus
CN107141346B (zh) * 2017-06-22 2020-04-14 浙江辉肽生命健康科技有限公司 一种生物活性多肽atledspevi及其制备方法和应用
CN107814837B (zh) * 2017-12-07 2020-04-14 浙江辉肽生命健康科技有限公司 一种生物活性多肽qqqtedelqdkihpf及其制备方法和应用
CN107880103B (zh) * 2017-12-07 2020-05-12 浙江辉肽生命健康科技有限公司 生物活性多肽qpevmgvskvkeamapkqkempfpky及其制备和应用
CN107880102A (zh) * 2017-12-07 2018-04-06 浙江辉肽生命健康科技有限公司 一种生物活性多肽peviesppein及其制备方法和应用
CN107759682B (zh) * 2017-12-07 2021-03-02 浙江辉肽生命健康科技有限公司 一种生物活性多肽pigsensgkttmpl及其制备方法和应用
CN107880107B (zh) * 2017-12-11 2020-05-12 浙江辉肽生命健康科技有限公司 一种生物活性多肽qvlsntvpa及其制备方法和应用
CN107973847B (zh) * 2017-12-11 2020-05-12 浙江辉肽生命健康科技有限公司 一种生物活性多肽vateevkitvd及其制备方法和应用
CN107880104A (zh) * 2017-12-11 2018-04-06 浙江辉肽生命健康科技有限公司 一种生物活性多肽sppeintvqvt及其制备方法和应用
CN108017704B (zh) * 2017-12-12 2020-05-12 浙江辉肽生命健康科技有限公司 一种生物活性多肽terqsltltdve及其制备方法和应用
CN107892716A (zh) * 2017-12-12 2018-04-10 浙江辉肽生命健康科技有限公司 一种生物活性多肽lppll及其制备方法和应用
CN107903316B (zh) * 2017-12-12 2020-05-12 浙江辉肽生命健康科技有限公司 一种生物活性多肽lpypyya及其制备方法和应用
CN107814842B (zh) * 2017-12-12 2020-04-14 浙江辉肽生命健康科技有限公司 一种生物活性多肽sqskvlpvpe及其制备方法和应用
CN107814841B (zh) * 2017-12-12 2020-04-14 浙江辉肽生命健康科技有限公司 一种生物活性多肽qqpvlgpvrgp及其制备方法和应用
CN108017705A (zh) * 2017-12-12 2018-05-11 浙江辉肽生命健康科技有限公司 一种生物活性多肽tvmfppq及其制备方法和应用
WO2019200184A1 (fr) * 2018-04-12 2019-10-17 Life Technologies Corporation Système d'expression de baculovirus et milieu de culture cellulaire chimiquement définis
JPWO2021059755A1 (fr) * 2019-09-26 2021-04-01

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5024947A (en) * 1987-07-24 1991-06-18 Cetus Corporation Serum free media for the growth on insect cells and expression of products thereby
EP1130027A1 (fr) * 2000-02-29 2001-09-05 Aventis Pharma Deutschland GmbH Peptides memnoique, procédé de preparation et ses utilisations

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0230958A3 *

Also Published As

Publication number Publication date
DE10050091A1 (de) 2002-04-25
WO2002030958A3 (fr) 2002-12-12
US20040086965A1 (en) 2004-05-06
JP2004535152A (ja) 2004-11-25
WO2002030958A2 (fr) 2002-04-18

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