Generation of a vector system facilitating cloning of DMBT1 variants and recombinant expression of functional full-length DMBT1.

Caroline End, Stefan Lyer, Marcus Renner, Cordula Stahl, Jutta Ditzer, Andreas Holloschi, Hella-M Kuhn, Heiko T Flammann, Annemarie Poustka, Mathias Hafner, Jan Mollenhauer, Petra Kioschis

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Udgivelsesdato: 2005-Jun
OriginalsprogEngelsk
TidsskriftProtein Expression and Purification
Vol/bind41
Udgave nummer2
Sider (fra-til)275-86
Antal sider11
ISSN1046-5928
DOI
StatusUdgivet - 1. jun. 2005

Fingeraftryk

Organism Cloning
Scavenger Receptors
Saliva
Lactoferrin
Genetic Polymorphisms
Virus Diseases
Cricetulus
Glycosylation
Ovary
Complementary DNA
Epithelial Cells
Ligands
Cell Line
Research

Citer dette

End, Caroline ; Lyer, Stefan ; Renner, Marcus ; Stahl, Cordula ; Ditzer, Jutta ; Holloschi, Andreas ; Kuhn, Hella-M ; Flammann, Heiko T ; Poustka, Annemarie ; Hafner, Mathias ; Mollenhauer, Jan ; Kioschis, Petra. / Generation of a vector system facilitating cloning of DMBT1 variants and recombinant expression of functional full-length DMBT1. I: Protein Expression and Purification. 2005 ; Bind 41, Nr. 2. s. 275-86.
@article{ab13f26057f811ddb1a1000ea68e967b,
title = "Generation of a vector system facilitating cloning of DMBT1 variants and recombinant expression of functional full-length DMBT1.",
abstract = "Deleted in malignant brain tumours 1 (DMBT1) codes for a approximately 340kDa glycoprotein with highly repetitive scavenger receptor cysteine-rich (SRCR) domains. DMBT1 was implicated in cancer, defence against viral and bacterial infections, and differentiation of epithelial cells. Recombinant expression and purification of DMBT1 is an essential step for systematic standardized functional research and towards the evaluation of its therapeutical potential. So far, DMBT1 is obtained from natural sources such as bronchioalveolar lavage or saliva, resulting in time consuming sample collection, low yields, and protein preparations which may substantially vary due to differential processing and genetic polymorphism, all of which impedes functional research on DMBT1. Cloning of DMBT1 cDNAs is hampered because of the size and the 13 highly homologous SRCR exons. In this study, we report on the setup of a vector system that facilitates cloning of DMBT1 variants. We demonstrate applicability of the vector system by expression of the largest DMBT1 variant in a tetracycline-inducible mammalian expression system using the Chinese hamster ovary cell line. Yields up to 30 mg rDMBT1 per litre of cell culture supernatant could be achieved with an optimized production procedure. By harnessing the specific bacteria-binding property of DMBT1 we established an affinity purification procedure which allows the isolation of more than 3 mg rDMBT1 with a purity of about 95{\%}. Although the glycosylation moieties of rDMBT1 are different from DMBT1(SAG) isolated from saliva, we demonstrate that rDMBT1 is functionally active in aggregating Gram-positive and Gram-negative bacteria and binding to C1q and lactoferrin, which represent two known endogenous DMBT1 ligands.",
keywords = "Agglutinins, Animals, CHO Cells, Chromatography, Affinity, Cloning, Molecular, Cricetinae, Cricetulus, Culture Media, Conditioned, Gene Expression Regulation, Genetic Vectors, Receptors, Cell Surface, Recombinant Proteins, Variation (Genetics)",
author = "Caroline End and Stefan Lyer and Marcus Renner and Cordula Stahl and Jutta Ditzer and Andreas Holloschi and Hella-M Kuhn and Flammann, {Heiko T} and Annemarie Poustka and Mathias Hafner and Jan Mollenhauer and Petra Kioschis",
year = "2005",
month = "6",
day = "1",
doi = "10.1016/j.pep.2005.02.010",
language = "English",
volume = "41",
pages = "275--86",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Heinemann",
number = "2",

}

End, C, Lyer, S, Renner, M, Stahl, C, Ditzer, J, Holloschi, A, Kuhn, H-M, Flammann, HT, Poustka, A, Hafner, M, Mollenhauer, J & Kioschis, P 2005, 'Generation of a vector system facilitating cloning of DMBT1 variants and recombinant expression of functional full-length DMBT1.', Protein Expression and Purification, bind 41, nr. 2, s. 275-86. https://doi.org/10.1016/j.pep.2005.02.010

Generation of a vector system facilitating cloning of DMBT1 variants and recombinant expression of functional full-length DMBT1. / End, Caroline; Lyer, Stefan; Renner, Marcus; Stahl, Cordula; Ditzer, Jutta; Holloschi, Andreas; Kuhn, Hella-M; Flammann, Heiko T; Poustka, Annemarie; Hafner, Mathias; Mollenhauer, Jan; Kioschis, Petra.

I: Protein Expression and Purification, Bind 41, Nr. 2, 01.06.2005, s. 275-86.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Generation of a vector system facilitating cloning of DMBT1 variants and recombinant expression of functional full-length DMBT1.

AU - End, Caroline

AU - Lyer, Stefan

AU - Renner, Marcus

AU - Stahl, Cordula

AU - Ditzer, Jutta

AU - Holloschi, Andreas

AU - Kuhn, Hella-M

AU - Flammann, Heiko T

AU - Poustka, Annemarie

AU - Hafner, Mathias

AU - Mollenhauer, Jan

AU - Kioschis, Petra

PY - 2005/6/1

Y1 - 2005/6/1

N2 - Deleted in malignant brain tumours 1 (DMBT1) codes for a approximately 340kDa glycoprotein with highly repetitive scavenger receptor cysteine-rich (SRCR) domains. DMBT1 was implicated in cancer, defence against viral and bacterial infections, and differentiation of epithelial cells. Recombinant expression and purification of DMBT1 is an essential step for systematic standardized functional research and towards the evaluation of its therapeutical potential. So far, DMBT1 is obtained from natural sources such as bronchioalveolar lavage or saliva, resulting in time consuming sample collection, low yields, and protein preparations which may substantially vary due to differential processing and genetic polymorphism, all of which impedes functional research on DMBT1. Cloning of DMBT1 cDNAs is hampered because of the size and the 13 highly homologous SRCR exons. In this study, we report on the setup of a vector system that facilitates cloning of DMBT1 variants. We demonstrate applicability of the vector system by expression of the largest DMBT1 variant in a tetracycline-inducible mammalian expression system using the Chinese hamster ovary cell line. Yields up to 30 mg rDMBT1 per litre of cell culture supernatant could be achieved with an optimized production procedure. By harnessing the specific bacteria-binding property of DMBT1 we established an affinity purification procedure which allows the isolation of more than 3 mg rDMBT1 with a purity of about 95%. Although the glycosylation moieties of rDMBT1 are different from DMBT1(SAG) isolated from saliva, we demonstrate that rDMBT1 is functionally active in aggregating Gram-positive and Gram-negative bacteria and binding to C1q and lactoferrin, which represent two known endogenous DMBT1 ligands.

AB - Deleted in malignant brain tumours 1 (DMBT1) codes for a approximately 340kDa glycoprotein with highly repetitive scavenger receptor cysteine-rich (SRCR) domains. DMBT1 was implicated in cancer, defence against viral and bacterial infections, and differentiation of epithelial cells. Recombinant expression and purification of DMBT1 is an essential step for systematic standardized functional research and towards the evaluation of its therapeutical potential. So far, DMBT1 is obtained from natural sources such as bronchioalveolar lavage or saliva, resulting in time consuming sample collection, low yields, and protein preparations which may substantially vary due to differential processing and genetic polymorphism, all of which impedes functional research on DMBT1. Cloning of DMBT1 cDNAs is hampered because of the size and the 13 highly homologous SRCR exons. In this study, we report on the setup of a vector system that facilitates cloning of DMBT1 variants. We demonstrate applicability of the vector system by expression of the largest DMBT1 variant in a tetracycline-inducible mammalian expression system using the Chinese hamster ovary cell line. Yields up to 30 mg rDMBT1 per litre of cell culture supernatant could be achieved with an optimized production procedure. By harnessing the specific bacteria-binding property of DMBT1 we established an affinity purification procedure which allows the isolation of more than 3 mg rDMBT1 with a purity of about 95%. Although the glycosylation moieties of rDMBT1 are different from DMBT1(SAG) isolated from saliva, we demonstrate that rDMBT1 is functionally active in aggregating Gram-positive and Gram-negative bacteria and binding to C1q and lactoferrin, which represent two known endogenous DMBT1 ligands.

KW - Agglutinins

KW - Animals

KW - CHO Cells

KW - Chromatography, Affinity

KW - Cloning, Molecular

KW - Cricetinae

KW - Cricetulus

KW - Culture Media, Conditioned

KW - Gene Expression Regulation

KW - Genetic Vectors

KW - Receptors, Cell Surface

KW - Recombinant Proteins

KW - Variation (Genetics)

U2 - 10.1016/j.pep.2005.02.010

DO - 10.1016/j.pep.2005.02.010

M3 - Journal article

VL - 41

SP - 275

EP - 286

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

IS - 2

ER -