Expression, purification and characterization of the cancer-germline antigen GAGE12I: a candidate for cancer immunotherapy

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

GAGE cancer-germline antigens are frequently expressed in a broad range of different cancers, while their expression in normal tissues is limited to the germ cells of the immune privileged organs, testis and ovary. GAGE proteins are immunogenic in humans, which make them promising targets for immunotherapy and candidates for cancer vaccines. Recombinant proteins may be superior to peptides as immunogens, since they have the potential to prime both CD4(+) and CD8(+) T cells and are not dependent on patient HLA-type. We have developed a method for production of highly pure recombinant GAGE12I-His by intracellular expression in yeast (Pichia pastoris) and nickel affinity, ion exchange and gel filtration purification. The identity of the purified protein was confirmed by mass spectrometry. This strategy yielded a total of 48 mg of highly pure (>98%) GAGE12I from 8 L of culture (6 mg/l). Interestingly, gel filtration and formaldehyde cross-linking indicated that GAGE12I forms tetramers. The purified recombinant GAGE12I represents a candidate molecule for vaccination of cancer patients and will form the basis for further structural analysis of GAGE proteins.
OriginalsprogEngelsk
TidsskriftProtein Expression and Purification
Vol/bind73
Udgave nummer2
Sider (fra-til)217-22
Antal sider6
ISSN1046-5928
DOI
StatusUdgivet - 1. okt. 2010

Fingeraftryk

Gel Chromatography
Neoplasms
Cancer Vaccines
Proteins
Pichia
Ion Exchange
Nickel
Recombinant Proteins
Germ Cells
Formaldehyde
Ovary
Peptides

Citer dette

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title = "Expression, purification and characterization of the cancer-germline antigen GAGE12I: a candidate for cancer immunotherapy",
abstract = "GAGE cancer-germline antigens are frequently expressed in a broad range of different cancers, while their expression in normal tissues is limited to the germ cells of the immune privileged organs, testis and ovary. GAGE proteins are immunogenic in humans, which make them promising targets for immunotherapy and candidates for cancer vaccines. Recombinant proteins may be superior to peptides as immunogens, since they have the potential to prime both CD4(+) and CD8(+) T cells and are not dependent on patient HLA-type. We have developed a method for production of highly pure recombinant GAGE12I-His by intracellular expression in yeast (Pichia pastoris) and nickel affinity, ion exchange and gel filtration purification. The identity of the purified protein was confirmed by mass spectrometry. This strategy yielded a total of 48 mg of highly pure (>98{\%}) GAGE12I from 8 L of culture (6 mg/l). Interestingly, gel filtration and formaldehyde cross-linking indicated that GAGE12I forms tetramers. The purified recombinant GAGE12I represents a candidate molecule for vaccination of cancer patients and will form the basis for further structural analysis of GAGE proteins.",
author = "Gjerstorff, {Morten F} and H{\"u}seyin Besir and Larsen, {Martin R} and Ditzel, {Henrik J}",
note = "Copyright 2010 Elsevier Inc. All rights reserved.",
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Expression, purification and characterization of the cancer-germline antigen GAGE12I: a candidate for cancer immunotherapy. / Gjerstorff, Morten F; Besir, Hüseyin; Larsen, Martin R; Ditzel, Henrik J.

I: Protein Expression and Purification, Bind 73, Nr. 2, 01.10.2010, s. 217-22.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Expression, purification and characterization of the cancer-germline antigen GAGE12I: a candidate for cancer immunotherapy

AU - Gjerstorff, Morten F

AU - Besir, Hüseyin

AU - Larsen, Martin R

AU - Ditzel, Henrik J

N1 - Copyright 2010 Elsevier Inc. All rights reserved.

PY - 2010/10/1

Y1 - 2010/10/1

N2 - GAGE cancer-germline antigens are frequently expressed in a broad range of different cancers, while their expression in normal tissues is limited to the germ cells of the immune privileged organs, testis and ovary. GAGE proteins are immunogenic in humans, which make them promising targets for immunotherapy and candidates for cancer vaccines. Recombinant proteins may be superior to peptides as immunogens, since they have the potential to prime both CD4(+) and CD8(+) T cells and are not dependent on patient HLA-type. We have developed a method for production of highly pure recombinant GAGE12I-His by intracellular expression in yeast (Pichia pastoris) and nickel affinity, ion exchange and gel filtration purification. The identity of the purified protein was confirmed by mass spectrometry. This strategy yielded a total of 48 mg of highly pure (>98%) GAGE12I from 8 L of culture (6 mg/l). Interestingly, gel filtration and formaldehyde cross-linking indicated that GAGE12I forms tetramers. The purified recombinant GAGE12I represents a candidate molecule for vaccination of cancer patients and will form the basis for further structural analysis of GAGE proteins.

AB - GAGE cancer-germline antigens are frequently expressed in a broad range of different cancers, while their expression in normal tissues is limited to the germ cells of the immune privileged organs, testis and ovary. GAGE proteins are immunogenic in humans, which make them promising targets for immunotherapy and candidates for cancer vaccines. Recombinant proteins may be superior to peptides as immunogens, since they have the potential to prime both CD4(+) and CD8(+) T cells and are not dependent on patient HLA-type. We have developed a method for production of highly pure recombinant GAGE12I-His by intracellular expression in yeast (Pichia pastoris) and nickel affinity, ion exchange and gel filtration purification. The identity of the purified protein was confirmed by mass spectrometry. This strategy yielded a total of 48 mg of highly pure (>98%) GAGE12I from 8 L of culture (6 mg/l). Interestingly, gel filtration and formaldehyde cross-linking indicated that GAGE12I forms tetramers. The purified recombinant GAGE12I represents a candidate molecule for vaccination of cancer patients and will form the basis for further structural analysis of GAGE proteins.

U2 - 10.1016/j.pep.2010.05.010

DO - 10.1016/j.pep.2010.05.010

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JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

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