Improved Purification of Human Granzyme A/B and Granulysin Using a Mammalian Expression System.
HEK293T
cytotoxic granular proteins
granulysin
granzyme A
granzyme B
isotonic buffer
lipofectamine 3000
mammalian expression
Journal
Frontiers in immunology
ISSN: 1664-3224
Titre abrégé: Front Immunol
Pays: Switzerland
ID NLM: 101560960
Informations de publication
Date de publication:
2022
2022
Historique:
received:
06
12
2021
accepted:
08
02
2022
entrez:
18
3
2022
pubmed:
19
3
2022
medline:
3
5
2022
Statut:
epublish
Résumé
Cytotoxic lymphocytes release proteins contained within the cytoplasmic cytolytic granules after recognition of infected or tumor target cells. These cytotoxic granular proteins (namely granzymes, granulysin, and perforin) are key immunological mediators within human cellular immunity. The availability of highly purified cytotoxic proteins has been fundamental for understanding their function in immunity and mechanistic involvement in sepsis and autoimmunity. Methods for recovery of native cytotoxic proteins can be problematic leading to: 1) the co-purification of additional proteins, confounding interpretation of function, and 2) low yields of highly purified proteins. Recombinant protein expression of individual cytolytic components can overcome these challenges. The use of mammalian expression systems is preferred for optimal post-translational modifications and avoidance of endotoxin contamination. Some of these proteins have been proposed for host directed human therapies (e.g. - granzyme A), or treatment of systemic infections or tumors as in granulysin. We report here a novel expression system using HEK293T cells for cost-effective purification of high yields of human granzymes (granzyme A and granzyme B) and granulysin with enhanced biological activity than previous reports. The resulting proteins are free of native contaminants, fold correctly, and remain enzymatically active. Importantly, these improvements have also led to the first purification of biologically active recombinant human granulysin in high yields from a mammalian system. This method can be used as a template for purification of many other secreted cellular proteins and may lead to advances for human medicine.
Identifiants
pubmed: 35300343
doi: 10.3389/fimmu.2022.830290
pmc: PMC8921980
doi:
Substances chimiques
Perforin
126465-35-8
Granzymes
EC 3.4.21.-
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
830290Subventions
Organisme : NHLBI NIH HHS
ID : F30 HL151136
Pays : United States
Organisme : NIAID NIH HHS
ID : R01 AI048391
Pays : United States
Informations de copyright
Copyright © 2022 Rasi, Hameed, Matthey, Bera, Grandgenett, Salentinig, Walch and Hoft.
Déclaration de conflit d'intérêts
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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