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His-FLAG Tag as a Fusion Partner of Glycosylated Human Interferon-Gamma and Its Mutant: Gain or Loss?
Joint Authors
Krachmarova, Elena
Tileva, Milena
Lilkova, Elena
Petkov, Peicho
Maskos, Klaus
Ivanov, Ivan
Litov, Leandar
Nacheva, Genoveva
Ilieva, Nevena
Source
Issue
Vol. 2017, Issue 2017 (31 Dec. 2017), pp.1-12, 12 p.
Publisher
Hindawi Publishing Corporation
Publication Date
2017-06-08
Country of Publication
Egypt
No. of Pages
12
Main Subjects
Abstract EN
In order to obtain glycosylated human interferon-gamma (hIFNγ) and its highly prone to aggregation mutant K88Q, a secretory expression in insect cells was employed.
To facilitate recombinant proteins purification, detection, and stability the baculovirus expression vectors were constructed to bear N-terminal His6-FLAG tag.
Although the obtained proteins were glycosylated, we found that their biological activity was 100 times lower than expected.
Our attempts to recover the biological properties of both proteins by tag removal failed due to enterokinase resistance of the tag.
Surprisingly, the tag was easily cleaved when the proteins were expressed in E.
coli cells and the tag-free proteins showed fully restored activity.
To shed light on this phenomenon we performed molecular dynamics simulations.
The latter showed that the tags interact with the receptor binding domains and the flexible C-termini of the fusion proteins thus suppressing their complex formation with the hIFNγ receptor.
We hypothesize that in the case of glycosylated proteins the tag/C-terminal interaction positions the FLAG peptide in close proximity to the glycans thus sterically impeding the enterokinase access to its recognition site.
American Psychological Association (APA)
Krachmarova, Elena& Tileva, Milena& Lilkova, Elena& Petkov, Peicho& Maskos, Klaus& Ilieva, Nevena…[et al.]. 2017. His-FLAG Tag as a Fusion Partner of Glycosylated Human Interferon-Gamma and Its Mutant: Gain or Loss?. BioMed Research International،Vol. 2017, no. 2017, pp.1-12.
https://search.emarefa.net/detail/BIM-1135637
Modern Language Association (MLA)
Krachmarova, Elena…[et al.]. His-FLAG Tag as a Fusion Partner of Glycosylated Human Interferon-Gamma and Its Mutant: Gain or Loss?. BioMed Research International No. 2017 (2017), pp.1-12.
https://search.emarefa.net/detail/BIM-1135637
American Medical Association (AMA)
Krachmarova, Elena& Tileva, Milena& Lilkova, Elena& Petkov, Peicho& Maskos, Klaus& Ilieva, Nevena…[et al.]. His-FLAG Tag as a Fusion Partner of Glycosylated Human Interferon-Gamma and Its Mutant: Gain or Loss?. BioMed Research International. 2017. Vol. 2017, no. 2017, pp.1-12.
https://search.emarefa.net/detail/BIM-1135637
Data Type
Journal Articles
Language
English
Notes
Includes bibliographical references
Record ID
BIM-1135637