Separation of the Epitopes in a Multi-Epitope Chimera: Helical or Flexible Linkers.
Multi-epitope chimera
expression level
helical and flexible linkers
immobilized metal ion affinity
chromatography
molecular modeling
purified chimera
Journal
Protein and peptide letters
ISSN: 1875-5305
Titre abrégé: Protein Pept Lett
Pays: Netherlands
ID NLM: 9441434
Informations de publication
Date de publication:
2020
2020
Historique:
received:
03
07
2019
revised:
25
09
2019
accepted:
29
09
2019
pubmed:
14
11
2019
medline:
16
1
2021
entrez:
14
11
2019
Statut:
ppublish
Résumé
The engineered chimeric peptides including functional multi-epitope structures fused by various peptide linkers are widely applied in biotechnological research to improve the expression level and biological activity of chimera. The aim of our study was to evaluate the effect of helical and flexible linkers on solubility, expression level and folding of multi-epitope chimera containing four epitopes of Human T Lymphotropic Virus Type 1 (HTLV-1). For this purpose, the chimera sequences connected by the helical or flexible linker were inserted into different plasmid vectors and expressed in E. coli strains. The expressed products were analyzed using SDS-PAGE and Western blot techniques. Additionally, the molecular modeling study of the chimera with helical or flexible linker was performed using iterative threading assembly refinement (I-TASSER) to attain their three-dimensional structures. Comparison of the chimera expression indicated that the insertion of a flexible (GGGGS)3 linker among chimera epitopes could significantly enhance the level of expression, whereas, the low-level of chimera expression was observed for chimera containing the contiguous helical (EAAAK)5 linker. According to the results of sequence alignment and plasmid stability test, the structure and function of a consecutive helical linker among chimera epitopes were similar to porins as the outer-membrane pore-forming proteins. The molecular modeling results confirmed our experimental study. This investigation illustrated the key role of linker design in determining the expression level of multi-epitope chimera and conformational folding.
Sections du résumé
BACKGROUND
BACKGROUND
The engineered chimeric peptides including functional multi-epitope structures fused by various peptide linkers are widely applied in biotechnological research to improve the expression level and biological activity of chimera.
OBJECTIVE
OBJECTIVE
The aim of our study was to evaluate the effect of helical and flexible linkers on solubility, expression level and folding of multi-epitope chimera containing four epitopes of Human T Lymphotropic Virus Type 1 (HTLV-1).
METHODS
METHODS
For this purpose, the chimera sequences connected by the helical or flexible linker were inserted into different plasmid vectors and expressed in E. coli strains. The expressed products were analyzed using SDS-PAGE and Western blot techniques. Additionally, the molecular modeling study of the chimera with helical or flexible linker was performed using iterative threading assembly refinement (I-TASSER) to attain their three-dimensional structures.
RESULTS
RESULTS
Comparison of the chimera expression indicated that the insertion of a flexible (GGGGS)3 linker among chimera epitopes could significantly enhance the level of expression, whereas, the low-level of chimera expression was observed for chimera containing the contiguous helical (EAAAK)5 linker. According to the results of sequence alignment and plasmid stability test, the structure and function of a consecutive helical linker among chimera epitopes were similar to porins as the outer-membrane pore-forming proteins. The molecular modeling results confirmed our experimental study.
CONCLUSION
CONCLUSIONS
This investigation illustrated the key role of linker design in determining the expression level of multi-epitope chimera and conformational folding.
Identifiants
pubmed: 31721685
pii: PPL-EPUB-102273
doi: 10.2174/0929866526666191112124602
doi:
Substances chimiques
Epitopes
0
Recombinant Fusion Proteins
0
Viral Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
604-613Informations de copyright
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