| RRC ID |
38220
|
| 著者 |
Yu HH, Nakase I, Pujals S, Hirose H, Tanaka G, Katayama S, Imanishi M, Futaki S.
|
| タイトル |
Expressed protein ligation for the preparation of fusion proteins with cell penetrating peptides for endotoxin removal and intracellular delivery.
|
| ジャーナル |
Biochim Biophys Acta
|
| Abstract |
Expressed protein ligation (EPL) is a useful method for the native chemical ligation of proteins with other proteins or peptides. This study assessed the practicability of EPL in the preparation of fusion proteins of enhanced green fluorescent protein (EGFP) with chemically synthesized cell-penetrating peptides (CPPs) for intracellular delivery. Using intein-mediated purification with an affinity chitin-binding tag (IMPACT) system, the thioester of EGFP (EGFP-SR) was prepared. Optimization of the ligation of EGFP-SR with arginine 12-mer (R12) produced the fusion protein in high yield. The EPL procedure also allows the preparation of EGFP-R12 containing a low level of endotoxin (ET), via the satisfactory ET removal of EGFP-SR prior to ligation with the R12 peptide. Fusion proteins of EGFP with R12 and the d-isomer of R12 prepared by EPL showed similar levels of cellular uptake compared to the fusion protein directly expressed in Escherichiacoli.
|
| 巻・号 |
1798(12)
|
| ページ |
2249-57
|
| 公開日 |
2010-12-1
|
| DOI |
10.1016/j.bbamem.2010.02.003
|
| PII |
S0005-2736(10)00043-X
|
| PMID |
20170629
|
| MeSH |
Animals
Cell-Penetrating Peptides / biosynthesis
Cell-Penetrating Peptides / chemistry
Cell-Penetrating Peptides / genetics
Cell-Penetrating Peptides / isolation & purification
Cell-Penetrating Peptides / pharmacology
Chromatography, Affinity / methods
Endotoxins*
Escherichia coli
Gene Expression
Green Fluorescent Proteins / biosynthesis
Green Fluorescent Proteins / chemistry
Green Fluorescent Proteins / genetics
Green Fluorescent Proteins / pharmacology
HeLa Cells
Humans
Inteins
Recombinant Fusion Proteins* / biosynthesis
Recombinant Fusion Proteins* / chemistry
Recombinant Fusion Proteins* / genetics
Recombinant Fusion Proteins* / isolation & purification
Recombinant Fusion Proteins* / pharmacology
|
| IF |
3.411
|
| 引用数 |
11
|
|
WOS 分野
|
BIOPHYSICS
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
| リソース情報 |
| ヒト・動物細胞 |
HeLa(RCB0007) |
