title: Expressed protein ligation for the preparation of fusion proteins with cell penetrating peptides for endotoxin removal and intracellular delivery.
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.
authors.affiliation: Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Hao-Hsin
authors.initials: HH
authors.lastname: Yu
authors.forename: Ikuhiko
authors.initials: I
authors.lastname: Nakase
authors.forename: Sílvia
authors.initials: S
authors.lastname: Pujals
authors.forename: Hisaaki
authors.initials: H
authors.lastname: Hirose
authors.forename: Gen
authors.initials: G
authors.lastname: Tanaka
authors.forename: Sayaka
authors.initials: S
authors.lastname: Katayama
authors.forename: Miki
authors.initials: M
authors.lastname: Imanishi
authors.forename: Shiroh
authors.initials: S
authors.lastname: Futaki
mesh_terms: D000818:Animals; D057846:Cell-Penetrating Peptides / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D002846:Chromatography, Affinity / Q000379:methods; D004731:Endotoxins; D004926:Escherichia coli; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D047668:Inteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: D057846:Cell-Penetrating Peptides; D004731:Endotoxins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins