Expression and purification of PEP-1-EGFP fusion protein and its transduction into human umbilical vein endothelial cells / 南方医科大学学报
Journal of Southern Medical University
;
(12): 1114-1117, 2006.
Article
Dans Chinois
| WPRIM
| ID: wpr-334983
ABSTRACT
<p><b>OBJECTIVE</b>To construct the expression vector pET15b-pep-1-EGFP and purify the fusion protein PEP-1-EGFP expressed in E. coli BL21(DE(3)) for evaluating the cell-penetrating capability of the cell-penetrating peptide PEP-1.</p><p><b>METHODS</b>Two oligonucleotides encoding PEP-1 was synthesized and annealed to generate PEP-1-encoding DNA. The recombinant plasmid pET15b-pep-1-EGFP was constructed by inserting PEP-1-encoding DNA and enhanced green fluorescent protein (EGFP) cDNA into pET15b. The fusion protein PEP-1-EGFP expressed in E. coli BL21(DE(3)) was purified with Ni(2+)-resin affinity chromatography and transduced into human umbilical vein endothelial cells.</p><p><b>RESULTS</b>Sequence analysis confirmed successful construction of the expression vector pET15b-pep-1-EGFP, and the fusion protein PEP-1-EGFP was expressed and purified efficiently with a yield of approximately 14.15 mg/100 ml bacteria medium. SDS-PAGE and Western blotting identified the purified protein as PEP-1-EGFP, and the cell-penetration assay verified that the fusion protein could be transduced into human umbilical vein endothelial cells.</p><p><b>CONCLUSION</b>The successful expression and purification of PEP-1-EGFP and its efficient transduction into human umbilical vein endothelial cells provides a basis for PEP-1-mediated biomacromolecular transduction in protein therapy.</p>
Texte intégral:
Disponible
Indice:
WPRIM (Pacifique occidental)
Sujet Principal:
Peptides
/
Plasmides
/
Veines ombilicales
/
Protéines de fusion recombinantes
/
Données de séquences moléculaires
/
Séquence nucléotidique
/
Transfection
/
Cellules cultivées
/
Technique de Western
/
Mercaptamine
Limites du sujet:
Humains
langue:
Chinois
Texte intégral:
Journal of Southern Medical University
Année:
2006
Type:
Article
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