Study on the penetrability of PEP-1-P27mt for cell membranes in vitro.
Double-stranded oligomeric nucleotide encoding PEP-1 peptides was synthesized, prokaryotic expression pET15b-pep-1-p27mt recombinant constructed, E. coli BL21 (DE3)pLysS transformed and induced with IPTG to highly express fusion protein PEP-1-P27mt. Fusion protein with an N-terminal His-tag could be purified by Ni2+-resin affinity chromatography and identified by SDS-PAGE and Western blotting. Cultured EC9706 cells treated with PEP-1-P27mt revealed that PEP-1-P27mt was transduced into cells after 15 min and reached maximal intracellular concentrations in 2 h. PEP-1-P27mt of 1 micromol/L final concentration could most strongly suppress the growth. It was suggested that PEP-1 can carry P27mt across membrane, which provides a new biological protocol for using cyclin dependent kinase inhibitors p27mt in suppressing the growth of tumor cells.
Yan
Shirong
S
Department of Clinical Laboratory, Shiyan People's Hospital Affiliated to Yunyang Medical College, Shiyan 442000, China. graceyan@163.com
Zhu
Minglei
M
Qiu
Fangcheng
F
Wang
Lilin
L
Qu
Shen
S
D000595:Amino Acid Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D002463:Cell Membrane Permeability; D049109:Cell Proliferation; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics* / Q000378:metabolism; D003543:Cysteamine / Q000031:analogs & derivatives* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000493:pharmacokinetics*; D014162:Transfection
C503924:Pep-1 peptide; D010455:Peptides; D011993:Recombinant Fusion Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003543:Cysteamine