title: Protein  transduction domain-hA20 fusion protein protects endothelial cells against high  glucose-induced injury.
abstract: We  constructed a plasmid containing a protein transduction domain (PTD) and a  human A20 (hA20) gene fragment; the fusion protein was obtained by highly  expressing this plasmid in the yeast Pichia pastoris GS115. The plasmid  was obtained by adding 9xArg and EcoRІ recognition sites to the end of the primer, and  6xHis-Tag and NotІ  recognition sites to its end. After sequencing, the hA20 gene fragment was  inserted into plasmid pPIC9k to construct expression vector pPIC9k-PTD-hA20;  then, we transfected GS115 with the vector and induced PTD-hA20 protein  expression. We purified protein from the yeast fermentation supernatant using a  nickel column. Human umbilical vein endothelial cells (HUVECs) were cultured in  high glucose medium (30 mM glucose) and in high glucose medium containing  different concentrations of protein. Apoptosis of HUVECs was assayed by TUNEL  72 h later. The biological activity tests indicated that the fusion protein not  only passed through the cell membrane freely, but also inhibited apoptosis of  HUVECs induced by high glucose levels. We conclude that the fusion protein  PTD-hA20 has potential for clinical use.
authors.affiliation: Key Lab of Biomechanics, Department of Anatomy, Third Military Medical University, Chongqing, China. houchunliwf@163.com
authors.forename: C L
authors.initials: CL
authors.lastname: Hou
authors.forename: Q
authors.initials: Q
authors.lastname: Huang
authors.forename: Y
authors.initials: Y
authors.lastname: Wei
authors.forename: W
authors.initials: W
authors.lastname: Zhang
authors.forename: J H
authors.initials: JH
authors.lastname: Mi
authors.forename: D J
authors.initials: DJ
authors.lastname: Ying
authors.forename: Z H
authors.initials: ZH
authors.lastname: Zhou
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D019610:Cytoprotection / Q000187:drug effects*; D004268:DNA-Binding Proteins / Q000494:pharmacology*; D004587:Electrophoresis, Agar Gel; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D016681:Genome, Fungal / Q000235:genetics; D005947:Glucose / Q000633:toxicity*; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D016254:Mutagenesis, Insertional / Q000235:genetics; D009687:Nuclear Proteins / Q000494:pharmacology*; D010843:Pichia / Q000187:drug effects / Q000235:genetics; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D011995:Recombination, Genetic / Q000235:genetics; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3
chemical_list: D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; C076529:TNFAIP3 protein, human; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D005947:Glucose