title: Cloning, expression, and purification of a recombinant Tat-HA-NR2B9c peptide.
abstract: To design a peptide disrupting the interaction between N-methyl-d-aspartate receptors-2B (NR2B) and postsynaptic density protein-95 (PSD-95), a gene fragment encoding a chimeric peptide was constructed using polymerase chain reaction and ligated into a novel expression vector for recombinant expression in a T7 RNA polymerase-based expression system. The chimeric peptide contained a fragment of the cell membrane transduction domain of the human immunodeficiency virus type1 (HIV-1) Tat, a influenza virus hemagglutinin (HA) epitope-tag, and the C-terminal 9 amino acids of NR2B (NR2B9c). We named the chimeric peptide Tat-HA-NR2B9c. The expression plasmid contained a gene fragment encoding the Tat-HA-NR2B9c was ligated to the C-terminal fragment of l-asparaginase (AnsB-C) via a unique acid labile Asp-Pro linker. The recombinant fusion protein was expressed in inclusion body in Escherichia coli under isopropyl β-d-1-thiogalactopyranoside (IPTG) and purified by washing with 2M urea, solubilizing in 4M urea, and then ethanol precipitation. The target chimeric peptide Tat-HA-NR2B9c was released from the fusion partner following acid hydrolysis and purified by isoelectric point precipitation and ultrafiltration. SDS-PAGE analysis and MALDI-TOF-MS analysis showed that the purified Tat-HA-NR2B9c was highly homogeneous. Furthermore, we investigated the effects of Tat-HA-NR2B9c on ischemia-induced cerebral injury in the rats subjected to middle cerebral artery occlusion (MCAO) and reperfusion, and found that the peptide reduced infarct size and improved neurological functions.
authors.affiliation: Laboratory of Cerebrovascular Disease, Key Laboratory of Cardiovascular Disease and Molecular Intervention, Nanjing Medical University, Nanjing, China.
authors.forename: Hai-Hui
authors.initials: HH
authors.lastname: Zhou
authors.forename: Ai-Xia
authors.initials: AX
authors.lastname: Zhang
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.forename: Dong-Ya
authors.initials: DY
authors.lastname: Zhu
mesh_terms: D000818:Animals; D002545:Brain Ischemia / Q000188:drug therapy; D002544:Cerebral Infarction / Q000188:drug therapy; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D008297:Male; D051379:Mice; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D020011:Protective Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D010455:Peptides; D020011:Protective Agents; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus