PMID- 22944204
OWN - NLM
STAT- MEDLINE
DCOM- 20130430
LR  - 20121002
IS  - 1096-0279 (Electronic)
IS  - 1046-5928 (Linking)
VI  - 85
IP  - 2
DP  - 2012 Oct
TI  - Cloning, expression, and purification of a recombinant Tat-HA-NR2B9c peptide.
PG  - 239-45
LID - S1046-5928(12)00220-3 [pii]
LID - 10.1016/j.pep.2012.08.011 [doi]
AB  - 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 beta-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.
CI  - Copyright (c) 2012 Elsevier Inc. All rights reserved.
FAU - Zhou, Hai-Hui
AU  - Zhou HH
AD  - Laboratory of Cerebrovascular Disease, Key Laboratory of Cardiovascular Disease 
      and Molecular Intervention, Nanjing Medical University, Nanjing, China.
FAU - Zhang, Ai-Xia
AU  - Zhang AX
FAU - Zhang, Yu
AU  - Zhang Y
FAU - Zhu, Dong-Ya
AU  - Zhu DY
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20120825
PL  - United States
TA  - Protein Expr Purif
JT  - Protein expression and purification
JID - 9101496
RN  - 0 (Hemagglutinin Glycoproteins, Influenza Virus)
RN  - 0 (Peptides)
RN  - 0 (Protective Agents)
RN  - 0 (Receptors, N-Methyl-D-Aspartate)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 0 (tat Gene Products, Human Immunodeficiency Virus)
SB  - IM
MH  - Animals
MH  - Brain Ischemia/drug therapy
MH  - Cerebral Infarction/drug therapy
MH  - Cloning, Molecular
MH  - Escherichia coli/genetics/metabolism
MH  - Hemagglutinin Glycoproteins, Influenza Virus/genetics
MH  - Male
MH  - Mice
MH  - Peptides/genetics/*isolation & purification/*metabolism/pharmacology
MH  - Protective Agents/chemistry/isolation & purification/pharmacology
MH  - Rats
MH  - Rats, Sprague-Dawley
MH  - Receptors, N-Methyl-D-Aspartate/biosynthesis/genetics/*isolation & purification
MH  - Recombinant Fusion Proteins/biosynthesis/genetics/*isolation & 
      purification/pharmacology
MH  - tat Gene Products, Human Immunodeficiency Virus/genetics/metabolism
EDAT- 2012/09/05 06:00
MHDA- 2013/05/01 06:00
CRDT- 2012/09/05 06:00
PHST- 2012/08/03 00:00 [received]
PHST- 2012/08/15 00:00 [revised]
PHST- 2012/08/16 00:00 [accepted]
PHST- 2012/09/05 06:00 [entrez]
PHST- 2012/09/05 06:00 [pubmed]
PHST- 2013/05/01 06:00 [medline]
AID - S1046-5928(12)00220-3 [pii]
AID - 10.1016/j.pep.2012.08.011 [doi]
PST - ppublish
SO  - Protein Expr Purif. 2012 Oct;85(2):239-45. doi: 10.1016/j.pep.2012.08.011. Epub 
      2012 Aug 25.