Recombinant Plasmid Construction, Expression And Purification Of Tat-HA-NR2B9C Fusion Protein

Brain damage of stroke or CNS trauma occurs as a result of overstimulation of N-methyl-D-aspartate (NMDA) receptor by L-glutamate,resulting in dysfunction of downstream signaling systems. This is the hypothesisof excitotoxicity. In theory, excitotoxic glutamate signaling can be disrupted byblockage of NMDA receptor. However, NMDA receptor antagonists failed toshow efficacy in clinical trials of stroke or traumatic brain injury as these causeddisruption of normal brain function and adverse side effects. An alternativeapproach to block NMDA receptor focuses on the downstream signalingpathways of NMDA receptor.At excitatory synapses of central neurons, ionotropic glutamate receptorsare organized into multiprotein signaling complexes within the postsynaptic density (PSD). A prominent organizing protein is PSD-95, which couples theNMDAR to intracellular proteins and signaling enzymes. Through its secondPDZ domain (PDZ2), PSD-95 binds the COOH-terminus tSXV motif ofNMDAR NR2 subunits as well as neuronal nitric oxide synthase (nNOS). Basedon the findings that linked NMDA receptor activation to downstream nitricoxide (NO) toxicity through the postsynaptic density protein PSD-95, thepeptide fluorescent-labelled Tat-NR2B9C was transduced in to neurons todisrupte the interaction of NMDA receptors with the PSD-95. This proceduredissociated NMDA receptors from downstream neurotoxic signaling withoutblocking synaptic activity or calcium influx. In vivo experiments show that thepeptide reduced focal ischemic brain damage in rats, and improved theirneurological function. This approach circumvents the negative consequencesassociated with blocking NMDARs and may constitute a practical stroketherapy.In this study, we expressed the fusion peptide Tat-HA-NR2B9C throughprokaryotic expression, which is relative to the Tat-NR2B9C. The peptideconsists of three parts: 1) Tat, Protain transduction domain (PTD) of the humanimmunodeficiency virus-type 1 (HIV-1). 2) HA (hemagglutinin), an epitope tag.3) NR2B9c, the nine COOH-terminal residues of NR2B. Tat renders the fusionpeptide cross Blood Brain Barrier and cell membrane. HA tag is an epitope tagdesigned for detection in western blot assay. NR2B9c disrupts the interaction ofNMDA receptors with PSD-95. The purpose of our experiment is to express and purify the fusion peptideTat-HA-NR2B9C. We designed the DNA construct of Tat-HA-NR2B9C basedon its amino acid sequence using E. coli preferenced code. Four Primers (P1-P4)was sythesised. The DNA constructs encoding Tat-HA-NR2B9C is fused to theC-terminal encoding sequence of asparaginase, encompassing residues 199 to326, through an acid-labile aspartyl-prolyl linker. The whole fragment codingAnsB-C-Tat-HA-NR2B9C was generated by three times add-PCR amplificationwith upstream common primer P1 and with downstream primer P2 for the firststep, P3 for the second step, P4 for the third step, respectively. The PCRfragment with an EcoRⅠsite located at the 5' end next to the coding sequenceof ansB-C and an NcoⅠsite located at the 3' end. The whole gene was clonedinto pET28a prokaryotic expression vector. The recombinant plasmid,pED-Tat-HA-NR2B9C, was transformed in to E.coli BL21(DE3). Theexpression of AnsB-C-Tat-HA-NR2B9C was induced at the final concentrationof 5mM lactose when the A550 of culture is 1.3-1.5. In LB medium, the fusionprotein was expressed, and the amount is below 5% of the whole cell protein inE.coli. Then we optimize the LB medium and induction time. We added0.1%(W/V) Arg in the LB medium, called Arg-Rich LB medium. In theArg-Rich LB medium, after induction for 4h, the fusion protein was highlyexpressed. The amount of fusion protein reached 30% of the whole cell protein.By means of washing the inclusion bodies and precipitation by 2 volume ofethanol, the fusion protein AnsB-C-Tat-HA-NR2B9C was purified. The Tat-HA-NR2B9C was released from the fusion protein by cleavage with80mmol/L HC1 at 50℃for 72h, then purified through chromatography on aCM-cellulose column.The experiment constructed the recombinant expression vector ofpED-Tat-HA-NR2B9C, expressed and purified the peptide Tat-HA-NR2B9C.We set up a new method of preparation of recombinant Tat fusion peptide byfusing the protein to the C-terminal of asparaginase through an acid-labileaspartyl-prolyl linker. This method may provide a new strategy for preparing aseries of mutant peptides of Tat-HA-NR2B9C, which will provide us moreunderstanding of protein-protein interactions. In addition, the use ofTat-HA-NR2B9C peptide in vivo may gain more avidence for theNMDAR/PSD-95/nNOS coupling in neurongenesis and fuction of memory andlearning.