NMR samples were prepared by dissolving the lyophilized proteins in 450 μL of 20 mM phosphate buffer (pH 6.0) for the NgBR ectodomain or Milli-Q water for the cytoplasmic domain and full-length proteins of NgBR (pH 4.5), with an addition of 50 μL of D2O for NMR spin-lock. HSQC experiments were acquired on an 800 MHz Bruker Advanced spectrometer equipped with pulse field gradient units at 298 K as previously described [14], [15] and [16]. Spectral processing and analysis were carried out using NMRpipe [17] and NMRview [18].
Results
Cloning and ASP3026 of NgBR and its dissected domains
The full-length NgBR, as well as its cytoplasmic domain and ectodomain were successfully cloned into pGEX 4T-1 or pET32a vector with BamHI/XhoI restriction sites and expressed as GST-fused or His-tagged proteins. The NgBR ectodomain was expressed as a GST fusion protein which was purified under native condition (Fig. 1B). After removing the GST by in-gel thrombin cleavage, the NgBR ectodomain was further purified by HPLC. On the other hand, the His-tagged cytoplasmic domain and full-length NgBR were found to exist in the inclusion body. As such the two recombinant proteins were first purified by Ni2+-affinity column under denaturing condition (Fig. 1C and D) followed by HPLC purification. The molecular weights of all three recombinant proteins determined by MALDI-TOF mass spectrometry matched those predicted from their amino acid sequences.
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