Expression, Purification And Biochemical Properties Of Protein Interacting With PKCα (PICK1)

PICK1, consisting of 416 amino acid reidues, is defined as a protein interacting with PKCa, originally identified by the yeast two-hybrid test. As the only known protein which involves both PDZ and BAR domains, PICK1 plays an important role in neurotransmitter vesicle docking, receptor cycling and regulating AMPA receptor trafficking. In the above processes,α-SNAP mediates the ATP hydrolysis of NSF closely related to membrane fusion. In this report, gene cloning, expression, purification and partially biological properties of PICK1 were conducted. Furthermore, the interaction between PICK1 and a-SNAP was performed as well by the binding assay and spectrosconic methods.The open reading frame (ORF) of PICK1 was cloned into vertor pET32M or pGEX-6p-1 to generate the recombinant plasmid pET-pickl or pG-pick1 respectively. Then engineered strains pET-pick1/E.coli BL21 and pG-pick1/E.coli BL21 were incubated in LB medium and induced at 16℃overnight under the final concentration of 0.2 mmol/L IPTG The results show that the target protein PICK1 was expressed as inclusion bodies in the former strain, while as a soluble form in the latter strain. The yield of fusion protein GST-PICK1 reaches about 20 % of the total proteins in cells. The fusion protein was loaded on a GST-Sepharose 4B affinity column and cleaved with PreScission Protease (PPase) on column. The pure PICK was polished by Superose 12 size-exclusion chromatography, if necessary. The molecular weight of PICK1 is about 50 kDa as evaluated by SDS-PAGE and the functional unit is about 100 kDa as determined by both size-exclusion chromatography and native-PAGE, indicating that the PICK1 protein is a dimer. In the fluorescence analysis, the Ca leads to a reduction in molar fluorescence intensity to about 30 %, compared with the control sample. The 2:1 stoichiometry of Ca²⁺ to PICK1 was confirmed by the fluorescence titration and the binding constant was calculated to be 1.84×10⁸ mol^-1·L.The ORF ofα-SNAP(α-SNAP₂₉₅) and its mutants(α-SNAP₁₁₅ ,α-SNAP_116-295) were cloned into vector pE32M and the corresponding recombinants were expressed in E.coli BL21. The results show that either the wild-typeα-SNAP or its mutants are soluble. Their yields can reach about 35 %- 40 % of total proteins in cells. The above proteins were purified by NTA agarose affinity column and Sephacryl S-200 size-exclusion chromatography respectively.To examine the interaction between PICK1 andα-SNAP, the immobilized NTA agarose-α-SNAP includingα-SNAP₂₉₅ orα-SNAP_116-295 was mixed with the partner, the PICK1 or GST-PDZ. At the same conditions, the immobilized amylose beads harboring MBP-BAR was mixed with the partner, solubleα-SNAP₂₉₅ orα-SNAP_116-295. SDS-PAGE analysis shows that the interaction region between PICK1 andα-SNAP was mapped into the PDZ domain of PICK1 and the C terminus ofα-SNAP(116-295aa). The interaction of PICK1 and its receptorα-SNAP labeled with FITC was also studied by fluorescence spectra. The fluorescence intensity of FITC-α-SNAP at 517 nm was increased in the presence of the PICK1 or its PDZ domain, while the fluorescence change of FITC-α-SNAP combined with BAR domain was not observed. The binding constant of PICK1 toα-SNAP₂₉₅ is calculated to be 4.5×10⁵mol^-1·L, while the binding constant of PDZ toα-SNAP₂₉₅ orα-SNAP_116-295 is 3.0×10⁵ mol^-1·L or 2.9×10⁵ mol^-1·L as determined by the fluorescence titration, indicating that the binding site of PICK1 is dominantly at PDZ domain, which is fitted with the C terminal region of a-SNAP at 116-295 amino acid residues.