【作者】 王丽丽；

【导师】 石亚伟；

【作者基本信息】 山西大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 蛋白激酶Ca相互作用蛋白（protein interacting with Cαkinase1，PICK1）是蛋白激酶Cα（protein kinase Cα，PKCα）的靶蛋白之一，是衔接PKCα和膜上众多受体的关键蛋白，在人和动物的细胞或组织内均可表达。小鼠来源的PICK1由416氨基酸残基组成，其N-末端含有PDZ（（?）SD95 （?）iscs large and （?）ol）结构域，C-末端含有一个BAR（Bin／amphiphysin／RVS）结构域，靠近C-末端含有一个酸性氨基酸区。α-SNAP蛋白（Soluble NSF Attachment Proteins）是膜泡转运中开启NSF的ATP水解酶活性的重要调节蛋白之一。在PICK1调节突触后膜上AMPA受体的胞吞作用引起LTD的过程中，α-SNAP协助参与不同阶段蛋白复合体的形成，但目前对PICK1蛋白和α-SNAP蛋白相互作用研究的报道比较少。本文对PICK1蛋白进行了基因克隆，重组蛋白的大量表达和纯化，并对其溶液中分子状态、钙离子结合性质，以及与α-SNAP蛋白之间的相互作用进行了初步研究。根据报道的PICK1序列，设计PCR引物，以重组质粒pRK5-pick1为模板，经PCR扩增获得PICK1基因，分别克隆到pET32M和pGEX-6p-1载体中，并在E．coliBL21中进行表达。重组子pET-pick1表达产物为包涵体，而重组子pG-pick1表达产物为可溶性融合蛋白GST-PICK1，可溶性产物占细胞总蛋白的20％，经GST-Sepharose 4B亲和层析和PreScission Protease（PPase）酶切，以及Sephacryl S-200凝胶层析获得了电泳纯的PICK1蛋白，经SDS-PAGE分析，其单体分子量约为50kDa。天然聚丙烯酰胺凝胶电泳和分子排阻层析结果表明，PICK1蛋白在溶液中主要以二聚体形式存在，分子量约为100 kDa。我们用荧光实验初步分析了PICK1蛋白与钙离子的结合特性，发现随着Ca²⁺浓度的增加，PICK1的内源荧光强度逐渐下降，经计算每个PICK1蛋白中可结合2个Ca²⁺，结合常数为1.84x10⁸ mol^-1·L。将α-SNAP基因及突变体α-SNAP₁₁₅，α-SNAP_116-295基因克隆到表达载体pE32M中，并在E．coli BL21中进行表达，目的蛋白以可溶形式存在，表达量可达到细胞总蛋白的20％-30％。经Ni²⁺-NTA agrose亲和层析和Sephacryl S-200凝胶层析获得电泳纯的目的蛋白。利用亲和层析法对α-SNAP蛋白和PICK1蛋白及其PDZ结构域，BAR结构域的相互作用进行比较，初步推测α-SNAP蛋白可能主要与PDZ结构域结合。在此基础上，用FITC标记α-SNAP蛋白，研究PICK1蛋白与α-SNAP不同截短型之间的相互作用，进一步证实PICK1蛋白与α-SNAP蛋白的相互作用区域可能主要位于PICK1蛋白的N-末端PDZ结构域和α-SNAP的C-末端含116-295氨基酸残基部分。并求得PICK1蛋白与α-SNAP₂₉₅的结合常数为4.5x10⁵ mol^-1·L，而PDZ蛋白与α-SNAP₂₉₅和α-SNAP_116-295的结合常数分别为3.0x10⁵ mol^-1·L和2.9x10⁵ mol^-1·L。更多还原

【Abstract】 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.更多还原