Auxiliary subunit KChIP4 and Kv4 kalium channel interactional structure and use of function site
A functional, self-amino-based technology, applied in the preparation methods of peptides, chemical instruments and methods, material analysis by optical means, etc.
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Embodiment 1
[0032] Embodiment 1, expression and purification of KChIP4 protein
[0033] 1. Construction of recombinant expression vector
[0034] 1. Construction of recombinant expression vector pET28a-KChIP4 of KChIP4 protein
[0035] RNA was extracted from the whole mouse brain tissue, and cDNA was obtained by reverse transcription. Using the cDNA as a template, the upstream primer 5'-6CACATATGAACTTGGAGGGGCTTGAAAT-3' and the downstream primer 5'-AGCTCCTCGAGCTAGATCACATTTTCAAAGAGCTGCATG-3' were used for PCR amplification to obtain the complete KChIP4 gene. Long cDNA fragments. The obtained KChIP4 gene full-length cDNA fragment was ligated with the pET28a plasmid (Novagen Company) after digestion with restriction endonucleases Nde I and Xho I, and the obtained recombinant expression vector was named pET28a-KChIP4. Both upstream and downstream of the multiple cloning site in the pET28a plasmid contain six histidine tags (His-tag) to facilitate purification in subsequent experiments.
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Embodiment 2
[0047] Example 2, analyzing the crystal structure of KChIP4, clarifying the structural basis of the interaction between Kv4 channel and KChIP4 protein
[0048] 1. Crystal growth
[0049] Mix 1 μl of the KChIP4 protein solution with a concentration of 6 mg / ml in Example 1 with equilibration buffer (25 mM Tris, pH 7.4, 100 mM NaCl, 10 mM DTT and 1 mM ZnCl 2 ) were mixed in equal volumes, and the initial crystallization conditions were screened by the hanging drop diffusion method. Screening of initial crystallization conditions used Hampton's Crystal Screening Kit (Hampton Research Company). The final crystals were placed in the mother liquor (2.0M NaH 2 PO 4 、2.0M K 2 HPO 4 , 0.2M Li 2 SO 4 and 0.1M 3-(cyclohexylamine)-1-propanesulfonic acid (CAPS), pH 10.5) to obtain single crystals of KChIP4.
[0050] 2. Crystal data collection
[0051] The protein crystals were transferred into the above mother liquor containing 30% (volume percentage) glycerol, and cooled in liquid...
Embodiment 3
[0057] Example 3. Based on the structural characteristics of KChIP4, wild-type KChIP4 and three mutants KChIP4-V11E-V14E-I15E, KChIP4-F18E-L21E-F25E and KChIP4-F61E-I68E generated based on the structure were used for gel filtration column layer Analytical method to detect the aggregation state of KChIP4 protein
[0058] By reading the number of milliliters corresponding to the highest absorption peak of gel filtration and comparing it with the standard curve of the protein molecular weight calibrated by the protein standard, it is determined that the aggregation state of the wild-type KChIP4 and its three mutant proteins is monomer or dimer bodies and tetramers. The results showed that the three mutants KChIP4-V11E-V14E-I15E, KChIP4-F18E-L21E-F25E and KChIP4-F61E-I68E could change the aggregation form of KChIP4 protein from multimer to monomer, indicating that KChIP4-V11- The amino acid sites V14-I15, KChIP4-F18-L21-F25 and KChIP4-F61-I68 are important parts of KChIP4 functio...
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