Expression, Purification And Biochemical Properties Of PICK1 And Its Domains

PICK1 (protein interacting with Cαkinase 1) is a protein kinase C (PKC)α-binding protein firstly identified by using yeast two-hybrid system. PICK1is composed of 416 amino acid residues, involving PDZ (PSD-95 Discs largeand Zol) domain at N-terminus as well as BAR (Bin/amphysin/RVS) domainranking the most part of the central region and an acidic amino acid domainclose to C-terminus. The PDZ domain is regarded as the main fragment tomediate the interaction with various proteins, including receptor tyrosinekinases, ionotropic glutamate receptors of the L-α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate subtypes,metabotropic glutamate receptors, ion channels, G protein-coupled receptors,aquaporins, trans-membrane transporters, and ADP-ribosylation factors etc.The BAR domain which is closely related to the function of membrane, suchas endocytosis is also occurred in the molecular structure of many proteins.And the acidic amino acid domain may regulate metal ion binding, Ca²⁺ inparticular. In conclusion, PICK1 plays an important role in clustering severalligand proteins in the plasma membrane, or in targeting them to theperinuclear region of cells. In this report, the main respects are about genecloning, expression, purification and some features of PICK1 as well as itsBAR and PDZ domains. The open reading frame (ORF) of PICK1 gene was cloned to vectorspET32M and pGEX-6p-1, to form recombinant plasmids pET-pick1 andpG-pick1 respectively. Subsequently, each construct was transferred intoE.coli BL 21 to generate the engineered strain E.coli BL 21/pET-pick1 orE.coli BL 21/pG-pick1. The experimental results show that the target proteinexpressed in strain E. coli BL 21/pET-pick1 is as the form of inclusion bodies,whilst the target protein expressed in strain E. coli BL21/pG-pick1 is as asoluble form. The fusion protein was purified by GST-Sepharose 4B affinitycolumn and then cleaved to be a single PICK1 by protease PreScissionProtease (PPase). For this product PICK1, the molecular weight is about 50kDa, as evaluated by SDS-PAGE and the functional unit is about 103 kDa asdetermined by both size-exclusion chromatography and native SDS-PAGE,meaning that the PICK1 protein is a dimer.The ORF of BAR gene was cloned into various vectors containingpE32M, pGEX-6P-1, pHGB, pMAL-p2X and the correspondingrecombinant was expressed in E. coli BL21 or E. coli JM109. The resultsindicated that the engineered strain E. coli JM109/pM-bar can express thefusion protein MBP-BAR in the soluble form, while other strains form thetarget protein in the form of inclusion bodies. The expression amount ofMBP-BAR was about 30% of total proteins in cells. Because the modifiedvector pMAL-s was designed by substituting the base-pair sequence of Factor Xa cleavage site in pMAL-p2X with that of PreScission Protease (PPase) site,the fusion protein can be cleaved by PPase to achieve the BAR domain.In order to separate the enzymatic mixture, several methods were testedas follows: 1, based on MBP amylose affinity, the beads harbored MBP-BARwas directly cleaved by PPase; 2, the enzymatic mixture was applied on toion-exchange resin or hydrophobic support; 3, the enzymatic mixture wasloaded on Sephacryl S-200. Unfortunately, all results were negative. In suchcase, the classic method to precipitate proteins by (NH₄)₂SO₄ was adopted.Fortunately, the cleavage mixture was easily separated to two parts, MBP inthe supernatant and BAR in the precipitate, at 1 mol/L (NH₄)₂SO₄. Theresoluble BAR reached ca.95% purity as analyzed by SDS-PAGE. However,when the BAR protein was run on a Pre-packed Sepharose 12 column, theretention volume was appeared in void volume, imply that the BAR could beas a polymer beyond the separation range of the column.The binding ability of BAR with lipids was confirmed by Protein LipidOverlay (PLO) assay. In addition, CD spectra of the BAR domain combinedwith bovine brain lipid extracts show that there are a few different from thecontrol, revealing that the helix content of the complex is increased and thebeta-sheet content is decreased. Based on the acidic amino acid regioninvolved in this BAR, the regulation of calcium ion was also analyzed duringthe process of the interaction between lipid and BAR domain. The CDspectra reveal that 1mM Ca²⁺ increased the random coil content of BAR domain without seriously affecting the helix content, while both Ca²⁺ andbrain lipid extracts were simultaneously added to the BAR solution, the helixcontent of BAR domain was increased. The fact may explain that theinteraction between BAR domain and lipid regulated by Ca²⁺.The ORF of PDZ gene was cloned vector pE32M and introduce into E.coli BL21. The engineered stain can express the target protein in the form ofinclusion bodies only. After 8 mol/L urea denaturation and then, refoldingwith step-wise dialysis, the protein recovery of the soluble PDZ is about 60ï¼….To conduct the interaction between BAR and PDZ domains, the immobilizedMBP-BAR on amylose beads was mixed with the partner, a soluble PDZdomain, and on contrary, the immobilized NTA-Agrose-PDZ was mixed withthe partner, a soluble BAR protein. After SDS-PAGE analysis, the interactionbetween BAR and PDZ domain was confirmed by in vitro binding assays. Inaddition, a quantitative analysis of the binding capacity between BAR andPDZ was performed to further confirm their interaction. The result shows thatthe actually amount of PDZ bound to Amylose-MBP-BAR reaches 16％±0.5ï¼…for the molar ratio of two proteins, as determined by ELISA. Althoughthe quantitative analysis seems to be lower, the interaction between PDZ andBAR domains was actually occurred based on repeated tests. Meanwhile, theinteraction between BAR domain and PDZ domain was also examined byusing yeast two-hybrid system. The same result as described above was achieved.The interaction of PICK1 and its receptor GluR2 C-terminal regionlabeled with FITC (FITC-GluR2C) was studied by fluorescence spectra. Thefluorescence intensity of the complex FITC-GluR2C at 517 nm wasdecreased in the presence of the PICK1 or its PDZ domain. Thestoichiometric ratio of GluR2C to PICK1 or PDZ domain is 1:1, asdetermined by the fluorescence titration. On the basis of the experimentaldata, the conditional binding constant in 0.05M Hepes, pH 7.2 at 25℃wascalculated to be K_PICK1-GluR2C=1.0×10⁷ M^-1 and K_PDZ-GluR2C=3.0×10⁶ M^-1respectively. Moreover, the fluorescence change of FITC-GluR2C combinedwith BAR domain was not observed. Therefore, the above results show thatthe binding site of GluR2C on PICK1 prefers the PDZ domain to BARdomain, predicting that the BAR domain may facilitate the interactionbetween the PDZ domain and GluR2C.