Overexpression, purification, and characterization of PDZ domain proteins NHERF and E3KARP in Escherichia coli.
NHERF (Na(+)/H(+) exchanger regulatory factor) and E3KARP (NHE3 kinase A regulatory protein or NHERF2) are structurally related adapter proteins that contain two tandem PDZ (PSD-95/Dlg-1/ZO-1) domains. Recent studies suggest that these proteins play important roles in the membrane targeting, trafficking, and sorting of several ion channels, transmembrane receptors, and signaling proteins in many tissues. Both NHERF and E3KARP interact with NHE3 through their C-terminally extended second PDZ domain, and the last 30 amino acids of these PDZ domain proteins interact with ezrin. However, the structural bases of the full-length human NHERF and E3KARP, in their physiological roles on the regulation of NHE3 trafficking, are still unknown. To obtain pure and soluble proteins for crystallization and X-ray studies, NHERF and E3KARP were subcloned into pET-30b and pET-30a vectors, and overexpressed in Escherichia coli strains of BL21(DE3). The soluble NHERF and E3KARP proteins were purified using Ni-NTA, anion-exchange column and gel filtration chromatography. The purity, molecular mass, and the conformation of the proteins were determined by high-performance liquid chromatography, matrix-assisted laser desorption-ionization-time-of-flight mass spectroscopy and circular dichroism studies, respectively.
Park
Kyoung Sun
KS
Korea Nanobiotechnology Center, Pusan National University, Jangjeon-dong, Keumjeong-gu, Busan 609-735, Republic of Korea.
Jeong
Mi Suk
MS
Kim
Jae Ho
JH
Jang
Se Bok
SB
D002942:Circular Dichroism; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000302:isolation & purification; D017923:Sodium-Hydrogen Exchangers; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003598:Cytoskeletal Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor