Figure 1.
ItkPH shows poor soluble expression compared with TecPH and BtkPH. (A) Domain architecture of constructs used in this study: full-length Itk and three PH-TH domain constructs with N-terminal purification/solubility tags as indicated. Numbering refers to the mouse Itk sequence. (B-D) Itk, Tec, and Btk PH-TH domain constructs with different N-terminal purification/solubility tags were expressed in BL21(DE3) cells, and the pellet (P) and soluble (S) fractions were analyzed by SDS-PAGE; the boxes indicate the bands corresponding to the PH-TH domain on each gel. (E) Superposition of one-dimensional 1H NMR spectra for His6-GB1-TecPH (black spectrum) and His6-GB1-ItkPH (red spectrum) in 100% D2O at identical concentrations (90 μM). The non-exchangeable, aliphatic region is expanded for clarity.