Purification and Characterization of HTRA Proteins

The protease domains of human HTRA1 (HTRA1^PD; D161-K379) and mouse HTRA1 (mHTRA1^PD; D161-K379) contained an N-terminal His6 tag followed by a TEV protease cleavage sequence as described recently^{47 (link)}. The chimeric HTRA1^PD-A202Y was made by introducing an A202Y mutation to the human HTRA1^PD construct. The catalytically inactive S328A form HTRA1^PD(SA) and HTRA1^PD/PDZ (D161-P480) had an N-terminal His6 followed by a thrombin cleavage sequence^{38 (link)}. The protease-PDZ domains of human HTRA3 (HTRA3^PD/PDZ; L130-M453) and HTRA4 (HTRA4^PD/PDZ; G153-N476) contained a C-terminal thrombin cleavage sequence followed by a His6 tag^{44 (link)}. The chimeric HTRA4^PD/PDZ-Y200A was made by introducing a Y200A mutation to the HTRA4^PD/PDZ construct. These constructs were expressed in E. coli and purified as described^{38 (link),44 (link),47 (link)}. Human full-length HTRA1 (HTRA1^FL, Q23-P480) with an N-terminal His6 tag and TEV protease cleavage sequence^{38 (link)} was expressed in Trichoplusia ni cells and purified as described^{38 (link),47 (link)}. The purity of the trimeric HTRA proteins after size exclusion chromatography was assessed by SDS-PAGE and LC/MS. The N- or C-terminal His6 tags of all HTRA proteins were not removed for the biochemical assays (enzyme assays, SPR, active site labeling). Human HTRA2^PD/PDZ (A134-E458) was from R & D Systems (catalog #1458-HT-100) and contained a C-terminal His6. Human HTRA3^FL (M1-M453, catalog #ab134450) and HTRA4^FL (M1-N476, catalog #ab134444) containing a C-terminal His6 tag were from Abcam.