Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation

Ting Gao; Mingdong Hu; Xiaopeng Zhang; Hongzhen Li; Lin Zhu; Hainan Liu; Qincai Dong; Zhang Zhang; Zhongyi Wang; Yong Hu; Yangbo Fu; Yanwen Jin; Kaitong Li; Songtao Zhao; Yongjiu Xiao; Shuping Luo; Lufeng Li; Lingfang Zhao; Junli Liu; Huailong Zhao; Yue Liu; Weihong Yang; Jing Peng; Xiaoyu Chen; Ping Li; Yaoning Liu; Yonghong Xie; Jibo Song; Lu Zhang; Qingjun Ma; Xiuwu Bian; Wei Chen; Xuan Liu; Qing Mao; Cheng Cao

Recombinant protein expression and renaturation were performed as described (24, 41) . In brief, pET22b-MASP-2 was transformed into the expression host strain BL21 (DE3). After induction with 1 mM IPTG, cells were harvested and sonicated. The inclusion bodies were solubilized in 6 M GuHCl, 0.1 M Tris-HCl (pH 8.3), and 100 mM DTT at room temperature; the 5 solubilized proteins were then diluted into refolding buffers containing 50 mM Tris-HCl, 3 mM reduced glutathione (Sigma), 1 mM oxidized glutathione (Sigma), 5 mM EDTA, and 0.5 M arginine. The protein samples were then cooled to 4°C. The renatured protein was dialyzed against 20 mM Tris, 140 mM NaCl, pH 7.4 at 4°C, concentrated with PEG8000, aliquoted, and stored at -70°C. 10 To obtain high-activity MASP-2, Flag-tagged MASP-2 was expressed in 293T cells, precipitated with anti-FLAG magnetic beads, and eluted with Flag peptide (Sigma). The concentration of MASP-2 was assessed using a BCA kit and immunoblot analysis, with the purified prokaryotic-expressed MASP-2 as a standard control.