Expression and Purification of NRRV P[4] Antigens

A total of three different NRRV P[4] fusion-protein antigens were used in these studies including E. coli expressed parent protein P[4], K. phaffii (P. pastoris) expressed parent protein P[4], and K. phaffii (P. pastoris) expressed P[4] variant (C173S), which are hereafter referred as E. coli P[4], Pp P[4] and Pp P[4]-C173S, respectively. The detailed description of the design of these P[4] mutants as well as the procedures for their expression and purification are described elsewhere^{31 ,32} (link) and in the companion paper.²³ The purity of these three P[4] samples was estimated to be >90% as determined by SDS-PAGE analysis and densitometry (Supplementary Fig. S1A). Sodium phosphate dibasic heptahydrate and sodium chloride (NaCl) were purchased from Fisher Chemicals (Hampton, NH). Sodium phosphate monobasic monohydrate, 8-anilino-1-naphthalenesulfonic acid (ANS), thimerosal (TH), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Slide-A-Lyzer mini dialysis devices, HPLC vials, LC-MS grade mobile phases and isopropanol were purchased from Thermo Fisher Scientific (Waltham, MA). NRRV P[4] specific mAb was developed by PATH and obtained from Precision Antibody (Columbia, MD) as described elsewhere.²² (link)^,33 (link)