Pet30a vector

The DNA sequence that encodes the wild-type rat IgA-Fc segment of CH2-CH3-TP was cloned into PET30a vector (Invitrogen) with an N-terminus 6×His-tag. Corresponding point mutations of C471S, C311S, and C311/471S were generated by site-directed mutagenesis. Human IgA1-Fc CH2-CH3-TP cDNA and its mutant for C471S were fused to sequences encoding IL-2 signal peptide and 6×His-tag at the 5′-end in pcDNA3 vector (Invitrogen). Methods for protein expression and purification were described previously (17 (link)). Briefly, rat IgA analogs were expressed in the BL21 DE3 strain of E. coli, and recombinant protein were purified using Histrap HP columns (GE Healthcare), whereas human rIgA mimetics were produced from human embryo kidney (HEK293) cells by transfection of the plasmids. Description of native IgA purification from plasma is in the Supplemental Methods.

INMAP was cloned into the pET30a (+) vector (Invitrogen, USA). The resulting construct was transformed into the bacterial strain BL21 (TransGen Biotech, China) for expression. His-tagged protein was purified on Ni²⁺ beads (GE, USA) from BL21 lysates. For His Pulldown assays, Ni²⁺ beads with INMAP were incubated with HeLa cell extract for 2 h, isolated by centrifugation, washed, and eluted by boiling in Laemmli sample buffer.

FintCSP1 and FoccCSP DNA were released from pEASY-T1/FintCSP1 and pEASY-T1/FoccCSP, respectively, by restriction digestion with BamHI and XhoI and cloned into the pET-30a vector (Invitrogen). The resulting construct was transformed into Transetta (DE3) Escherichia coli competent cells (Transgen). The production of recombinant FintCSP1 and FoccCSP was induced by addition of isopropyl-β-D-1-thiogalactopyranoside (IPTG, 0.2 mM) at 15°C and purified by Ni ion affinity chromatography (Sangon Biotech, Shanghai, China). The target protein was verified by 15% SDS-PAGE, and its concentration was determined with a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). The recombinant purified protein was stored at −20°C until use.