Ni sepharose 6 fast flow

The three plasmids pET-espP1, pET-espP2 and pET-apd were used to transform E. coli BL21 (DE3). EspP1, EspP2, and Apd were expressed as His fusion proteins in E. coli BL21 (DE3) and purified using Ni Sepharose 6Fast Flow (GE Healthcare Biosciences, Pittsburgh, PA, USA). The three purified protein concentrations were measured using a BCA kit (Bioshap, Hefei City, China).

Expression host strain E. coli BL21(DE3) and plasmid pET22b(+) were purchased from Novagen (San Diego, CA, USA). E. coli strain TOP10F’ was obtained from Invitrogen (San Diego, CA, USA). QIAprep spin miniprep kit was from Qiagen (Germantown, MD, USA). Wizard SV Gel and PCR Clean-Up DNA Purification System for elution of DNA fragments from agarose gel was purchased from Promega (Madison, WI, USA). Enzymes and other reagents for DNA manipulation were from New England Biolabs (Ipswich, MA, USA). Ni Sepharose^™ 6 Fast Flow was from GE Healthcare (Uppsala, Sweden). The GKY20 peptide was chemically synthesized by INBIOS s.r.l. (University of Naples, Italy). Difco Nutrient Broth was from Becton-Dickenson (Franklin Lakes, NJ). All other chemicals were from Sigma-Aldrich (Milano, Italy).

Ig sequences were cloned into human IgH Fab and IgK plasmids, and Fabs were expressed by transient transfection of HEK293-6E cells and purified with Ni Sepharose 6 Fast Flow (GE Healthcare). After buffer exchange in PBS, the yield was determined by measurement with nanodrop analysis and PAGE.

A Ni²⁺ affinity column (10 i.d. × 50 mm, Ni Sepharose 6 Fast Flow) used for the purification of the recombinant MA was purchased from GE Healthcare (Chicago, IL, USA). Isopropyl β-d-1-thiogalactopyranoside (IPTG), 1,1-diphenyl-2-picrylhydrazine (DPPH), dimethyl sulfoxide (DMSO), and maltodextrin (dextrose equivalent 4.0–7.0) were bought from Sigma (St. Louis, MO, USA). α-CD, β-CD, γ-CD, soluble starch, and pullulan were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Restriction enzymes and DNA-modified enzymes were obtained from New England Biolabs (Ipswich, MA, USA). All kits for molecular cloning, including the Geno Plus Genomic DNA Extraction Midiprep System, Mini Plus Plasmid DNA Extraction System, Gel Advanced Gel Extraction Miniprep System, and Midi Plus Ultrapure Plasmid Extraction System, were purchased from Viogene (Taipei, Taiwan). Other reagents and solvents used are commercially available.

AtBIN2 full‐length cDNA was cloned into the pGEX4T‐1 vector with the GST‐tag, and AtJAZ1 full‐length cDNA was cloned into the pET32a vector with the His‐tag. The resulting plasmids were transformed into E. coli strain BL21 (DE3). The recombinant proteins AtBIN2‐GST and AtJAZ1‐His were purified with Glutathione Sepharose™ 4 Fast Flow and Ni Sepharose™ 6 Fast Flow (GE Healthcare, Pittsburgh, PA), respectively, according to the manufacturers’ instructions. The pull‐down assays were performed as described in previous studies (Wang et al., 2011 (link)). Proteins retained on the beads were analysed by immunoblotting with an anti‐GST or anti‐His antibody.

MHCII monomers were produced in High Five insect cells (Trichoplusia ni BTI-Tn-5B1-4 cells, Invitrogen) using the baculovirus expression system^{40 (link),54 (link),55 (link)}. DNA encoding the I-A^b α- and β-chains and the mouse MPO_415–428 (₄₁₅KLYQEARKIVGAMV₄₂₈), fused to the N-terminus of the β-chain via a flexible linker (SGGSGSGSAS), were cloned into pFastBac Dual vector and recombinant baculovirus propagated in Sf9 insect cells (Spodoptera frugiperda, Invitrogen). The C-termini of the I-A^b α- and β-chains contained enterokinase cleavable Fos and Jun leucine zippers, respectively, to promote correct heterodimeric pairing. The C-terminus of the β-chain also contained a BirA ligase recognition sequence for biotinylation and poly-histidine tag for purification, immediately following the Jun leucine zipper sequence. MPO:I-A^b monomers were purified from baculovirus infected High Five insect cell supernatants through immobilized metal ion affinity (Ni Sepharose 6 Fast-Flow, GE Healthcare), size exclusion (S200 Superdex 16/600, GE Healthcare) and anion exchange (HiTrap Q, HP, GE Healthcare) chromatography. MPO:I-A^b tetramers were assembled by the addition of Streptavidin-PE (BD Biosciences)^{54 (link),55 (link)}.