Expi293 expression system kit

Eculizumab Drug Substance was used throughout this study. Ravulizumab Drug Substance was used in all studies to characterize immune complexes by size exclusion HPLC. Laboratory grade ravulizumab was highly purified by chromatography over a HiTrap Protein A HP column (GE Healthcare, cat# 17-0403-01). C5-binding mAb N19-8 [15 (link)], included as a control in some experiments, was purified from the parent hybridoma. MAb TPP-2799, was generated in-house using the sequences of a Chugai Pharmaceutical mAb designated as RO7112689 (also known as crovalimab) and described in CAS Registry Number 1917321-26-6. MAb TPP-3544 was generated in-house based on the sequences described in US Patent #10633434 (heavy chain, SEQ ID NO 353; light chain, SEQ ID NO 354; Regeneron Pharmaceuticals, Inc.) [16 ], later known as REGN3918 and more recently named as pozelimab. Sequences were synthesized and cloned separately into a mammalian expression vector (GeneArt Gene Synthesis, Thermo Fisher Scientific, Waltham MA), and transiently expressed in an Expi293 Expression System kit. The respective mAbs were purified using MabSelect SuRe^TM chromatography (GE Healthcare, cat# 11-0034-95)).

Recombinant UBR5 (and mutants thereof) for biochemical and structural studies were produced in HEK293F cells via transient expression using the Expi293 Expression System Kit (Thermo Fisher) following the manufacturer’s protocol. Recombinant nuclear receptor proteins (RARA, RXRA, GR, ER, VDR as LBD or DBD-LBD constructs) were produced in E. coli BL21-CodonPlus(DE3)-RIL cells grown in LB broth media. Recombinant NCOA1 proteins were produced in Trichoplusia ni and Spodoptera frugiperda insect cells cultured at 27°C in SF4 Baculo Express Media (BioConcept). Functional genomic screens were performed in U937-Cas9 cells provided by the Genetic Perturbation Platform, Broad Institute. Chromatin-immunopreciptiation sequencing (ChIPseq) and RNA sequencing experiments were performed in NB4 cells, provided by the Genetic Perturbation Platform, Broad Institute. A549 cells were purchased directly from ATCC. Cells were authenticated by the Molecular Diagnostics Laboratory (MDL) at Dana-Farber Cancer Institute via high resolution small tandem repeat (STR) profiling compliant with American National Standards Institute (ASN-0002) recommendations. U937 (male), NB4 (female), A549 (male) cell lines were grown in RPMI with 10% FBS and 1% glutamine and penicillin-streptomycin at 37°C and 5% CO2.

His/Avi-tagged bacterial expression constructs encoding the cytoplasmic tails of ephrinB1, ephrinB2, ephrinB3, and integrin αIIb or β3 were expressed in Escherichia coli (DE3), and the biotinylated recombinant proteins were purified as described previously (Pfaff et al, 1998 (link)). A mammalian expression construct encoding his-tagged human full-length kindlin2 was generated in gWIZ vector (Genlantis) by PCR. Recombinant kindlin2 protein was expressed in Expi293F cells and purified according to the instructions of manufacturer using Expi293 Expression System Kit (A14635; Thermo Fisher Scientific). 5 µg of purified cytoplasmic tails immobilized on NeutrAvidin (Pierce Biotechnology) was incubated with EGFP-kindlin2-transfected cell lysate in lysis buffer (50 mM Tris-Cl, 150 mM NaCl, 0.5% NP-40, 0.5% Triton X-100, 0.5 mM CaCl₂, 0.5 mM MgCl₂, 1 µM calpeptin, protease inhibitor cocktail, and PhosSTOP phosphatase inhibitors, pH 7.4) at 4°C for 1 h. Bound proteins were washed with lysis buffer, fractionated on SDS–PAGE, and recognized by Western blotting with anti-GFP antibody.

Heavy and light chain sequences of BCRs of interest were synthesized as gene fragments (IDT, Gene Universal) and cloned into human IgG, IgK, and IgL expression vectors (Human IgG Vector Set, Sigma) according to the manufacturer’s instructions. Recombinant expression of antibodies was performed as previously described using the Expi293 Expression System Kit (Thermo Fisher Scientific) (3.63 (link)) or the ExpiCHO Expression System (Thermo Fisher Scientific). Filtered cell culture supernatant was used for dot blot and VirScan characterization. Antibody purification was performed using NAb Protein A Plus Spin Column (Thermo Fisher Scientific) according to the manufacturer’s instructions.

An expression construct encoding amino acids 358-571 (VEQA…CPKL) of the MERS S glycoprotein and a carboxy terminal 6x HIS tag was synthesized by Twist Biosciences and cloned into the pTwist CMV BetaGlobin WPRE Neo expression vector. Plasmids were produced in NEB 5-alpha competent Escherichia coli (New England Biolabs, Ipswich, MA, USA) and transfected into Expi293 cells using the Expi293 Expression System Kit (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s instructions. Seven days following transfection, cleared supernatants were passed over a HisTrap HP column (Cytiva), and bound protein was eluted with 450 mM imidazole. Buffer exchange to PBS and protein concentration was performed using centrifugal protein concentrators with a 10 kDa MW cutoff (Pierce, Appleton, WI, USA). Final protein concentration was determined by BCA protein assay (Pierce). MW and protein purity were confirmed by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA, USA) (Figure 2).