Generation of recombinant Agrin

Recombinant human nonneuronal C-terminal Agrin (rAGRIN) was generated as follows. The C-terminal portion of Agrin (amino acids 1244 to 2045 from GenBank accession number BAD52440) was cloned by PCR from the backbone of the full-length human nonneuronal Agrin adenovirus and subcloned into a third-generation lentivirus gene expression vector backbone downstream of the cytomegalovirus (CMV) promoter, an immunoglobulin G (IgG) kappa signal peptide, and followed by an enterokinase cleavage site, thermostable alkaline phosphatase, Myc, and 10X His tags and lastly by a stop codon. The lentivirus backbone was transiently transfected into Expi293 cells (Thermo Fisher Scientific) using the Expi293 Expression System (Thermo Fisher Scientific) as per manufacturer’s instructions. At day 3 after transfection, the cell-free supernatant was collected, and recombinant Agrin was recovered using His SpinTrap columns (GE Healthcare), according to the manufacturer’s instructions.