Pvl1392 vector

DNA sequences coding for Pgm, Ku70a and Ku80c were obtained by gene synthesis (DNA 2.0 or Eurofins MWG/Operon). The synthetic PGM DNA sequence was first cloned into the pMAL-c2x vector (New England Biolabs). The MBP-PGM fusion, the MBP, the KU70a and the KU80c sequences were further introduced into the pVL-1392 vector (BD Biosciences). A HA tag was added to the C-terminus of Ku70a and the N-terminus of Ku80c. All plasmid sequences are provided in a supplementary information file (Text S1). Plasmids pVL1392-MBP-PGM, pVL1392-MBP, pVL1392-KU70a-HA and pVL1392-HA-KU80c were transfected individually into High Five cells together with the BD BaculoGold Linearized Baculovirus DNA (BD Biosciences).

The recombinant Rspo1 proteins (rRspo1) and HGF proteins (rHGF) are as previously described^{16 (link)}_. Basically, the complimentary DNAs (cDNAs) of human Rspo1 and HGF were amplified for the construction of 6-His fusion proteins, using the forward primer for Rspo1: 5′-TTGCGGCCGCATGCGGCTTGGGCTGTG-3′, and the reverse primer for Rspo1: 5′-GGGAATTCGGCAGGCCCTGCAGATGTGAGTGGCC-3′; the forward primer for HGF: 5′-TTGCGGCCGCATGTGGGTGACCAAACTCC-3′, and the reverse primer for HGF: 5′-GGGAATTCTGACTGTGGTACCTTATATG-3′. The inserts of Rspo1 and HGF were digested with NotI/EcoRI. They were ligated into the pVL1392 vector (BD Pharmingen).
Recombinant Rspo1 and HGF were expressed in Sf9 insect cells using the baculovirus expression system (BaculoGold; BD Pharmingen) and purified to homogeneity from the serum-free supernatant of Sf9 cells infected with their respective viral stocks (multiplicity of infection, 2 × 10⁸/ml) by Talon metal affinity chromatography (BD Clontech). Endotoxin levels of these isolated recombinant proteins were <0.1 U/mg of proteins measured by limulus amebocyte lysate (LAL) from Cape Cod. Eluted proteins were dialyzed into PBS buffer.