Ppicza expression vector

To screen the Prestwick library, recombinant DS-epi1 was purified from the supernatant of HEK293 cells that were transiently transfected with the full-size (amino acid 1–958) human DS-epi1-myc-HIS sequence inserted in the pcDNA.3 expression vector (Maccarana et al. 2006 (link)). The medium (serum-free) was concentrated with Centriprep Centrifugal Filter Units MWCO 30 kDa (Millipore) and buffer-exchanged to the DS-epi assay buffer by PD-10 column. For all tests following the primary screening, the truncated form (amino acid 23–775-HIS) recombinant human DS-epi1 was prepared as described (Tykesson et al. 2016 (link)). Recombinant human D4ST1-HIS (amino acid 61–376) was produced as described (Tykesson et al. 2018 (link)). Recombinant full-length human HSepi (GLCE) was cloned into pPICZa expression vector (Invitrogen) and was produced as fusion protein-myc-HIS in the medium of Pichia pastoris cultivation. The media was concentrated and dialyzed into assay buffer.

High-fidelity DNA polymerase, restriction endonuclease (NdeI, XhoI), and dNTP were purchased from TaKaRa (Otsu, Japan). pEASY-E2 expression vector and pMD19-T vector were purchased from TaKaRa (Otsu, Japan). pPICZA expression vector was purchased from Invitrogen (Shanghai, China). The plasmid mini-prep kit and DNA gel extraction kit were purchased from Omega (Taipei, USA). One Step cloning kit was purchased from Vazyme biotech (Nanjing, China). Fast MultiSite Mutagenesis System and Bradford protein assay kit were purchased from TransGen (Beijing, China). Escherichia coli Trans1-T1 cells, E. coli BL21 (DE3) cells, and E. coli DMT cells were purchased from TransGen (Beijing, China). All other chemicals were of analytical grade and commercially available.