Figure 2. Recombinant GST-TbFUT1 transfers [³ H]Fuc to a variety of sugar acceptors.

Each assay used 2 μg of purified GST-TbFUT1, GDP-[³H]Fuc, and 1 mM of acceptor. Reaction products were desalted and separated by silica High Performance Thin Layer Chromatography (HPTLC) and detected by fluorography. LNB, LNB-OMe, and LNB-terminating structures were the best acceptors tested. The acceptor abbreviations above each lane are defined in Table 1. -ctrl: negative control reaction without acceptor (lane 4) or with buffer alone (lane 8).

Figure 2—figure supplement 1. Purification of recombinant GST-TbFUT1.

The material encoded by the empty vector (lanes 1–4) and the GST-TbFUT1 (lanes 5–9) was expressed and purified following the same protocol (see Materials and methods). Aliquots from the lysis and affinity purification steps were run on a SDS-PAGE and stained with Coomassie. SF: soluble fraction; P: pellet (insoluble fraction); E1–E3: elutions.

Figure 2—figure supplement 2. TbFUT1 activity is independent of divalent metal cations.

We performed activity assays using lacto-N-biose (LNB) as acceptor (see experimental procedures) either using the complete reaction buffer (+, lanes 1 and 4), removing MgCl₂ and MnCl₂ from it (none, lane 3, 50 mM TrisHCl, 25 mM KCl pH 7.2) or adding EDTA to it (EDTA, lane 2, 50 mM TrisHCl, 25 mM KCl, 5 mM EDTA pH 7.2). A negative control missing the LNB acceptor (-ctr, lane 4) was also performed.