Fig. 1. Ulp1 processes the Pmt3 precursor. (A) Recombinant 6xHis-tagged Ulp1 expressed in E. coli. Extracts were made from cells transformed with empty pRSETB (lane 1) or pRSETB-Ulp1 (lane 2), separated by SDS PAGE (10% polyacrylamide) and western blotted using anti-His antisera. (B) Recombinant Ulp1 was tested for Pmt3-processing activity as described in Materials and Methods. Lanes 1-10, ³⁵S-labelled Pmt3 or Pmt3-GG produced in the TnT system. Lane 1, full-length Pmt3 with no additions; lane 2, Pmt3-GG with no additions. Lanes 3-9 full-length Pmt3 incubated with 0.72 µg of recombinant Ulp1. Lane 3, no inhibitors; lane 4, 1:1000 mammalian protease inhibitor cocktail; lane 5, 1:1000 yeast protease inhibitor cocktail; lane 6, 5 mM benzamidine; lane 7, 10 mM iodoacetamide; lane 8, 10 mM NEM; lane 9, 1 mM PMSF; lane 10, 2 µl elution fraction from E. coli BL21 + pRSETB. Lanes 11-13, full-length Pmt3 (600 ng) or Pmt3-GG (600 ng) purified from E. coli BL21 + pET15b-Pmt3 or E. coli BL21 + pET15b-Pmt3-GG respectively. Lane 11, full-length Pmt3, no additions; lane 12, Pmt3-GG no additions; lane 13 full-length Pmt3 incubated with 0.72 µg Ulp1. All samples were incubated at 30°C for 30 minutes.

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