Figure 7.
LH3 with Mutation 1 or Mutation 2 Produced in Insect Cells
(A) Immunoblot analysis of recombinant LH3, LH3mut1, and LH3mut2, stained with c-myc antibody.
(B) Immunoblot of EndoH-treated (1 hr at 37°C with 50 U EndoH from NEB) LH3 and LH3mut1 shows removal of asparagine-linked oligosaccrarides, seen as a mobility shift of the bands.
(C) Removal of N-linked glycans by Endo H has no effect on GGT activity of LH3mut1. We used 10 μg soluble protein of insect cell supernatant in the analysis. GGT activity result is a mean value of four independent experiments; SD is indicated by the bars. Expression of human LH3 cDNA was carried out in baculovirus transfer vector pFastBacI in the BAC-TO-BAC (Invitrogen) expression system.29 cDNA (coding amino acids 25–738) of LH3 was inserted to the vector in-frame downstream from ER signal sequence from LH1 and c-myc epitope tag. Insect cells were harvested 72 hr after infection and homogenized into a solution containing 0.1 M glycine, 1% Igepal CA-630 (Sigma), and 20 mM Tris-HCl (pH 7.4). The homogenate was sonicated and then centrifuged at 15,000 × g for 20 min and the soluble fraction was used for western-blot analysis. Proteins were transferred from SDS-PAGE gel to Hybond-LFP (GE Healthcare) PVDF membrane. Anti c-myc antibody 9E10 (Santa Cruz) and anti-mouse-IgG-AlexaFluor488 (Molecular Probes) were used with Typhoon 9400 (GE Healthcare) for fluorescence detection.