Figure 1

Shake flask analysis of rTf expression. (A) SDS-PAGE analysis for rTf with and without N-linked glycosylation. 30 μl of five-day BMMD SFC supernatant was analyzed per lane by SDS-PAGE (4-12% Bis-Tris NuPAGE^®, MOPS buffer, Invitrogen). Samples in lanes 2-4 were non-reduced; the corresponding samples in lanes 6-8 were reduced. Track loadings were: Lanes 1 & 5) 5 μl SeeBlue^® Plus2 protein standards (Invitrogen) used for comparing relative migration of Tf samples only. Lanes 2 & 6) DYB7 [pSAC35] supernatant, negative control. Lanes 3 & 7) DYB7 [pDB2506] supernatant, glycosylated transferrin. Lanes 4 & 8) DYB7 [pDB2536] supernatant, rTf (N413Q, N611Q). (B) Western blot analysis for rTf with and without N-linked glycosylation. Lane 1) 5 μl MagicMark™ XP western protein standards (Invitrogen). All other lanes as for (A) above. (C) Relative productivity of rTf (N413Q, N611Q) expression systems showing the effect of PDI1 copy number enhancement. 30 μl of five-day BMMD SFC supernatant was analyzed per lane by non-reducing SDS-PAGE (4-12% Bis-Tris NuPAGE^®, MOPS buffer, Invitrogen). Track loadings were: Lane 1) SeeBlue^® Plus2 protein standards (Invitrogen) used for comparing relative migration of Tf samples only. Lane 2) JRY188 [pDB2536] with one genomic PDI1 gene. Lane 3) JRY188 [pDB2711] with multiple copies of PDI1. Lane 4) DYB7 [pDB2536] with one genomic PDI1 gene. Lane 5) DP9 [pDB2536] with two genomic PDI1 genes. Lane 6) DYB7 [pDB2711] with multiple copies of PDI1.