Figure 2

The impact of multi-copy clones on titer levels. Expression or activity levels were determined from published data and are presented as a ratio compared to the expression or activity of a single copy strain (calculated by dividing by the equivalent value of a single copy clone). A star (*) indicates that values were estimated. Tetanus toxin fragment C utilized different integration sites (HIS4 or AOX1) by linearizing the vector with different restriction sites prior to transformation. Intracellular expression using the AOX1 promoter. Samples were grown in either shake flasks or bioreactors [5]. Mouse epidermal growth factor (mEGF) was expressed as a secreted protein using the AOX1 promoter. Samples were grown in shake flasks and bioreactors [19]. Hepatitis B surface antigen (HBsAg) was expressed intracellularly under the GAP promoter in shake flasks [12]. Trypsinogen (TRY1) was expressed extracellularly using the GAP promoter or AOX1 promoter [20]. Miniproinsulin (MPI) was expressed using the AOX1 promoter as a secreted protein [21]. Necator americanus secretory protein (Na-ASP1) was co-expressed with varying copies of protein disulfide-isomerase (PDI) to determine the impact of chaperone coexpression. All variants were secreted and expressed under the AOX1 promoter [22]. Human superoxide dismutase (hSOD) was expressed intracellularly under the GAP promoter. Integration occurred at the rDNA locus with multi-copy clones generated by PTVA. Stability was observed for 28 generations, indicated by the diagonal stripes [17]. Porcine insulin precursor (PIP), using PTVA to generate multi-copy clones, was expressed under the AOX1 promoter and secreted [16]. Instability was observed in clones (under inducible conditions) with a copy number above 6, indicated by the horizontal stripes [23]. Interleukin and human growth hormone proteins were fused with HSA, IL-HSA and HG-HSA respectively, and co-expressed with PDI or BiP. The fusion proteins were secreted under the control of the AOX1 promoter [24].