Technical Background

For several protein targets the initial task towards commercial manufacturing is the careful and correct choice of the best suited expression system. The Xplor Vector System, developed at the IPK, Gatersleben and provided by ARTES, is the ideal tool to allow for an initial selection of the optimal host from available yeast platforms. While quantitative questions are addressed at a later stage – applying strong homologous and inducible promoter elements – expression from the Xplor Vector System answers relevant qualitative aspects, such as processing, stability and post-translational modification.

The Xplor Vector System is composed of a fixed backbone for propagation in E. coli and individual modules for:

• integration into the yeast's rDNA
• selection of integrants (auxotrophic and dominant marker)
• transient or permanent autonomous replication
• recombinant gene expression (constitutive, wide range promoter and terminator)

From the resulting vector portfolio expression cassettes can be excised for single or multi-copy integration. The expression cassettes can furthermore be designed in such a way that the generated integrants are void of any bacterial sequences.

These expression cassettes are stably integrated into the rDNA of yeast genomes. The approach of comparative expression from a single vector source is designed for virtually any yeast platform and verified up to now for:
Hansenula polymorpha

• Arxula adeninivorans (budding and filamentous cells)
• Saccharomyces cerevisiae
• Pichia pastoris
• Yarrowia lipolytica
• Debaryomyces polymorphus and hansenii

Further yeast expression systems to be included are Kluyveromyces lactis, Candida boidinii, Pichia stipitis and Schwanniomyces occidentalis. Especially for the yeasts P. stipitis, S occidentalis and S.cerevisiae a general proof-of-principle for an autonomous wide range vector has already been successfully applied in the past (Piontek et al., 1998).