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Promosome LLC, Scientific Presentation Scientific Presentation

Promosome LLC, Scientific Presentation Scientific Presentation. Expression Technologies for Enhancing Protein Production. Promosome’s technologies enable increased protein production by increasing translation efficiencies. Background: Translation involves 3 stages. 1) initiation

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Promosome LLC, Scientific Presentation Scientific Presentation

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  1. Promosome LLC, Scientific Presentation Scientific Presentation Expression Technologies for Enhancing Protein Production

  2. Promosome’s technologies enable increased protein production by increasing translation efficiencies

  3. Background: Translation involves 3 stages 1) initiation 2) elongation 3) termination

  4. Translation initiation involves ribosomal recruitment and recognition of a start site

  5. Ribosomal recruitment can occur at the m7G cap via various eukaryotic initiation factors (eIFs)

  6. Ribosomal recruitment can also occur at an internal ribosome entry site (IRES)

  7. An initiation codon is then recognized and a ribosomal complex forms at this site

  8. Translation Enhancer Elements (TEEs)

  9. Translational Enhancer Elements (TEEs) • TEEs are short nucleotide sequences that increase translation efficiency • Some function as ribosomal recruitment sites • Powerful synthetic translational enhancers have been generated by linking together five or more individual TEEs

  10. Gtx TEE can dramatically boost protein synthesis in some cell lines without increasing mRNA levels

  11. Activities of synthetic translational enhancers with 5 copies of individual TEEs in CHO-DG44 cells

  12. TEEs can enhance production of recombinant antibodies Expression of this human antibody was enhanced maximally by TEEs in the 5’ leader of the L chain cistron In this example, secretion of L chain dimers was also observed when L chain expression was enhanced • ≈6-fold increase

  13. TEEs enhance expression of a hard-to-express mucin-Fc fusion protein (Recopharma) • ≈2-3-fold increase

  14. RESCUE

  15. RESCUE • Increases protein production by eliminating negative features in mRNAs that can decrease translation initiation • Primarily involves alterations in mRNA coding regions • but does not involve altering codon utilization, minimizing potential alterations to protein secondary structure • Applicable to multiple cell types

  16. RESCUE examples in CHO cells CAT enzyme (CHO-DG44) IgG L and H chain (CHO-K1) EPO (CHO-DG44)

  17. RESCUE-modification of mucin-Fc fusion protein (Recopharma) • ≈7-8 fold increase

  18. Summary • TEEs • Short sequences identified for ability to enhance protein expression in CHO cells • Enable more protein to by synthesized per unit mRNA • May avoid limitations associated with high mRNA levels and enable high expressing cell lines without multiple chromosomal copies • RESCUE • A novel technology is based on our new ideas about translation initiation • Potential advantages include increased protein expression and improved cell physiology • This technology appears to be broadly applicable to many cell types • RESCUE is expected to be compatible with other technologies

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