title: Small structural differences of targeted anti-tumor toxins result in strong variation of protein expression.
abstract: Targeted anti-tumor toxins consist of a toxic functional moiety that is chemically linked or recombinantly fused to a cell-directing ligand. Ribosome-inactivating proteins (RIPs), especially type I RIPs such as saporin or dianthin, are commonly used as toxin components. Although expression of type I RIP-based fusion proteins is well reported, the achievement of higher protein yields in heterologous expression systems through innovative strategies is of major interest. In the present study, the targeted toxins (his)saporin-EGF (SE) and (his)dianthin-EGF (DE) were expressed as fusion proteins under identical expression conditions. However, the total amount of DE was nearly two-times higher than SE. The identity of the heterologously expressed targeted toxins was confirmed by mass spectrometric studies. Their biological specific activity, monitored in real time, was almost equal. Sequence alignment shows 84% identity and a structural comparison revealed five major differences, two of which affect the secondary structure resulting in a loop (SE) to β-strand (DE) conversion and one introduces a gap in SE (after position 57). In conclusion, these structural variations resulted in different protein expression levels while codon usage and toxicity to bacteria were excluded as a cause. Minor structural differences identified in this study may be considered responsible for the protection of DE from bacterial proteases and therefore may serve as a lead to modify certain domains in type I RIP-based targeted toxins.
authors.affiliation: Institut Für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité - Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany.
authors.forename: Roger
authors.initials: R
authors.lastname: Gilabert-Oriol
authors.forename: Mayank
authors.initials: M
authors.lastname: Thakur
authors.forename: Christoph
authors.initials: C
authors.lastname: Weise
authors.forename: Jens
authors.initials: J
authors.lastname: Dernedde
authors.forename: Benedicta
authors.initials: B
authors.lastname: von Mallinckrodt
authors.forename: Hendrik
authors.initials: H
authors.lastname: Fuchs
authors.forename: Alexander
authors.initials: A
authors.lastname: Weng
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D000922:Immunotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D015203:Reproducibility of Results; D054789:Ribosome Inactivating Proteins, Type 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D000076985:Saporins; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
chemical_list: D000970:Antineoplastic Agents; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D000076985:Saporins
keywords: Dianthin; Protein expression; Protein structure; Saporin; Targeted toxins; Tumor therapy