RÉSUMÉ
We investigated the ability of signal peptides of eukaryotic origin (human, mouse, and yeast) to efficiently direct model proteins to the Escherichia coli periplasm. These were compared against a well-characterized prokaryotic signal peptide-OmpA. Surprisingly, eukaryotic signal peptides can work very efficiently in E. coli, but require optimization of codon usage by codon-based mutagenesis of the signal peptide coding region. Analysis of the 5' of periplasmic and cytoplasmic E. coli genes shows some codon usage differences.
Sujet(s)
Codon/génétique , Escherichia coli/génétique , Régulation de l'expression des gènes bactériens , Périplasme/métabolisme , Signaux de triage des protéines/génétique , Séquence d'acides aminés , Animaux , Séquence nucléotidique , Clonage moléculaire , Bases de données factuelles , Escherichia coli/métabolisme , Gènes bactériens/génétique , Code génétique , Humains , Fragments Fab d'immunoglobuline/génétique , Souris , Données de séquences moléculaires , Mutagenèse/génétique , Conformation d'acide nucléique , Plasmides/génétique , Transport des protéines , Stabilité de l'ARN , ARN messager/composition chimique , ARN messager/génétique , ARN messager/métabolisme , Analyse de séquence de protéine , Levures/génétiqueRÉSUMÉ
The peptide sequence (N)DKTH(C) was previously investigated as a site for efficient, specific cleavage of a fusion protein by cupric ions using a humanized gamma1 Fab' as a model protein. Here we show that conservative mutations to three of the residues in the introduced cleavage site resulted in cleavage sites that were significantly improved. They were cleaved more efficiently by Cu(2+), such that cleavage reactions could be shorter, of lower pH or at a lower temperature. Some were even found to be measurably cleaved by Ni(2+). Use of these new cleavage sequences along with cupric ions may provide a more rapid and less harsh method for cost-effective, large-scale proteolytic cleavage of fusion proteins and peptides.
Sujet(s)
Cuivre/pharmacologie , Fragments Fab d'immunoglobuline/composition chimique , Séquence d'acides aminés , Séquence nucléotidique , Sites de fixation , Séquence conservée , Humains , Concentration en ions d'hydrogène , Fragments Fab d'immunoglobuline/métabolisme , Cinétique , Données de séquences moléculaires , Mutagenèse dirigée , Nickel/pharmacologie , Oligodésoxyribonucléotides , Plasmides , Protéines de fusion recombinantes/composition chimique , Protéines de fusion recombinantes/métabolisme , ThermodynamiqueSujet(s)
Laparoscopie , Grossesse tubaire/chirurgie , Salpingostomie , Femelle , Humains , GrossesseRÉSUMÉ
Antibody-drug conjugates utilize the targetting potential of antibodies to improve the potential of cytostatic or cytocidal drugs. One such murine monoclonal antibody, CTM01 (mCTM01), which recognizes an epitope on breast epithelial mucin, has potential for the treatment of breast and ovarian cancers. We examine in this paper the comparative properties of mCTM01 against a number of other anti-mucin antibodies. We then describe the humanization and high level re-expression of humanized CTM01 (hCTM01), a process designed to avoid the immune response to administered murine antibodies in human patients and to produce sufficient material for clinical studies. We show that the humanized form has properties superior to mCTM01 in terms of binding affinity to antigen presented on tumour cells.