Improving protein pharmacokinetics by genetic fusion to simple amino acid sequences.
The role of primary amino acid sequences in protein pharmacokinetics, an issue of relevance in both basic knowledge and biotechnology, was addressed here using as a starting point two repetitive antigens from the hemoflagellate Trypanosoma cruzi that are known to stabilize their associated proteins in the bloodstream. A major drawback to their pharmacological application is that these repetitive sequences are highly immunogenic, being therefore the deletion of this characteristic desirable. Based on sequence homology and epitope mapping analyses, an artificial repetitive sequence (PSTAD) was engineered. This motif was tested by genetic fusion to the C terminus of both the trypanosomal trans-sialidase and the rat tyrosine aminotransferase and found to produce a 4.5-6-fold increase in the half-life of the associated proteins in blood while displaying significantly lower immunogenicity. Residues involved in the stabilizing properties of the novel peptide were mapped by a site-directed mutagenesis approach, allowing us to successfully identify another two motifs. Searching databases for sequences displaying some homology, embedded in proline frameworks and associated to shed virulence factors from unrelated microorganisms, resulted in the identification of four other protein extensions. Remarkably, three of them (from Streptococcus pneumoniae, Actinomyces viscosus, and Escherichia coli) revealed similar pharmacokinetic features, suggesting therefore an analogous evolutionarily acquired mechanism to ensure the biodistribution of their corresponding proteins. Our findings indicate that the insertion of defined motifs into a proline-rich framework constitutes a suitable alternative to construct a chimeric protein with extended half-life in blood.
Alvarez
Paula
P
Instituto de Investigaciones Biotecnológicas, Universidad Nacional de General San Martín, B1650WAB San Martín, Buenos Aires, Argentina.
Buscaglia
Carlos A
CA
Campetella
Oscar
O
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001709:Biotechnology / Q000379:methods; D018604:Epitope Mapping; D005821:Genetic Techniques; D006023:Glycoproteins; D008099:Liver / Q000201:enzymology; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009439:Neuraminidase / Q000737:chemistry; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000493:pharmacokinetics*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014444:Tyrosine Transaminase / Q000737:chemistry; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000737:chemistry
D006023:Glycoproteins; D011506:Proteins; D011994:Recombinant Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma; D011392:Proline; D014444:Tyrosine Transaminase; C069715:trans-sialidase; D009439:Neuraminidase