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J. Biol. Chem., Vol. 279, Issue 5, 3375-3381, January 30, 2004

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Improving Protein Pharmacokinetics by Genetic Fusion to Simple Amino Acid Sequences^*

Paula Alvarez, Carlos A. Buscaglia, and Oscar Campetella¶

From the Instituto de Investigaciones Biotecnológicas, Universidad Nacional de General San Martín, B1650WAB San Martín, Buenos Aires, Argentina

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.

Received for publication, October 15, 2003 , and in revised form, November 10, 2003.

^* This work was supported by grants from the Agencia Nacional de Promoción Científica y Tecnológica and the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) from Argentina and the World Bank/UNDP/WHO Special Program for Research and Training in Tropical Diseases. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY249142 (hydrophilic extension of the TS from T. carassii) and AY243566 (C terminus of the EspF protein from the enteropathogenic E. coli O145:NM strain).

Research Fellow of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET).

Current address: Michael Heidelberg Division of Immunology, Dept. of Pathology, New York University School of Medicine, 550 First Ave., New York, NY 10016.

^¶ Researcher of CONICET. To whom all correspondence should be addressed: Instituto de Investigaciones Biotecnológicas, Universidad Nacional de General San Martín, Predio INTI, Edificio 24, Av. General Paz y Constituyentes, B1650WAB San Martín, Buenos Aires, Argentina. Tel.: 54-11-45807255; Fax: 54-11-47529639; E-mail: oscar{at}iib.unsam.edu.ar.

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