Improving Protein Pharmacokinetics by Genetic Fusion to Simple Amino Acid Sequences

Abstract

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.

Construction and analysis of variants of a polyvalent Lyme disease vaccine: Approaches for improving the immune response to chimeric vaccinogens

“…In that case, it was speculated that structural issues or proteolytic degradation by carboxypeptidases was responsible [39][40][41]. Numerous methods have been proposed for protection of peptides and recombinant proteins from protease activity, including amidation or PEGylation of the C-terminus, acetylation of the amino (N)-terminus, and addition of protective amino acid motifs [42][43][44][45][46][47]. Two protective motifs, Gly-Gly and Pro-Pro-Ala, were assessed for their ability to enhance antibody responses to the ABKD vaccinogen.…”

“…Two protective motifs, Gly-Gly and Pro-Pro-Ala, were assessed for their ability to enhance antibody responses to the ABKD vaccinogen. The addition of neutral, hydrophilic Gly residues may reduce the activity of carboxypeptidases C and D, which are specific for hydrophobic and basic C-terminal amino acids, respectively [45,46,48]. The Pro-Pro-Ala motif may sterically hinder carboxypeptidase progression through the juxtaposed, bulky proline residues [47].…”

The intrinsic disorder alphabet. III. Dual personality of serine

“…129 An incomplete list of the disordered serine-containing tags shown to increase the half-life of a protein of interest inside the cell includes an unstructured recombinant polypeptide of 864 amino acids with the PESTAG composition, called XTEN, 130,131 serine-glycine-containing HAP 132 and proline-alanine-serine-containing PAS tags, 133 as well as tags containing PESAK, PASTDH, and PASTD repeats. 134,135 Some functions of serine-rich proteins To get a clue on what proteins with high serine content can be used for biologically, functions of several proteins with polyserine regions are outlined below. Although for a long time, polyserine sequences have been ignored as the non-functional result of transcription slippage, because these and other homopolymer sequences are typically encoded by trinucleotide repeats at the DNA level, 49 data overviewed below clearly shows that polyserine regions not only serve as flexible linkers, but have a rather diverse set of functions.…”

The Gp85 Surface Glycoproteins from Trypanosoma cruzi

“…The protein ends with a second α-helix which, in the members of Group I, is followed by the SAPA repeats, composed by 12 repeated amino acids (Frasch 1994 ) and associated with the stability of TS in the blood (Alvarez et al 2004 ). SAPA, a highly antigenic region (Frasch 1994 ;Alvarez et al 2004 ), is absent from the proteins of Group II. In TS, three putative glycosylation sites are localized at the N-terminus and two at the C-terminus domain (Buschiazzo et al 2000 ).…”