Construction and Selection of Affilin® Phage Display Libraries.

Affilin® molecules represent a new class of so-called scaffold proteins. The concept of scaffold proteins is to use stable and versatile protein structures which can be endowed with de novo binding properties and specificities by introducing mutations in surface exposed amino acid residues. Complex variations and combinations are generated by genetic methods of randomization resulting in large cDNA libraries. The selection for candidates binding to a desired target can be executed by display methods, especially the very robust and flexible phage display. Here, we describe the construction of ubiquitin based Affilin® phage display libraries and their use in biopanning experiments for the identification of novel protein ligands.

The bioactive dipeptide anserine is transported by human proton-coupled peptide transporters.

The bioactive dipeptide derivative anserine (beta-alanyl-1-N-methyl-L-histidine) is absorbed from the human diet in intact form at the intestinal epithelium. The purpose of this study was to investigate whether anserine is a substrate of the H(+)/peptide cotransporters 1 and 2 (PEPT1 and PEPT2). We first assessed the effects of anserine on [(14)C]glycylsarcosine ([(14)C]Gly-Sar) uptake into Caco-2 cells expressing human PEPT1 and into spontaneous hypertensive rat kidney proximal tubule (SKPT) cells expressing rat PEPT2. Anserine inhibited [(14)C]Gly-Sar uptake with K(i) values of 1.55 mM (Caco-2) and 0.033 mM (SKPT). In HeLa cells transfected with pcDNA3-hPEPT1 or pcDNA3-hPEPT2, K(i) values of 0.65 mM (hPEPT1) and 0.18 mM (hPEPT2) were obtained. We conclude from these data that anserine is recognized by PEPT1 and PEPT2. Carnosine also inhibited [(14)C]Gly-Sar uptake. Using the two-electrode, voltage-clamp technique at Xenopus laevis oocytes, strong hPEPT1-specific inward transport currents were recorded for Gly-Sar, anserine and carnosine, but not for glycine. We conclude that anserine and carnosine interact with the human intestinal peptide transporter and are transported by hPEPT1 in an active, electrogenic H(+) symport. As PEPT1 is the predominant transport system for di- and tripeptides at the intestinal epithelium, this transporter is most probably responsible for the intestinal absorption of anserine after food intake. In addition, anserine might be useful for the design of new substrates of peptide transporters, such as prodrugs, that can be administered orally.

Transport of the advanced glycation end products alanylpyrraline and pyrralylalanine by the human proton-coupled peptide transporter hPEPT1.

The glycation compound pyrraline, which originates from the advanced Maillard reaction, appears in urine after consumption of pyrraline-containing food. We hypothesized that the absorption of pyrraline occurs in the form of dipeptides rather than the free amino acid. The human intestinal peptide transporter hPEPT1 was transiently expressed in HeLa cells. In hPEPT1-transfected cells but not in cells transfected with empty vector, the uptake of [(14)C]glycylsarcosine was strongly inhibited by alanylpyrraline (Ala-Pyrr) and pyrralylalanine (Pyrr-Ala). Free pyrraline did not inhibit peptide uptake. In Xenopus laevis oocytes expressing human PEPT1, both Ala-Pyrr and Pyrr-Ala generated significant inward directed currents. In a third approach, uptake of the dipeptides into hPEPT1-transfected HeLa cells was analyzed by HPLC. Ala-Pyrr and Pyrr-Ala were taken up by hPEPT1-expressing cells at a 4- to 7-fold higher rate than by HeLa cells transfected with the empty vector. We conclude that pyrraline containing dipeptides are transported by hPEPT1 in an electrogenic manner into intestinal cells.