Peptide Aptamer Libraries

Abstract

Peptide aptamers are molecules that bind to protein targets and are able to interfere with their functions. In the past, important achievements have been made using such peptide aptamers in different approaches and for various purposes. Peptide aptamers are comprised of a variable peptide region of 8 to 20 amino acids in length, which is displayed by a scaffold protein. An overview of the numerous scaffold proteins that have been investigated for their suitability to present peptide aptamers will be given. To identify peptide aptamers efficiently and specifically binding to a predetermined target, two eukaryotic systems have been used in multiple studies: a modified version of the Gal4 yeast-two-hybrid system and the optimized LexA interaction trap system. The two yeast systems are compared and the design of high-complexity peptide aptamer libraries for these systems is described. Although the yeast-two-hybrid system is based on intracellular interactions mammalian screens, performed in cell culture experiments, are sometimes preferred or required. We will give an overview of the mammalian selection systems available, which are based on the expression of peptide aptamers in retroviral or lentiviral vectors. We will show that the isolation and use of peptide aptamers as inhibitors of individual signaling components represents a new challenge for drug development.

Peptide Aptamers: Development and Applications

“…The scaffold has to be soluble, non-toxic and capable of high-level expression in prokaryotic systems. The cloned scaffold gene should be engineered to accommodate functional protein elements such as localization signals, and epitope and purification tags [57]. It is highly desirable that permutations introduced into variable regions do not adversely affect solubility, folding and the aggregating properties of the resulting combinatorial product.…”

“…Employing the yeast two-hybrid method a number of peptide aptamers, such as PA against human cyclin-dependent kinase 2 (Cdk2) [37] and E2F transcription factor, were identified from corresponding random loop libraries, as well as several other targets, mostly inhibitors of signaling pathways involved in cancer development and progression. Owing to its remarkable characteristics, TrxA was the first widely used peptide aptamer scaffold (see Table 1 in review [57]). In 2012 Groner et al [67] described a PA against Stat3 and Survivin, selected using human Thioredoxin (hTrx) that was modified to provide high levels of expression and facilitate cellular delivery; in 2013 the same group developed a PA against Stat5 [68].…”

“…This general principle was employed by Brent [37] in designing the first peptide aptamer library using fusions of Thioredoxin-constrained peptides with Gal4 AD as a prey and Cdk2-fused BD as bait. This was later followed by construction and Y2H screening of more peptide aptamer libraries with scaffold-constrained and linear peptides [57]. …”

Increasing the range of drug targets

“…Peptide aptamers are short peptides, usually 12 to 20 amino acids in length, which can be selected from random, high-complexity peptide libraries in yeast two-hybrid screens 11,35 Our experiments have shown that a variant of the human thioredoxin (hTrx), devoid of cysteine residues, provides a favorable scaffold for the display of such target interacting peptides. Appropriately appended scaffolds allow the presentation of the peptide in a constrained conformation, the production as recombinant proteins and the cellular delivery of specific peptide aptamers.…”

“…35 If the inhibition of the target protein triggers a particular phenotype, these peptide sequences can be validated as functional inhibitors upon expression in cells 39 or upon transduction of recombinant proteins into cells. 13 These cell culture experiments, however, cannot easily be extrapolated to the in vivo situation.…”