Peptide Aptamers in Label-Free Protein Detection: 1. Characterization of the Immobilized Scaffold

Abstract

Protein microarray development is absolutely dependent upon the ability to construct interfaces capable of specific, stable, sensitive, and designable recognition of specific proteins. Peptide aptamers, being peptide recognition moieties presented and constrained by a robust scaffold protein, offer one possible solution. The relative uniformity of a scaffold protein across potentially many thousands of arrayed peptide aptamers is predicted to simplify the production of microarrays. This paper describes the generation and assaying characteristics of a scaffold protein adlayer. Orientational control of the scaffold protein STM, a triply mutated form of the stable intracellular protein inhibitor stefin A is achieved with a surface cysteine residue, which leads to the presentation of the scaffold recognition surface to solution. Operational stability of the system is excellent, with only a minor decrease in detection sensitivity over time (less than 1% h-1). We use this system to establish a surface plasmon resonance assay offering a limit of detection of 1 nM (150 ng mL-1) and determine the affinity constant of interaction of STM for a cognate antibody to be KD = 1.47 +/- 0.23 nM. Thus, we have established a solid foundation for the future creation of highly multiplexed peptide aptamer microarrays that will be compatible with a broad range of label-free detection technologies.

Label-free detection of interleukin-6 using electrolyte gated organic field effect transistors

“…6,7 The last years have witnessed a large number of studies attempting at developing label-free biosensors alternative to currently used platforms based on optical response. [8][9][10][11][12][13][14][15] As for IL-6, the latest reported biosensors that allow for a real-time monitoring of the cytokine levels without requiring secondary [33][34][35][36][37] .…”

Optimisation of a multivalent Strep tag for protein detection

“…The streptavidin binding tags were cloned into a modified pET30a+ vector (Novagen, USA; residue [26]), amino-terminal to the SQM peptide aptamer scaffold (pET30a+-SQM, [25]). The Strep-tag (WSHPGFEK) was generated by annealing the primers…”

“…In our system, the SQM protein is expressed in E coli with an amino-terminal tail designed to contain a variety of functional domains that enable subsequent purification or modification of the expressed protein. One of these domains introduces a unique cysteine residue [26] that allows the dimerisation of the SQM proteins under oxidising conditions. We use a subscript "2" to indicate where the SQM protein (and any attached Strep tags) are observed to be largely dimerised (Fig 1C).…”

Highly specific label-free protein detection from lysed cells using internally referenced microcantilever sensors

“…To enable the controlled immobilisation of recombinant peptide aptamers onto gold surfaces, a single cysteine residue was introduced into the amino terminal tail of fusion proteins expressed from pET30a+ (Novagen); we call the engineered vector pET30a+CYS+ (Davis et al, 2007). The protein scaffold, STM, pep2-STM, and pep6-STM (Woodman et al, 2005) were each subcloned from pJG4-5 into pET30a+CYS+.…”

“…They offer significant advan- tages over antibodies in low-cost production, regeneration and stability. Recently, peptide aptamers have been shown to be capable of directed immobilization and orientation on gold surfaces without loss of integrity (Davis et al, 2007) and to retain affinity and specificity for protein detection in simple mixtures (Xu et al, 2002;Evans et al, 2008;Johnson et al, 2008). Although a large number of engineered scaffold proteins exist (reviewed in Binz et al, 2005), here we focus on peptide aptamers presented by a scaffold designated Stefin A Triple Mutant (STM) (Woodman et al, 2005).…”