Insertional protein engineering for analytical molecular sensing

Abstract

The quantitative detection of low analyte concentrations in complex samples is becoming an urgent need in biomedical, food and environmental fields. Biosensors, being hybrid devices composed by a biological receptor and a signal transducer, represent valuable alternatives to non biological analytical instruments because of the high specificity of the biomolecular recognition. The vast range of existing protein ligands enable those macromolecules to be used as efficient receptors to cover a diversity of applications. In addition, appropriate protein engineering approaches enable further improvement of the receptor functioning such as enhancing affinity or specificity in the ligand binding. Recently, several protein-only sensors are being developed, in which either both the receptor and signal transducer are parts of the same protein, or that use the whole cell where the protein is produced as transducer. In both cases, as no further chemical coupling is required, the production process is very convenient. However, protein platforms, being rather rigid, restrict the proper signal transduction that necessarily occurs through ligand-induced conformational changes. In this context, insertional protein engineering offers the possibility to develop new devices, efficiently responding to ligand interaction by dramatic conformational changes, in which the specificity and magnitude of the sensing response can be adjusted up to a convenient level for specific analyte species. In this report we will discuss the major engineering approaches taken for the designing of such instruments as well as the relevant examples of resulting protein-only biosensors.

Figure 1

The biosensing principles of the constructs listed in Table 1 are summarized here as split in two groups. In a), the sensing principles underlying cleavable platforms are presented in which simple hydrolysis of protease target site-bearing hybrid proteins by an effector protease (P) result in a macroscopic signal. Among others, variations of the migration pattern, enzyme activation or inactivation, repressor inactivation, enhanced fluorescence by removal of a quencher or dual fluorescence emission by FRET modulation. In b), a ligand (L) promotes conformational modifications in the sensor either multimerization, correct folding or allosteric activation. A few enzyme biosensors are inactivated in presence of the ligand probably by steric hindrance of the active site.