Table 1 Representative examples of protein only biosensors obtained by insertional mutagenesis.
From: Insertional protein engineering for analytical molecular sensing
Holding protein | Strategy | Insert | Analyte | Sensing mechanism | Signal (factor, when activated) | Application (proved or suggested) | References |
|---|---|---|---|---|---|---|---|
β-galactosidase | Site directed insertion | FMDVa and HIV antigenic peptides | Anti-peptide antibodies and immune sera | Allosteric | Enzymatic activity up-shift (up to 12-fold) | Diagnosis | [38,39,43,47,48,49,59] |
β-galactosidase | Site directed insertion | HIV protease substrate | HIV protease | Cleavage mediated inactivation | Enzymatic activity down-shift or electrophoretic analysis | Antiviral drug design and screening | [25,26] |
Alkaline phosphatase | Site directed insertion | HIV antigenic peptide | Anti-peptide antibodies | Probably steric hindrance | Enzymatic activity down-shift | Diagnosis | [46] |
Alkaline phosphatase | Site directed insertion plus site directed mutagenesis of the active site | HIV and HCV antigenic peptide | Anti-peptide antibodies | Allosteric | Enzymatic activity up-shift (up to 2.5-fold) | Diagnosis | [40] |
GFP | Site directed insertion followed by random mutagenesis | TEM1 β-lactamase | TEM1 β-lactamase inhibitor | Allosteric | Fluorescence emission up-shift (not determined) | Drug design and screening | [41,42] |
EGFP | Amino acid replacement | LPS/LA-binding motif | Bacterial LPS | Quenching | Fluorescence emission down-shift | Quality control (endotoxin detection) | [60] |
TEM β-lactamase | Random insertion and phage-mediated selection | Random peptides | Anti PSA antibodies | Allosteric and steric hindrance upon the specific construct | Enzymatic activity down- or up-shift (up to 1.7-fold) | Diagnosis | [10] |
p53 | Site directed insertion plus site directed deletion | LF, HA and HSV antigenic peptides | Anti-peptide antibodies | Dimerization | Electrophoretic mobility up-shift (up to 100-fold) | Diagnosis and screening | [28] |
p53 | Site directed insertion | HIV and LF protease substrates | HIV protease and LF | Auto-inhibitory domain removal | Electrophoretic mobility up-shift (up to > 100-fold) or in situ hybridisation (2-fold) | Screening | [28] |
cI lambda repressor | Site directed insertion | HIV, HCV and SARS protease substrates | HIV, HCV and SARS proteases | Cleavage mediated inactivation | Phage plaques counting (up to 50-fold) | Antiviral drug design and screening | [32,33,61] |
MBP | Site directed insertion eventually followed by punctual mutagenesis | Zinc binding sites | Zinc | Allosteric | Fluorescence emission modulation (up to 8-fold) | Not specified, presumably wide | [62] |
MBP | Random insertion | TEM-1 beta-lactamase segment | Maltose and other sugars | Allosteric | Enzymatic activity up-shift (up to 1.7-fold) | Not specified, presumably wide | [11] |
DHFR | Site directed insertion eventually followed by punctual mutagenesis | FKBP macrolide- binding protein and ERα ligand binding domain | FK506 and estrogen | Binding-promoted thermostability and consequent genetic complementation | Growth of temperature-sensitive yeast under non-permissive temperatures (up to 2.5-fold) | Drug design and screening | [56] |
FynSH3 b | Deletion | none | Proline-rich peptide ligand | Ligand induced protein folding | Tryptophan fluorescence increase (up to 15-fold) | Not specified, presumably wide | [55] |
GFP-DsRed fusion b | Modular fusion | TEV protease substrate | TEV protease | Cleavage mediated fluorescent tag separation | Dual fluorescent emission yield | Antiviral drug design and screening | [29] |