Table 1

Representative examples of protein only biosensors obtained by insertional mutagenesis.
Holding protein	Strategy	Insert	Analyte	Sensing mechanism	Signal (factor, when activated)	Application (proved or suggested)	References

β-galactosidase	Site directed insertion	FMDV^aand 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]

^aAbbreviations are explained in the abbreviation list. ^bA few examples of protein sensors obtained by either deletion or end-to-end fusion approaches are also shown.
Ferraz et al. Microbial Cell Factories 2006 5:15 doi:10.1186/1475-2859-5-15