BioMed Research International

Review Article

Converting a Periplasmic Binding Protein into a Synthetic Biosensing Switch through Domain Insertion

Table 1

Selected examples of PBP-based protein switches generated by domain insertion.


Function modulated in vivo	Input domain (PBPs)	Output domain	Method of creating switch	Input signal	Kd (M)	Variant name: switch effect	Ref

	MBP	BLA	Random insertion of BLA into MBP	Maltose	3.2	T164-165: 1.6-fold	[54]
	MBP	BLA	Random insertion of BLA into MBP	Maltose	1.7	T164-165-H: 1.8-fold	[54]
	MBP	BLA	Circular permutation of BLA and random insertion into MBP	Maltose	5.5	RG13: 25-fold	[55]
	MBP	BLA	Iterative circular permutation and random insertion of BLA into MBP	Maltose	0.5	MBP317–347: 600-fold	[56]
			Five residues in the maltose binding site were randomized generating a switch variant that binds to sucrose	Maltose	23	MBP317–347/5-7: 86-fold
				Sucrose	0.7	MBP317–347/5-7: 82-fold
	GBP	BLA	cpBLA was randomly inserted into the PBP	Glucose	ND	MRD2col9: 2-fold	[33]
	RBP		cpBLA was randomly inserted into the PBP	Ribose	ND	P1. F10: 7.2 fold
	XBP		Random insertion of BLA into XBP and linker optimization	Xylose	ND	XBPBLA12: 4.4-fold
Antibiotic resistance	MBP	BLA	Site-directed mutagenesis of I329 residue of RG13	Maltose	0.67	I329A: 23-fold	[57]
					0.63	I329K: 32-fold
					25.6	I329P: 12-fold
					0.55	I329W: 20-fold
	MBP	BLA	Disulfide bonds were rationally introduced in RG13	Maltose	ND	RG13-AND2: 2-fold	[58]
					ND	RG13-ORN2: 8-fold
					ND	RG13-YES: 2-fold
				Maltose (+GSH)	ND	RG13-AND2: 8-fold
					ND	RG13-ORN2: 16-fold
					ND	RG13-YES: 4-fold
	MBP	BLA		Maltose	ND	RG13-AND2: 1.38	[59]
	MBP	BLA		Maltose (+ e^-)	ND	RG13-AND2: 3.59	[59]
	MBP	BLA	Linker modification of nonallosteric MBP-BLA (c4) to search for emergence of allostery through modulation of the conformational entropy	Maltose (heat or OH^-)	ND	c4-4G: 32-fold	[60]
	MBP	BLA	Point mutations at selected residues of the maltose binding pocket of MBP317-347	Maltose	499	E153D: 2,360-fold	[61]

	MBP	GFP	Circular permutation of GFP and random insertion into MBP	Maltose	2.8	Mal-B2: 8.1-fold	[62]
	TMBP	GFP	Circular permutation of GFP and random insertion into MBP	Trehalose	0.053	Tre-C04: 6.3-fold	[62]
	PhnD	GFP	Circular permutation of GFP and insertion at four positions of PnBP based on structure. Mutagenesis of the inter-domain linkers	2AEP	37	EcPhnD90- cpGFP.L1AD. L297R,L301R: 1.5-fold	[63]
	MBP	GFP	cpGFP was inserted in selected places of MBP. Further linker optimization	Maltose	4.5	MBP165-cpGFP: 0.2-fold	[64]
					3	MBP165-cpGFP.PPYF: 2.5-fold
					1.3	MBP175-cpGFP.L1-HL: 0.5-fold
Fluorescence	MglB	YFP	CFP (FRET donor) insertion into selected sites of MglB,with YFP at either C-term or N-term of MglB	Glucose	600	FLII¹²Pglu-600μ: 2.66-fold	[17]
Fluorescence	GltI	YFP	CFP (FRET donor) insertion into selected sites of GltI,with YFP at C-term of GltI	Glutamate	1	FLII81PE-1μ: 3.8-fold	[17]
	QBP	CFP	QBP coding sequence was amplified as two fragments. One was inserted at the linker region of YFP-CFP cassette and the other at the N-term yielding QBP-YFP-QBP-CFP	Arginine	2100	QBP/Citrine/CFP: 1.3-fold	[65]
	QBP	CFP		Ornithine	2000	QBP/Citrine/ECFP:1.1-fold	[65]
	GltI	GFP	cpGFP was inserted in a selected site of GltI. Further linker optimization	Glutamate	107	GltI253.L1LV/L2NP: 4.5-fold	[66]
	GltI	GFP		Aspartate	145	GltI253.L1LV/L2NP: 2-fold	[66]
	GltI	RFP	Nonpermuted RFP was inserted in a selected site of cpGltI. Further linker optimization and directed evolution	Glutamate	0.9	-iGluSnFR1 4.8-fold	[67]

Enzymatic activity	XBP	XynA	XynA was inserted into selected places of XBP. Further linker variation	Xylose	0.16	2621B: 1.49-fold	[22]
Enzymatic activity	XBP	XynA	XynA was randomly inserted in XBP	Xylose	ND	XynA–XBP271: 1.5-fold	[23]

Control of gene expression	MBP	ZFP	The zinc finger protein BCR-ABL1 was inserted into MBP	Maltose	ND	SP: 3-fold	[68]
	MBP	ZFP	BCR-ABL1 was randomly inserted into MBP. Further linker optimization	Maltose	ND	316R: 4-fold	[69]
						277A: 1.2-fold
						270A: 2-fold
						335P: 2.4-fold

Studies in which many variants were constructed, and only those displaying the best switch effect were considered.
MBP, maltose binding protein; GBP, glucose binding protein; RBP, ribose binding protein; XBP, xylose binding protein; TMBP, trehalose/maltose-binding protein; PhnD, phosphonate-binding protein; MglB, glucose/galactose-binding protein; QBP, glutamine binding protein; GltI, glutamate-binding protein.
BLA, TEM1 β-lactamase; GFP, green fluorescent protein; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; XynA, xylanase from Bacillus subtilis; ZFP, zinc finger protein.
in vitro or in vivo assay values in the presence of the effector divided by the value in the absence of the effector.