WO1999034212A1

WO1999034212A1 - Biosensor

Info

Publication number: WO1999034212A1
Application number: PCT/US1998/027882
Authority: WO
Inventors: Homme W. Hellinga
Original assignee: Duke University
Priority date: 1997-12-31
Filing date: 1998-12-31
Publication date: 1999-07-08
Also published as: AU760743B2; EP1044374B1; DE69840107D1; ES2314996T3; CA2316199C; JP4472169B2; DK1044374T3; EP1044374A1; US6521446B2; US20020004217A1; CA2316199A1; JP2002500361A; EP1044374A4; US6277627B1; AU2210199A; ATE410683T1

Abstract

The present invention relates to a glucose biosensor comprising a genetically engineered Glucose Binding Protein (GBP). In a specific embodiment, the invention relates to a GBP engineered to include mutations that allow site specific introduction of environmentally sensitive reporter groups. The signal of these prosthetic groups changes linearly with the degree of glucose binding. Thus, the glucose sensor of the invention can be used, for example, for detection of glucose in blood or industrial fermentation processes.

Description

BIOSENSOR

This application claims priority from Provisional Application 60/070,293, filed December 31, 1997, the entire contents of that application being incorporated herein by reference.

TECHNICAL FIELD

The present invention relates, in general, to biosensors and, in particular, to a glucose biosensor comprising a genetically engineered Glucose Binding Protein.

BACKGROUND

Biosensors couple highly specific biomolecular ligand binding events to

changes in physical signals, thereby providing analytical tools that can measure

the presence of single molecular species in complex mixtures. (Hall, Biosensors,

Prentice-Hall: Englewood Cliffs (1991)). Most biosensors are naturally

occurring macromolecules, such as enzymes or antibodies, which provide the

desired analyte specificity, but often are not well suited to simple signal

transduction mechanisms. (Griffiths et al, Tr. Biotech. 11 :122-130 (1993)). One

solution to this problem is to use protein engineering techniques to integrate signal transduction functions directly into proteins, adapting them to

straightforward detection technologies, rather than developing instrumentation

specific to the properties of a particular protein (Adams et al, Nature 39:694-697

(1991); Braha et al, Chem. Biol. 4:497-505 (1997); Brennan et al, Proc. Natl.

Acad. Sci. U.S.A. 92:5783-5787 (1995); Brune et al, Biochemistry 33:8262-8271

(1994); Cornell et al, Nature 387:580-583 (1997); Gilardi et al, Anal. Chem.

66:3840-3847 (1994); Godwin et al, J. Am. Chem. Soc. 118:6514-6515 (1996);

Marvin et al, Proc. Natl. Acad. Sci. U.S.A. 94:4366-4371 (1997); Post et al, J.

Biol. Chem. 269: 12880-12887 (1994); Romoser, J. Biol. Chem. 272: 13270-13274

(1997); Stewart et al, J. Am. Chem. Soc. 116:415-416 (1994); Thompson et al, J.

Biomed. Op. 1 :131-137 (1996); Walkup et al, J. Am. Chem. Soc. 119:5445-5450

(1997)). A simple approach to building such integrated signal transducers is to

exploit optical detection strategies based on changes in fluorescent reporter groups

which respond to ligand binding (Guiliano et al, Annu. Rev. Biophys. Biomolec.

Struct. 24:405-434 (1995); Czarnik, Chem. Biol. 2:432-438 (1995)).

Fluorophores can be site-specifically introduced into a protein by using total

synthesis, semi synthesis, or gene fusions. In this way pairs of fluorophores can

be arranged for detection of binding by fluorescence energy transfer, or a single,

environmentally-sensitive fluorophore can be positioned to respond to conformational changes accompanying binding events. (See references cited

above.)

Ideally, the structural relationship between ligand binding site and reporter

group is such that each can be manipulated independently, allowing a modular

approach to the optimization of the properties of the binding site or the

fluorophore. (Marvin et al, Proc. Natl. Acad. Sci. U.S.A. 94:4366-4371 (1997);

Walkup et al, J. Am. Chem. Soc. 1 19:3443-3450 (1997); Cheng et al, J. Am.

Chem. Soc. 1 18: 11349-1 1356 (1996); Ippolito et al, Proc. Natl. Acad. Sci. U.S.A.

92:5017-5021 (1995); Elbaum et al, J. Am. Chem. Soc. 1 18:8381-8387 (1996)).

One way to achieve such modularity is to spatially separate the two sites to

minimize steric interference between them. Spatial separation of the reporter

group and the binding site requires that the behavior of the fluorophore remain

coupled to the degree of occupancy of the ligand binding site via an allosteric

linkage mechanismn. Recently, it has been shown that it is possible to engineer

such integrated fluorescent allosteric signal transducer (FAST) functions in the

Maltose Binding Protein (MBP) of E. coli by taking advantage of the large

conformational changes that occur upon ligand binding in this protein, using a

structure-based rational design approach (Marvin et al, Proc. Natl. Acad. Sci.

U.S.A. 94:4366-4371 (1997)). The present invention relates, in one embodiment, to a Glucose/ Galactose

Binding Protein with engineered FAST functions and to a new class of fluorescent

glucose sensors with applications in the food industry (Suleiman et al,

In:Biosensor Design and Application, Matthewson and Finley, Eds. American

Chemical Society, Washington, D.C., Vol. 511 (1992)), and clinical chemistry

(Wilkins et al, Med. Eng. Phys. 18:273-278 (1996); Pickup, Tr. Biotech. 1 1 :285-

291 (1993); Meyerhoff et al, Endricon 6:51-58 (1996)).

SUMMARY OF THE INVENTION

The present invention relates to a glucose biosensor comprising a

genetically engineered Glucose Binding Protein (GBP). In a specific

embodiment, the invention relates to a GBP engineered to include mutations that

allow site specific introduction of environmentally sensitive reporter groups. The

signal of these prosthetic groups changes linearly with the degree of glucose

binding. Thus, the glucose sensor of the invention can be used, for example, for

detection of glucose in blood or industrial fermentation processes.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Comparison of the closed forms of MBP (Spurlino et al, J. Biol.

Chem. 266:5202-5219 (1991)) (top; Protein Data Bank ref 2MBP) and GBP

(Vyas et al, Nature, 327:635-638 (1987); Vyas et al, Science 242:1290-1295

(1988); Vyas et al, Biochemistry 33:4762-4768 (1994)) (bottom: PDB ref IGLG)

complexed with their respective substrates, showing position. Attachment sites of

conjugated fluorophores are indicated by spheres. The sphere located in the "flap"

region of MBP indicates the position of the fluorophore that was found to give the

best allosteric response (Marvin et al, Proc. Natl. Acad. Sci. USA 94:4366-4371

(1977)) (attached to Asp95Cys). On the basis of this result, four sites in the

analogous region of GBP (255, 257, 294, 296) are predicted to be potentially

allosterically linked to the glucose binding pocket. Sites 15 and 152, located in

the glucose binding pocket, are positions for potential nonallosteric reporter

groups. The ribbon diagrams were produced with Molscript (Krollis, J. Appl.

Crystal. 24:946-950 (1991)).

Figures 2A and B. Binding of glucose to the L255C-acrylodan (Fig. 2A)

and H152C-IANBD (Fig. 2B) conjugates. The binding curve is the average of

three separate titrations (error bars are smaller than the circles shown). Insert shows changes in the emission spectra upon addition of saturating glucose: no

glucose (dashed line), 10 mM glucose (solid line).

Figure 3. Solute quenching by iodide of the L255C-acrylodan conjugate.

F₀/F fractional change in fluoresence emission at 498 nm upon addition of iodide.

Quenching was determined for apoprotein (circles) and in the presence of 10 mM

glucose (diamonds), and is plotted for both emission maxima at 498 nm (closed

symbols) and 520 nm (open symbols).

Figure 4. Glucose binding pocket in GBP (Protein Databank Ref 2GBP).

Figure 5. Blue LED-based fluorometer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel glucose-sensing protein

engineered from GBP to include amino acid residues that allow site-specific

introduction of environmentally sensitive reporter groups, such as fluorophores.

The invention results, at least in part, from the identification of regions in GBP

that are either placed directly within or are allosterically coupled to the glucose

binding site.

Prior to the production of the glucose sensors of the invention, allosteric

sites had been identified in the Maltose Binding Protein (MBP) (Marvin et al,

Proc. Natl. Acad. Sci. USA 94:4366-4371 (1997)). Direct identification of the allosteric sites was possible as high resolution X-ray structures of MBP were

known in both the maltose-bound ("closed") and maltose-free ("open") states. By

contrast, GBP had been crystallized only in the "closed" (ie, glucose-bound)

conformation and thus direct calculation of potentially allosteric site (PAS)

locations was precluded. While MBP and GBP share little homology, have

different molecular weights and even somewhat different secondary structure

topology, it proved possible to exploit the rough structural similarity between the

two proteins to predict the location of allosteric sites in GBP (Vyas et al. Nature

327:635-638 (1987); Vyas et al, Science 242:1290-1295 (1988); Vyas et al,

Biochemistry 33:4762-4768 (1994)).

While glucose sensors of the invention are described in some detail in the

Examples that follow, it will be appreciated that the particular engineered GBP's

described represent only individual embodiments. Further, it will be appreciated

that as GBP is a member of a superfamily of receptor proteins, the invention

includes engineered GBP's other than engineered E. coli GBP's, such as

engineered thermophilic bacteria GBP (see Tarn et al, Microbiol. Rev. 57:320

(1993)).

Engineered proteins of the invention can be produced by site-specifically

introducing a reporter group(s) by total synthesis, semi synthesis, or gene fusions

(see Godwin et al (1996), Walkup et al (1997), Adams et al (1991), Brune et al (1994), Gilardi et al (1994), Marvin et al (1997), Post et al (1994), Thompson et al

(1996), Romoser et al (1997), all cited above)).

A variety of reporter groups can be used, differing in the physical nature of

signal transduction (e.g., fluorescence, electrochemical, nuclear magnetic

resonance (NMR), and electron paramagnetic resonance (EPR)) and in the

chemical nature of the reporter group. For example, a variety of different

fluorophores can be used. Fluorophores that operate at long excitation and

emission wavelengths (e.g., >600 nm) are preferred when the molecular sensor is

to be incorporated into a transdermal biosensor (the skin being opaque below

600 nm). Presently, there are few environmentally sensitive probes available in

this region of the spectrum and none with thiol-reactive functional groups

(Thompson, R.B., Red and near-infrared fluorometry, in Topics in Fluorescence

Spectroscopy, J.R. Lackowicz, Editor 1994, Plenum Press: New York, p. 151-

181). However, thiol-reactive derivatives of osmium(II) bisbipyridyl complexes

and of the dye Nile Blue can be prepared (Geren et al, Biochem. 30:9450 (1991)).

Conjugates containing these fluorophores, for example, attached at various

cysteine mutants constructed in the hinge region of GBP, can be screened to

identify which results in the largest change in fluorescence upon glucose binding.

Os(II) bisbipyridyl complexes have absorbances at wavelengths longer than

600 nm with emission maxima in the 700 to 800 nm region (Demas et al, Anal. Chem. 63: 829 A (1991)) and the life-times are long (in lOOnsec range),

simplifying the fluorescence life-time instrumentation. The absorbance and

luminescence properties of these complexes can be tuned readily by ligand

substitution. In these complexes, Os toxicity is reduced because the Os(II) state is

exchange inert (greatly reducing any potential loss of metal from the complex),

and the redox potential ~1 V so that it is unlikely to be oxidized to Os(III) under

physiological conditions (Demas et al, Anal. Chem. 63:829A (1991)). Redox

cofactors can also be used as reporter groups, e.g., ferrocene and thiol-reactive

derivatives thereof. Thiol-reactive derivatives of organic free radicals such as

2,2,6,6-tetramethyl-l-piperinoxidy (TEMPO) and 2,2,5,5-tetramethyl-l-

piperidinyloxy (PROXYL) can also be used and changes in the EPR spectra of

these probes in response to ligand binding monitored.

The reporter group(s) can be positioned in the binding pocket of the GBP

(Vyas et al, Nature 327:635-638 (1987); Vyas et al, Science 242:1290-1295

(1988); Vyas et al, Biochemistry 33:4762-4768 (1994)), so that changes in

reporter signal are a consequence of direct interactions with the bound glucose.

This approach may be disadvantageous in that it can be accompanied by

unfavorable steric interactions between the reporter group and glucose which

lower the glucose affinity or substrate selectivity. Alternatively, the reporter

group(s) can be positioned in locations distant from the binding site where the reporter group(s) senses glucose binding indirectly via an allosteric coupling

mechanism based on detection of the domain movements. Appropriate allosteric

sites are located in regions of GBP that undergo a local conformational change in

concert with the interdomain bending motion. In the case of MBP, the positions

of such sites were deduced by comparison of the experimentally determined

structures of both protein conformations (ie, "open" and "closed"). As indicated

above, it proved possible to extend this analysis to GBP on the basis of the rough

structural homology between the two proteins. The allosteric sensing mechanism

has the advantage that there is no direct interaction between the reporter group and

glucose, so that the original properties of the binding site are essentially

unaffected. The allosteric design strategy is modular in nature, that is, the ligand-

binding site can be altered without destroying the reporting properties of the

attached reporter, and vice versa.

In a further aspect of the present invention, mutations are introduced both

at an allosteric site (for purposes of reporter group linkage) and in the binding site

in order to alter affinity of the protein for glucose without destroying, or unduly

impacting, allosteric linkage with the reporter group. This aspect of the invention

can find application, for example, when the biosensor is used for determining

blood glucose concentrations. Blood glucose is typically about 7 mM and

fluctuates between 5-15 mM in diabetic conditions (Tietz Textbook of Clinical Chemistry, 2^nd ed., (1986) Burtis and Ashwood (eds.) Saunders, London). In

order to accurately determine the glucose concentration without the necessity for

sample dilution, the binding constant of the engineered biosensor be adjusted so

as to match the physiological (and pathological) operating range. Wild type GBP

has a K_d (glucose)=0.8 μm. Advantageously, a biosensor of the invention has a

weaker binding constant (about four orders of magnitude weaker) but is still

specific for glucose. For example, biosensors having a binding constant for

glucose in the range of 0.8 μM to 20 mM can be used. Advantageously, the

binding constant is in the range of 1 mM to 20mM when the biosensor is to be

used for clinical purposes. Binding constants can be determined, for example, by

measuring the fluorescence change of an engineered protein in response to known

quantities of glucose (see Examples that follow). Similarly, binding constants can

be "tuned" to match the operating conditions in industrial processes.

Mutations that change the binding constant of GBP without altering the

sugar specificity can be identified by examination of the three-dimensional

structure of the glucose binding pocket in GBP (Figure 4). Three categories of

interactions between the protein and the bound sugar can be altered: (A) direct

hydrogen bond interactions, (B) direct van der Waals interactions, (C) indirect

van der Waals or hydrogen-bond contacts. Class (A ) contacts are most likely to

contribute to specificity and therefore may be left unaltered. Class (B) and (C) contacts can be altered by site-directed mutagenesis with specific residues being

mutated, in the first instance, to alanine. W183A and Y10A single mutants, as

well as the W183AY10A double mutant, have been constructed in an

allosterically signaling construct designated 255C. These mutations change the

binding constant for glucose from 0.2μM to 20μM, 50μM and 650μM,

respectively, demonstrating that the binding constant can be "tuned" without

destroying the allosteric signaling mechanism. The D154AW183A double mutant

described in Example 5 (also in a 255C background) has a binding constant of

7.2. Other mutations involving, for example, Phel6, Lys92, Glul47, Lys9,

Aspl84, Asnl4, Asn91, Hisl52, Aspl54, Argl58, Asn21 1, Asp236, Asn256;

TyrlO, Metl7, Asn66, Serl 12, Serl 15, Trpl83, Asn210, Met214, Gln261 and/or

Tyr295, can be made. In addition to changing specific positions to alanine, the

effect of other amino acids can also be determined. Advantageously, all

mutations will also be examined in the non-allosteric 152C background.

The biosensor of the invention can be used in essentially any setting where

glucose detection is required. For example, the present biosensor can be used in

the food industry (Suleiman et al, In: Biosensor Design and Application:

Mathewson and Finley Eds; American Chemical Socieity, Washington, DC 1992,

vol. 51 1) and in clinical chemistry (Wilkins et al, Med. Eng. Phys. 18:273-288

(1996); Pickup, Tr. Biotech. 1 1 :285-291 (1993); Meyerhoff et al, Endricon 6:51- 58 (1966); Riklin et al, Nature 376:672-675 (1995); Willner et al, J. Am. Chem.

Soc. 118:10321-10322 (1996)). The biosensor of the invention can be used, for

example, as the basis for the construction of a fluorescent flow cell containing

immobilized GBP-FAST conjugates. (See Wilkins et al, Med. Eng. Phys. 18:273-

288 (1966), Pickup, Tr. Biotech. 11 :285-291 (1993); Meyerhoff et al, Endricon.

6:51 (1966); Group, New Engl. J. Med. 329:977-986 (1993); Gough et al,

Diabetes 44:1005-1009 (1995)). The present biosensor can be used in an

implantable device suitable for use as an artificial pancreas.

Certain aspects of the present invention are described in greater detail in

the non-limiting Examples that follows. (See also Marvin et al, J. Am. Chem.

Soc. 120:7 (1998)).

Examples

The following experimental details relate to the specific Examples that

follow.

Mutagenesis. The gene for the cytoplasmic form of GBP (Scholle et al,

Mol. Gen. Genet. 208:247-253 (1987)) (i.e., lacking the leader sequence peptide)

was amplified from E. coli genomic DNA using the Polymerase Chain Reaction

(PCR) with flanking primers designed to introduce an EcoRI restriction site 5' to the start codon N-Terminus (5' CCG GAA TTC GGA GAT ACC ATG GCT

GAT ACT CGC ATT GGT GTA AC A ATC TAT 3'; restriction site and start

codon are underlined), and a BamHl site just before the stop codon (5' AAG CTT

TCA TTA GGA TCC TTT CTT GCT GAA CTC AGC CAG GTT GCT TTT 3').

The resulting fragment was cloned into the pKK223-3 expression vector

(Pharmacia). An oligonucleotide cassette coding for a His₅ oligopeptide was

subsequently cloned into the BamHl site to allow single-step purification by

Immobilized Metal Affinity Chromatography (Hochuli et al, Bio/Technol.

6:1321-1325 (1988)) (IMAC). Individual cysteine mutants were made by

overlapping PCR fragment mutagenesis.

Protein Expression and Purification. A colony of E. coli XLI -blue cells

(Stratagene) freshly transformed with a plasmid expressing a mutant GBP protein

was grown at 37°C overnight in 20 mL of 2XYT medium containing 100 μg/mL

ampicillin. 2XYT medium (1 L) supplemented with 0.2% (w/v) glucose was

inoculated with 10 mL of the overnight culture and grown with vigorous shaking

at 37°C until OD₆₀₀ - 0.5. Protein expression was induced with 1 mM 1PTG and

grown for a further 4 h. Cells were harvested by centrifugation at 3000g,

resuspended in 40 mL of high salt buffer (1 M NaCl, 50 mM phosphate, pH 7.0;

HSB), and stored frozen at -80°C. The cells were thawed and lysed in a chilled

French press at 1200 psi. Cellular debris was removed by centrifugation at 6000g for 10 min. DNA was precipitated by addition of polyenimine (pH8) to 5% (w/v)

and removed by centrifugation at 6000g for 30 min. GBP was purified with a

single-step IMAC (Hochuli et al, Bio/Technol. 6:1321-1325 (1988)) procedure.

Cleared lysate was diluted to 100 mL with HSB onto a 30 mL iminodiacetate/zinc

column (Pharmacia) and extensively washed with HSB to remove unbound

proteins (and bound glucose), followed by elution of mutant GBP with use of a

200 mL, 0-100 mM imidazole gradient in HSB. GBP was located in a single late

peak that revealed only one protein band on an overloaded SDS/polyacrylamide

gel stained with Coomassie Blue. Yields were typically 5 mg/L growth.

Fluorophore Coupling. Fluorophores were conjugated to the mutant

GBP proteins via the single free cysteine. Acrylodan (Prendergast et al, J. Biol.

Chem. 259:7541-7544 (1983)) and (((2-(iodoacetoxy)ethyl)methyl)amino)-7-

nitrobenz-2-oxa-l,3-diazole (Ghosh et al, Biochem. J. 108: 155 (1968)) (IANBD)

were purchased from Molecular Probes and used without further purification. The

fluorophores were dissolved in acetonitrile and reacted with freshly purified

cysteine mutants (5:1 molar ratio) of GBP (~1 mg) in 1 M NaCl, 50 mM

phosphate buffer (pH 7.0), for 3-5h at room temperature. Unreacted fluorophore

was separated from protein by gel filtration. The extent of coupling was measured

both by determining the remaining free thiol concentration with use of Ellman' s

reagent (Ellman, Arch. Biochem. Biophy. 82:70-77 (1959)) and by using the ratio of the absorbances of the major protein and fluorophore chromophores (e₂₈₀(GBP)

= 37 mM^"1 cm^"1 (this study); e₄₆₉(IANBD) = 23 mM^-1 cm^'1; e₃₉₂(acrylodan) = 20

mM^-1 cm^-1). Coupling was always found to be greater than 95%. The conjugates

were stable at 4°C for a period of months, as determined by glucose binding

assays.

Measurement of Glucose and Galactose Binding. Sugar binding was

determined by measuring changes in fluorescence of the conjugated fluorophores

on a SLM-Aminco-Bowman series-2 fluorimeter at 25 ± 1°C. Glucose or

galactose (Sigma) was titrated into a 50 nM conjugated protein solution in 0.1 M

NaCl, 50 mM phosphate (pH 7.0), which was continuously mixed with a magnetic

stirrer. For titrations, the excitation and emission slit widths were set to 4 and 16

nm, respectively (IANBD: λ_ex = 469 nm, λ_cm = 540 nm; acrylodan: λ_ex = 392 nm,

λ_em = 520 nm). Under these conditions the instrument noise was <1% of the

fluorescence signal observed in saturating solutions of glucose. Experimentally

observed binding curves were fit to a binding isotherm:

ΔF = ΔF_max (1+K_d/S)-1

0) where ΔF is the change in fluorescence, ΔF_max the fluorescence change at

saturating concentrations of ligand, K_d the binding constant, and S the

concentration of ligand. Example 1

Identification of Allosterically Linked Reporter Sites

The conformational differences of the high-resolution X-ray structures of

the open (Spurlino et al, J. Biol. Chem. 266:5202-5219 (1991) and closed (Sharff

et al, Biochemistry 31 :10657-10663 (1992)) forms of MBP were analyzed, and

regions that were predicted to be allosterically linked to the binding site were

analyzed (Marvin et al, Proc. Natl. Acd. Sci. USA 94:4366-4371 (1977)). Site-

specific attachment of environmentally sensitive fluorophores demonstrated that

these regions are allosterically coupled to maltose binding. GBP has been

crystallized only in the closed conformation, precluding direct calculation of

potentially allosteric site (PAS) locations. Instead, the results obtained on MBP

were used, and the rough structural similarity between MBP and GBP relied upon

to predict the location of analogous PASs in the latter, even though the two

proteins share little sequence homology and are of different molecular weight and

somewhat different secondary structure topology (Hsiao et al, J. Mol.. Biol.

262:225-242 (1996)).

The site that gave the most pronounced allosteric signaling in MBP is

located in a region that forms a mobile "flap" covering the actual hinge. This flap

is formed by two unconnected halves, each confined to one of the domains. Their relative movement changes the environment of an attached fluorophore which is

completely separated from the binding pocket by the hing β-sheet. The equivalent

flap region in GBP is much smaller, with only one-half truly retained, which

limits the attached positions in GBP to the hinge itself (Figure 1).

Faced with the limited amount of structural information available for GBP,

the search for PAS locations was restricted to this region. Since it is impossible to

predict which of the residues in the flap region is likely to give the most

pronounced allosteric response to ligand binding, the β-sheet portion of the flap

was scanned and four sites for reporter group attachment (L255, D257, P294,

V296) were identified. These positions are all located in one side of the hinge β-

sheet, forming a surface onto which the flap α-helix is packed. Their

microenvironment is therefore predicted to change if the flap region rearranges

upon ligand binding.

EXAMPLE 2

Nonallosteric (Peristeric) Reporter Sites

In addition to the PAS mutations, two sites for attachment of reporter

groups in the binding site itself were identified (N15, H152). Fluorophores placed

in these positions are predicted to respond to changes in their microenvironment

by direct interaction with the ligand, by protein conformational changes as the "jaws" of the binding site close around the ligand, or by changes in solvation.

This strategy has been used successfully to introduce nonallosteric signal

transducing fluorescent reporter groups in MBP (Gilardi et al, Anal. Chem.

66:3840-3847 (1994)) and Phosphate Binding. Protein (Brune et al, Biochemistry

33:8262-8271 (1994)) (PBP), another member of the periplasmic binding protein

family, which binds to inorganic phosphate. In both cases the ligand binding

constant of the conjugated protein is significantly increased relative to wild-type,

indicating a significant degree of steric interference between the ligand and the

fluorophore, which may also account for the change in the microenvironment of

the fluorophore (Gilardi et al, Prot. Engin. 5:479-486 (1997)). This strategy

therefore loses the steric independence between reporter group and binding site

inherent in the allosteric approach.

EXAMPLE 3

Signal Transduction Properties of Mutants

The six GBP variants with single cysteines introduced for site-specific

covalent attachment of fluorophores were constructed by a PCR mutagenesis

strategy. Table 1 shows the results of the acrylodan (Prendergast et al, J. Biol.

Chem. 259:7541-7544 (1983)) or (((2-(iodoacetoxy)ethyl)methyl)amino)-7-

nitrobenz-2-oxa-l,3-diazole (Ghosh et al, Biochem. J. 108:155 (1968)) (IANBD) conjugates of these mutant proteins. These two fluorophores have been selected

because of their known sensitivity to the polarity of their microenvironments.

TABLE 1

Binding Properties of Fluorescent Conjugates of the Mutant Proteins

IANBD acrylodan

K_d(Glc) K_d(Gal) K_d(Glc) K_d(Gal) mutant R μM μM R μM μM

N15C 0.8 0.13 0.1 0.7 0.17 0.15

H152C 4.0 20 160 1.0 nd nd

L255C 0.8 0.32 0.49 0.5 0.43 0.62

D257C 1.6 0.80 1.5 0.8 0.5 0.5

P294C 1.0 nd nd 0.9 1 1

V296C 0.7 0.1 0.3 1.0 nd nd

R: ratio of fluorescence of fully saturated GBP (10 mM glucose) to apoprotein (R = 1.0 indicates no change; R < 1.0 indictes a decrease upon glucose binding; R > indicates an increase). K_d(Glc), K_d(Gal): binding constant (μM) for glucose and galactose, respectively (nd: not done; binding constants were determined for all cases where R ≠ 1.0). Wild-type has a K_d(Glc) = 0.2 μM, and K_d(Gal) = 0.4 μM (Miller et al, J. Biol. Chem. 258:13665-13672 (1983)). N15C and H152C are the positions for nonallosteric reporter groups, the other four mutants are located in the hinge region of the allosteric flap.

All four mutants in the hinge region showed a change in fluorescence of

their acrylodan or IANBD conjugates upon ligand binding. The acrylodan

conjugate at position 255 gives the largest change (2-fold decrease; Figure 2A).

In all cases, a single-site hyperbolic binding curve could be constructed by

measuring the change in fluorescence as a function of glucose or galactose concentration, from which we conclude that the fluorophores attached to the hinge

region are allosterically linked to the sugar binding pocket, as predicted.

Furthermore, the sugar binding constants are affected by no more than a factor of

4, indicating that the FAST and ligand binding sites are sterically separated, as

intended.

Two cysteine mutations were also constructed in the binding pocket itself

(Figure 1). H152C interacts directly with the sugar, since it replaces hisl52 which

forms a hydrogen bond with the 06 oxygen of both galactose and glucose. (Vyas

et al, Biochemistry 33:4762-4768 (1994)). The largest change in fluorescence of

all the variants explored in this study (4-fold increase) was observed with IANBD

attached at this position. However, this conjugate shows a large increase in the

binding constants for glucose (~100-fold) and galactose (~500-fold) as would be

expected both from the loss of the hydrogen bond to the 06 oxygen and from

direct steric interference with the bound sugar.

Fluorophores attached to Nl 5C are intended to respond to changes in the

interdomain distance, rather than by direct interaction with the sugar, since Asnl5

points away from the sugar binding pocket. Both the acrylodan and the IANBD

conjugates show a change upon sugar binding, though not as large as IANBD at

the 152 position. However, the conjugates at the 15 position do not greatly perturb the sugar binding constants, indicating that there is no direct interaction

with the sugar.

EXAMPLE 4

Microenvironment of the Fluorophore Conjugates

Both acrylodan and IANBD are sensitive to changes in the polarity of their

microenvironment (Ghosh et al, Biochem. J. 108:155 (1968); Macgregor et al,

Nature 319:70-73 (1986); Weber et al, J. Biochemistry 18:3075-3078 (1979)).

which may result from changes in solvent accessibility, probe mobility, and

changes in the steric interactions with the surrounding protein (or ligand, in the

case of the H152C-NBD conjugate). Such microenvironmental changes may

manifest themselves as differences in emission intensity as well as shifts in the

wavelengths of their maxima. The emission maxima of acrylodan are known to

be particularly dependent on the polarity of the environment, showing a

significant blue shift in nonpolar relative to aqueous environments (Macgregor et

al, Nature 319:70-73 (1986); Weber et al, Biochemistry 18:3075-3078 (1979)).

The responses of the different sites vary widely. In several cases only one

of the two conjugates coupled at a particular site responds to binding of the sugar

(see Table 1). Furthermore, none of the conjugates show an appreciable shift in

emission maxima upon ligand binding. The behavior of the best allosteric signal transducer in the hinge region, L255C-acrylodan (Figure 3), was examined in

more detail. The emission spectrum of this conjugate has two maxima (498 and

520 nm; Figure 2A), suggesting that the attached acrylodan is present in two

distinct environments differing in their polarities which are intermediate between

water and ethanol based on their blue shifts relative to water (Prendergast et al, J.

Biol. Chem. 259:7541-7544 (1983)). Both peaks are present in the apo and sugar-

bound forms, although their relative intensity changes somewhat upon ligand

binding, suggesting a slight redistribution between the two states. To examine the

potential contribution of differences in the solvent accessibility of the attachment

site to changes in dipolar relaxation of the fluorophore that occurs upon ligand

binding, we determined the effect of iodide on the fluorescence in the presence

and absence of glucose. Iodide selectively quenches solvent-exposed

fluorophores. The degree of quenching follows the Stern-Volmer equation

describing steady-state collisional quenching (Lehrer et al, Methods Enzymol.

49:222-236 (1978)):

F./F = 1 + K[F]

(2) where F₀/F is the fractional decrease in fluorescence, and K the Stern-Volmer

quenching constant (K>1.0 for a solvent-exposed fluorophore) which is related to

the degree of solvent accessibility. It was found that the quenching constants measured for both emission maxima of the acrylodan are approximately the same,

do not change upon glucose addition and are >1 (Figure 3), indicating that both

acrylodan conformations are partially solvent exposed, and the change in the

microenvironment is unlikely to involve a change in solvent accessibility. Similar

observations were made on other conjugates.

These results suggest that the mechanism by which the conformational

changes affect the dipolar relaxation of the attached fluorophore does not involve

change in the local solvent-accessibility of the attachment position, but is

dependent on the detailed interaction of the fluorophore with its

microenvironment, as was also observed for IANBD attached in the binding site

of MBP (Gilardi et al, Prot. Engin. 5:479-486 (1997)).

EXAMPLE 5

Alteration of Binding Constant for Glucose

Examination of the experimentally determined three-dimensional structure

of GBP (see Fig. 4) indicates that there are two types of residues that form the

glucose binding site, those that make direct contact with glucose through

hydrogen bonds or van der Waals interactions (primary binding surface) and those

that serve to orient the residues in the primary binding surface (secondary binding

surface). (Primary binding surface in E. coli GBP: Asnl4, Asn91, Hisl52,

Aspl54, Argl58, Asn21 1, Asp236, Asn256; secondary binding surface in E. coli GBP: TyrlO, Phelό, Metl7, Asn66, Serl 12, Serl 15, Trpl83, Asn210, Met214,

Gln261, Tyr295). In order to change the binding constant for glucose, residues in

both surfaces have been systematically mutated, either singly or in pairs. The data

obtained are shown in Table 2. The Aspl54Ala, Trpl83Ala (D154A + W183A)

double mutant has a binding constant of 7.2 mM. The dynamic range is thus

matched for operating in physiologically relevant glucose concentration ranges.

EXAMPLE 6

Optical Instrumentation

Since the emission wavelengths of the blue LED (maximum at 470 nm,

half-power +30nm) are perfectly matched to the excitation wavelength of Nile

blue dye (NBD) fluorophore (maximum at 469 nm), a simple prototype special-

purpose fluorometer was constructed based on inexpensive, simple, readily available electronic components (see Fig. 5). A 1 ml cuvette is illuminated with a

Nichia Chemical silicon-mixed gallium nitride blue LED (3 cd at 20 mA). A

double convex lens is used to focus light into the sample. A high-pass glass filter

(515 nm cutoff) is used to separate excitation from emission light. A large

(100mm²) planar diffuse Si PIN photodiode (Photonic Detectors) is used to detect

the emitted light. The photodiode is operated in an unbiased (photovoltaic) mode,

optimized for low-noise and low-frequency operation. The signal is low-pass

filtered (5 Hz cutoff), and subsequently passed through a variable summer and

variable gain amplifier to achieve the desired low- and high-end calibration.

LT1028 (ultra-low noise) operational amplifiers are used for all analog signal

processing. A 12-bit dual-slope integrating analog-to-digital converter with a

total conversion time of -150 ms is used to average out noise from the analog

input. The digital output is fed through a 27C64 EPROM. The reading appears

on a four seven-segment green LED display. This instrument can detect changes

in NBD fluorescence upon ligand binding to sufficient accuracy to construct a

hyperbolic binding curve.

* * * * *

All documents cited above are hereby incorporated in their entirety by

reference. One skilled in the art will appreciate from a reading of this disclosure that

various changes in form and detail can be made without departing from the true

scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. A glucose biosensor comprising a glucose binding protein (GBP) and a reporter group that transduces a detectable signal, wherein said reporter group is attached to said GBP so that a signal transduced by said reporter group when said GBP is bound to glucose differs from a signal transduced by said reporter group when said GBP is not bound to glucose.

2. The biosensor according to claim 1 wherein said reporter group is attached to said GBP at a site distant from a glucose binding site of said GBP.

3. The biosensor according to claim 1 wherein said reporter group is attached to a glucose binding site of said GBP.

4. The biosensor according to claim 1 wherein said reporter group is a fluorophore.

5. The biosensor according to claim 1 wherein said reporter group is a redox cofactor.

6. The biosensor according to claim 1 wherein said GBP has a binding constant for glucose in the range of 0.8 μM to 20 mM.

7. The biosensor according to claim 6 wherein said GBP has a binding constant for glucose in the range of 1 mM to 20 mM.

8. The biosensor according to claim 1 wherein said GBP is a mutant bacterial protein.

9. The biosensor according to claim 8 wherein said GBP has a binding constant for glucose in the range of 0.8 μM to 20 mM.

10. The biosensor according to claim 9 wherein said GBP has a binding constant for glucose in the range of 1 mM to 20 mM.

11. The biosensor according to claim 9 wherein said mutant protein is a mutant E. coli protein.

12. The biosensor according to claim 11 wherein said GBP differs from wild type E. coli GBP at positions 154 and 183.

13. The biosensor according to claim 12 wherein alanines are present at positions 154 and 183.

14. The biosensor according to claim 13 wherein said reporter group is a fluorophore.

15. The biosensor according to claim 13 wherein said reporter group is attached to a cysteine residue at position 255.

16. A method of detecting the presence of glucose in a test sample comprising contacting said biosensor according to claim 1 with said test sample under conditions such that said biosensor can bind to glucose present in said test sample and comparing the signal transduced by said reporter group when said biosensor is contacted with said test sample with the signal transduced by said reporter group when said biosensor is contacted with a glucose-free control sample, wherein a difference in the signal transduced by said reporter group when said biosensor is contacted with said test sample, as compared to when said biosensor is contacted with said control sample, indicates that test sample contains glucose.

17. The method according to claim 16 wherein said test sample is a physiological fluid.

18. The method according to claim 17 wherein said physiological fluid is blood, urine, interstitial fluid of saliva.

19. A method of determining the concentration of glucose in a test sample comprising contacting said biosensor according to claim 1 with said test sample under conditions such that said biosensor can bind to glucose present in said test sample, and comparing the signal transduced by said reporter group when said biosensor is contacted with said test sample with a standard hyperbolic glucose binding curve prepared by measuring the signal transduced by said reporter group when said biosensor is contacted with control samples containing known quantities of glucose, and thereby making said determination.

20. The method according to claim 20 wherein said test sample is a physiological fluid.

1. The method according to claim 20 wherein said physiological fluid , urine, interstitial fluid of saliva.