This document describes the development of an assay system to measure real-time phosphate (Pi) release by the enzyme phosphoenolpyruvate carboxylase (PEPC) using Escherichia coli phosphate binding protein (PBP) labelled with the fluorophore MDCC. PBP binds Pi tightly and its fluorescence increases ~7-fold upon Pi binding, making it suitable for sensing Pi. The assay aims to measure PEPC activity in the presence of its product oxaloacetate to better mimic physiological conditions. Key steps include cloning PBP into a vector, expressing and purifying the protein, and labeling it with MDCC to generate a stoichiometric Pi sensor for monitoring PEPC activity.

1. 1
Building a novel assay system for
phosphoenolpyruvate carboxylase
Hugh E G Thompson2
, Nathan B P Adams2*
and James D Reid1
1
Department of Chemistry, The University of Sheffield, Sheffield S3 7HF
2
Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, nathan.adams@sheffield.ac.uk
Introduction
Escherichia coli phosphate binding protein (PBP)
(figure 1) is a member of the ABC transporter
superfamily. It is expressed in the periplasm under
conditions of low inorganic phosphate (Pi). Under
these conditions, PBP binds Pi and transfers it to
an integral membrane protein for transport into the
cell cytoplasm.1
Structurally, PBP contains two
hinged domains which share a similar three-layer
αβα sandwich fold.2
A Pi binding cleft is situated
between the domains and the binding mechanism
has been described by the Venus flytrap model,
with large conformational changes of both domains
to enclose the Pi ligand.1,2
The assay system presented here was originally
developed by Webb and co-workers and employs
PBP labelled with the coumarin fluorophore N-[2-(1-
maleimidyl)ethyl]-7-(diethylamino) coumarin-3-
carboxamide (MDCC).3
The resultant labelled
protein (MDCC-PBP) is reported to give a ~7-fold
increase in fluorescence emission upon binding Pi
under saturating conditions.1
Moreover, MDCC-
PBP binds Pi rapidly (1.36 x 108
M-1
s-1
at 22 °C)
and tightly (Kd ≈0.1 µM) such that it is effective as a
stoichiometric Pi sensor at nanomolar Pi
concentrations.1
As MDCC-PBP binds Pi so tightly,
this system is easily contaminated by background
Pi (from glassware, pH metres etc) which is
ubiquitous in virtually any laboratory. To reduce this
Scheme 1 PNPase acting upon 7-MEG (1) to form α-ribose-1-
phosphate (2) and 7-methylguanine (3)
Figure 1 Crystal structure of PBP The structure
of PBP is represented in cartoon, showing bound
phsophate ion (red sticks), MDCC dye (yellow
sticks) bound to protein via engineered cystine
197 (blue sticks). Reproduced from Hirshberg et
al. 2

2. 2
contamination, a ‘Pi-Mop’ system is also used.1,3
This system employs 7-methylguanosine (1) (7-
MEG) and purine nucleoside phosphorylase
(PNPase; EC 2.4.2.1).1,3
PNP catalyzes
phosphorylysis of 7-MEG, irreversibly sequestering
background Pi as α-ribose-1-phosphate (2) (Keq =
100) (Scheme 1).3
In this work, PBP has been cloned into a pET14b
vector, overexpressed with an N-terminal Hexahis®
tag and purified using Ni-NTA affinity
chromatography. Purified PBP was labelled with
MDCC, and MDCC-PBP was titrations with Pi in
order to develop a stoichiometric sensor that can be
used to measure real-time Pi release by the
enzyme phosphoenolpyruvate carboxylase (PEPC;
EC 4.1.1.31) in future work. PEPC catalyses
formation of four carbon (C4) oxaloacetate (7)
(OAA) and Pi (6) from three carbon (C3)
phosphoenolpyruvate (5) (PEP) and bicarbonate
(4) (HCO3
-
) using Mg2+
or Mn2+
as a cofactor
(Scheme 2).4
Scheme 2 The reaction catalysed by phosphoenol pyruvate
carboxylase (PEPC).
PEPC has been extensively studied via coupled
assays with both malate and lactate
dehydrogenases.5
These assays remove OAA,
however in vivo the enzyme bathes in a pool of this
C4 product.4
Hence, construction of an assay
system which allows measurement of real-time Pi
formation by PEPC in the presence of OAA is
desirable owing to its greater physiological
relevance.
Materials
Reagents
All reagents were purchased from Sigma-Aldrich
unless otherwise stated.
 pET14b-PhoS plasmid (made in-house).
 Agar
 Chloramphenicol CAUTION! harmful if
swallowed, potential carcinogen, can cause
skin and eye irritation. Wear gloves when
handling.
 Amphicillin CAUTION! harmful if
swallowed, can cause skin and eye
irritation. Wear gloves when handling.
 NaCl (Fisher Scientific)
 Tryptone (Fisher Scientific)
 Yeast Extract (Fisher Scientific)
 Petri dishes
 E.coli Rosetta™ DE3 PLySs competent
cells (Merck-Millipore)
 50 ml sterile Falcon Tubes (Fisher
Scientific)
 Eppendorf tubes (Star Labs)
 Magnesium sulfate (MgSO4)
 Corning®
Costar®
Stripette®
serological
pipettes
 ZYM-5052 medium (see REAGENT
SETUP)
 Imidazole CAUTION! Imidazole is a
respiratory, skin and eye irritant. Use gloves
when handling.
 Tris (CalBiotech)
 Acrylamide (40% solution) CAUTION!
Acrylamide is a potent neurotoxin and
potential human carcinogen and teratogen.
Wear gloves and handle in a fume
cupboard.
 N,N,N’,N’-Tetramethylethylenediamine
(TEMED) CAUTION! Harmful if inhaled or
swallowed; can cause skin and eye
irritation.
 Ammonium persulfate (APS) CAUTION!
Harmful if swallowed.
CRITICAL Prepare fresh as APS decomposes
on exposure to water.
 4 x gel separating buffer (see Reagent
Setup)
 PD10 column Binding Buffer (see Reagent
Setup)
 PD10 column Wash Buffer (see Reagent
Setup)
 PD10 column Elution Buffer (see Reagent
Setup)

3. 3
 NiSO4 CAUTION! Can cause respiratory,
skin and eye irritation. Also a human
carcinogen, wear gloves when handling.
 Ethanol (20% solution) CAUTION! Ethanol
is highly flammable.
 N-[2-(1-maleimide acetyl]- 7-
(diethylamino)coumarin-3-carboxamide
(MDCC)
 CAUTION! Harmful if swallowed; can cause
skin and eye irritation.
CRITICAL – Once prepared, shield from light to
prevent photodegradation.
 7-methylguanosine (7-MEG)
 Purine nucleoside phosphorylase (PNP)
 Dimethylformamide (DMF) CAUTION! DMF
can cause respiratory, eye and skin
irritation. It is also a probable human
carcinogen. Wear gloves and handle in a
fume cupboard.
 Bradford Reagent CAUTION! Harmful if
swallowed. Also stains skin, wear gloves
when handling.
 Sodium Dodecyl Sulfate (SDS) CAUTION!
Harmful if inhaled or swallowed; can cause
skin and eye irritation.
 Dithiothreitol (DTT)
 Potassium phosphate (KPi)
Reagent Setup
Lysogeny Broth (LB) and Agar (500ml): Weigh
out 10 g Tryptone, 5 g Yeast Extract, 5 g NaCl and
12.5 g Agar. Add to a clean conical flask with 500
ml ddH2O and dissolve using a magnetic stirrer.
Sterilise by autoclave.
LB for cell growth: 10 g/L Tryptone, 5 g/L Yeast
Extract and 5 g/L NaCl. Weigh out appropriate
amounts of Tryptone, Yeast Extract and NaCl and
add to the appropriate volume of dd. H2O for the
volume of cells you wish to grow.
Chloramphenicol (Chlor) 1000 x stock: 35
mg/ml, dissolved in 100% ethanol. Sterile filter
using a 0.2 µM filter. Once made, the Chlor can be
aliquotted into Eppendorf tubes and stored at -20
°C until needed.
Amphicillin (Amp) 1000 x stock: 100 mg/ml,
dissolved in dd. H2O. Sterile filter using a 0.2 µM
filter. Once made, the Amp can be aliquoted into
Eppendorf tubes and stored at -20 °C until needed.
Pi stock: 50 ml, 200 mM KPi.
ZYM-5052 medium: For 1 litre: 938 ml ZY, 40 ml
25 x M, 20 ml 50 x 5052, 2 ml MgSO4 and 0.2 ml
trace elements (Table 1)
CRITICAL– Sterile filter and do not autoclave the
50 x 5052 component. Autoclave all other
components at 121 °C for 15 minutes.
Component of
ZYM-5052
medium
Ingredients Concentration
ZY Tryptone
Yeast Extract
10 g/L
5 g/L
25 x M Na2HPO4
KH2PO4
NH4Cl
Na2SO4
12.5 mM
12.5 mM
25 mM
2.5 mM
50 x 5052 Glycerol
D-Glucose
α-lactose
0.5 %
0.005 %
0.2 %
MgSO4 MgSO4 2 mM
1000 x Trace
elements
Trace
elements
0.2 ml/L
1000 ml PD10 column Binding Buffer*: 25 mM
Tris, 5 mM Imidazole, 500 mM NaCl, pH 7.4.
500 ml PD10 column Wash Buffer*: 25 mM Tris,
20 mM Imidazole, 500 mM NaCl, pH 7.4.
500 ml PD10 column Elution Buffer*: 25 mM Tris,
400 mM Imidazole, 100 mM NaCl, pH 7.4.
500 ml HEPES Buffer*: 50 mM HEPES, pH 8.0.
100 ml NiSO4 solution: 400 mM NiSO4.
500 ml 20 % Ethanol solution: 100 ml absolute
Ethanol (EtOH) and 400 ml dd. H2O.
100 ml 2 x SDS-PAGE Sample Buffer - 250 mM
Tris-HCl, pH 6.8, 2% SDS w/v, 20% Glycerol v/v,
0.01% Bromophenol Blue. Add 14 µl 2-
mercaptoethanol to 200 µl of 2 x Sample Buffer
CAUTION! 2-mercaptoethanol is toxic, wear
gloves.
4 x Separating Gel Buffer: 1.5 M Tris-HCl, pH 8.8,
0.4 % w/v SDS.
4 x Stacking Gel Buffer: 0.5 M Tris-HCl, pH 6.8,
0.4 % w/v SDS.
500 ml Desalting Buffer*: 20 mM Tris-HCl, pH 8.1.
*CRITICAL – To minimize background Pi
contamination, make up all these buffers in plastic
bottles and weigh out their components using
disposable spatulas and weighing boats. To

4. 4
determine their pH, aliquot ~ 5ml of buffer into a
sterile 50 ml Falcon tube and determine the pH of
this sample.
Procedure
Cloning of PhoS into pET14b vector
Using Q5 Hotstart Taq (New England Biolabs)
PhoS was cloned from E. coli genomic DNA using
the primers phoS_F 5’-
TTCCATATGGAAGCAAGCCTGACAGG-3’ and
phoS_R 5’-TTCGGATCCTTAGTACAGC-3’. This
removes the leading 27 N-terminal periplasm
targeting sequence and introduces NdeI and
BamHI and the 5’ and 3’ end respectively. The
gene was ligated into pET14b between NdeI and
BamHI and sequence verified (GATC
biotech). Using Quick Change Mutagenesis
(Stratagene) mutant A197C was generated and
sequence verified. The new truncated PhoS A197C
protein was renamed phosphate binding protein
(PBP).
To obtain a sample of the plasmid contact Dr
Nathan Adams, (nathan.adams@sheffield.ac.uk).
Transformation of E.coli PLySs competent cells
1. Rapidly defrost pET14bPBP DNA and E.coli
PLySs cells then store them on ice.
2. Add 1 µl of DNA to 30-50 µl of cells.
3. Incubate the mixture on ice for 30 minutes.
4. Incubate at 42 °C for 2 minutes.
5. Further incubate on ice for 2 minutes.
6. Aseptically add 800 µl of sterile LB to the mixture.
7. Incubate at 37 °C, 180 rpm for 60 minutes.
8. Centrifuge the mixture for 1 minute at 4000 rpm.
9. Discard the supernatant and resuspend the pellet
in 200 µl of sterile LB.
10. Plate onto LB-Agar Amp/Chlor plates
11. Incubate plate(s) overnight at 37 °C.
12. Remove plate(s) from incubator, inspect for
colonies, cover edges with Parafilm and store in a
refrigerator at 4 °C.
PAUSE POINT Plates can be stored at 4 °C until
used.
Overexpression of PBP - overnight starter
cultures
13. Aseptically add 20 µl of 1000 x stock Amp and
Chlor to 20 ml of sterile LB.
14. Inoculate 1 transformant colony into the LB
Amp/Chlor.
15. Incubate the overnight starter culture at 37 °C,
200 rpm for ~ 18 hours.
Overexpression of PBP - Large 1l overnight
cultures with autoinducing ZYM-5052 medium
16. To the ZY media add appropriate volumes of M
component, 5052 component and trace metals
17. Aseptically, add 1000 µl of 1000 x stock Amp
and Chlor to each l.5 l flask.
18. Aseptically, add 1 O/N starter culture to each
flask.
19. Incubate large 1 l overnight cultures for 21
hours, 180 rpm at 25 °C.
20. Harvest cells at 8000 x G for 30 minutes at 25
°C.
21. Discard the supernatant and retain the pellet in
a sterile 50 ml Falcon tube.
22. Resuspend each pellet in 40 ml of Binding
Buffer (See Reagent Setup).
PAUSE POINT Resuspended cells can be stored
at -20 °C until required.
'No stain' gel for SDS-PAGE
23. Wear gloves for this preparation. Prepare a
10% separating gel by adding following
components, in order, to a sterile 50 ml Falcon tube.
 2.5 ml of 4 x gel separating buffer
 4.9ml of dH2O
 2.5 ml of 40% acrylamide
 5 µl of TEMED
24. Vortex the solution briefly to mix components.
25. Add ~5 mg of Ammonium Persulfate to 1 ml of
dd. H2O in another sterile Falcon tube and vortex to
mix.
26. Add 50 µl of APS solution to the other
components of the 10% separating gel.

5. 5
27. Vortex this solution again to mix all the
components.
28. Apply the 10% separating gel using a syringe.
29. Add a thin layer of isopropanol to create a level
interface between the stacking and separating gels
30. Leave the 10% separating gel to set for 30 – 45
minutes.
31. Prepare a 17% stacking gel by adding the
following components, in order to a sterile 50 ml
Falcon tube.
 2.5 ml of 4 x stacking gel buffer
 6.6 ml of dH2O
 6.8 ml of 40 % acrylamide
 10 µl of TEMED
32. Vortex these components briefly to mix.
33. Add 100 µl of the APS solution to the 17%
stacking gel and vortex the components again
briefly to mix
34. Apply the 17 % stacking gel using a syringe
35. Leave the 17% stacking gel to set for 30 - 45
minutes.
36. Once the gel is set, wrap it in moist paper towel
and then aluminium foil.
PAUSE POINT – The gel can be stored in a
refrigerator at 4 °C until required.
Purification of PBP - preparation of Ni-NTA
PD10 column.
37. Pour a 10 ml chelating sepharose column and
prepare by as follows:
 Wash with 50 ml dd. H2O
 4 ml NiSO4 solution
 50 ml dd. H2O
 50 ml Binding Buffer
 50 ml 20 % ethanol solution
PAUSE POINT -. Once prepared, the Ni-NTA
column can be stored in 10 ml of 20 % ethanol at 4
°C until required.
Purification of PBP
38. Remove the 20% ethanol present in the Ni-NTA
column by applying 50 ml of binding buffer (see
Reagent Setup) to the column.
39. Obtain some ice and set up 2 x rows of 10-15
Eppendorf tubes. To one row add 10 µl of Bradford
Reagent and 100 µl of dd. H2O.
40. If previously frozen, rapidly defrost the
resuspended E.coli cells in hot water.
41. Add 1 mM of Pefabloc® serine protease
inhibitor to the defrosted cells and invert the
solution to mix.
42. Sonicate cells for 6-10 minutes in 30 second
'pulses' (30 seconds 'on', 30 seconds 'off'.
CRITICAL - Place falcon tube(s) containing the
cells in a plastic beaker of ice to prevent
overheating from sonication. Position the sonicator
probe in the middle of the resuspended cell solution
and check that this position has not changed as the
ice melts with sonication.
43. Centrifuge the resultant 'cell slurry' at 21 000
rpm for 20 minutes at 4 °C using a JA 25.5 rotor.
44. Retain the supernatant in a 50 ml Falcon tube
and store on ice. Discard the pellet.
45. Add the supernatant to the column. Collect the
eluent in a 50 ml Falcon tube labelled 'original
supernatant' and store on ice.
46. Apply 50 mls of binding buffer to the column.
Collect the eluent in a 50 ml Falcon tube labelled
'wash 1' and store on ice.
47. Apply 100 ml of wash buffer to the column.
Collect the eluent in a 50 ml Falcon tube labelled
'wash 2' and store on ice.
48. Apply 20-30 ml of elution buffer to the column.
Collect the eluent in numbered Eppendorf tubes as
2 ml fractions.
49. Add 10 µl of each 2 ml fraction to an Eppendorf
tube containing Bradford reagent and dd. H2O (see
step 39). In addition, take 10 µl samples from wash
1, wash 2 and the original supernatant.
CRITICAL - Look for a colour change from brown
to blue. This will determine which eluted 2 ml
fractions and whether or not Wash 1, 2 and original
supernatant contain protein.
50. Pool the 2 ml fractions which contain protein
(the eluted PBP) and discard the others.
51. Add 10 mM DTT to the pooled fractions from a
1 M stock.

6. 6
PAUSE POINT – Store the pooled fractions with
DTT at 4 °C overnight.
Verifying the presence of PBP by SDS-PAGE
52. Remove a pre-prepared 'no stain' gel from the
refrigerator.
53. Take a 10 µl aliquot of all samples and place in
fresh Eppendorf tubes with 10 µl of 2 x sample
buffer.
54. Incubate all samples at 70 °C for 20 minutes.
55. To Lane 2 of the gel load 5 µl of protein
molecular weight marker (BioRad).
56. Load all incubated samples into other lanes of
the gel.
57. Run the gel at 200 V for 45 minutes.
58. Image the gel, and verify the presence of PBP
(a band of ~ 35 kDa) in the pooled fractions eluted
from the Ni-NTA column (Figure 2).
Figure 2 SDS-PAGE of PBP purification Lane 1; supernatant,
Lane 2; molecular weight marker; Lane 3, unbound elution;
Lane 4, Wash 1 (5 mM Imidazole); Lane 5, Wash 2 (20 mM
Imidazole); Lane 6, Wash 3 (50 mM Imidazole); Lane 7, Elution
1; Lane 8, Elution 2; Lane 9, Elution 3; Lane 10, Elution 4; Lane
11, Elution 5; Lane 12, Elution 6.
Buffer exchange of purified PBP
59. Equilibrate a PD10 desalting column with 30 ml
of desalting buffer
60. Concentrate the PBP protein in a Vivaspin
column at 4000 rpm until the solution is >2.5 ml.
61. Remove the supernatant using a plastic Pasteur
pipette and transfer to a small Falcon tube.
62. Make the solution up to 2.5 ml with desalting
buffer.
63. Add this 2.5 ml solution to the equilibrated PD10
column.
64. Add 3 ml of desalting buffer to the column and
collect the eluent in a new 25 ml Falcon tube.
PAUSE POINT – The buffer exchanged PBP can
be frozen in liquid nitrogen and stored as 200 µl
aliquots at -80 °C until required.
Determining the concentration of unlabelled
PBP
65. Rapidly defrost a 200 µl aliquot of purified,
unlabelled PBP (section XXX)
66. Measure absorbance of the unlabelled protein
at 280 nm, ε = 60 880 M-1
cm-1
. Calculate the
protein concentration.
Preparation of MDCC
67. Dissolve 5 mg of MDCC in 2 ml of DMF in an
Eppendorf tube to give a 2.5 mg/ml solution.
CAUTION! DMF is a hazardous solvent (see
REAGENTS). Wear gloves.
68. Calculate the concentration of MDCC in moles.
CRITICAL – Wrap the Eppendorf tube in Aluminium
foil to protect the dissolved MDCC from
photodegradation.
PAUSE POINT – The dissolved MDCC can be
stored at -20 °C until required.
Preparation of 7-MEG stocks
69. Prepare a 4.2 ml, 10 mM stock of 7-MEG in 50
mM HEPES, pH 8.0. Make 150 µl aliquots.
PAUSE POINT – The aliquots of 10 mM 7-MEG
stock can be stored at -20 °C and used as required.
CRITICAL – Once defrosted, do not refreeze the
150 µl aliquots. Dispose of any excess aliquot
appropriately.
Buffer exchange of unlabelled PBP into HEPES
buffer
70. Defrost a 200 µl aliquot of unlabelled PBP then
store on ice.
71. Apply 300 µl of 50 mM HEPES buffer, pH 8.1 to
two Zeba™ spin desalting columns.

7. 7
72. Centrifuge both columns for 1 minute at 4000 x
g. Discard the flow-through.
72. Repeat steps 71 and 72 another seven times to
remove residual Pi from the columns.
73. Apply 100 µl of defrosted PBP to each Zeba™
column.
74. Centrifuge both columns for 2 minutes at 4000
x g and pool the flow-through in an Eppendorf tube.
Incubation of unlabelled PBP with 'Pi Mop'
system
75. Dilute the unlabelled PBP to 100 µM in 50 mM
HEPES, pH 8.1.
76.Defrost a 200 µl aliquot of 10 mM stock 7-MEG
and the PNP then store on ice.
77. Incubate the 100 µM unlabelled PBP with 0.2
unit/ ml PNP and 1 mM 7-MEG (from the 10 mM
stock) for 20-25 minutes at room temperature.
CRITICAL - Ensure to rapidly defrost the unlabelled
PBP, PNP and 7-MEG.
Labelling of PBP with MDCC
78. Defrost the 2.5 mg/ ml (5.2 mM) stock of MDCC.
79. Incubate 200 µM MDCC with the 'Pi mopped',
unlabelled PBP (a 2:1 molar equivalent of
MDCC:PBP) on an end-over-end mixer.
CRITICAL - Ensure the Eppendorf tube containing
the labelling reaction components is wrapped in
Aluminium foil to protect the MDCC from
photodegradation.
PAUSE POINT - This labelling reaction can be
incubated overnight at 4 °C or at room temperature
for 4 hours.
Removal of excess MDCC from the labelling
reaction
80. Set up a PD-10 desalting column as
appropriate.
81. Remove the top cap and discard the column
storage solution. Cut the bottom sealed end.
82. Equilibrate the column with 30 ml of desalting
buffer (see Reagent Setup) to remove any residual
Pi.
83. Apply all of the MDCC-PBP to the column, and
top this volume up to 2.5 ml with desalting buffer if
necessary.
84. Collect the eluent in a 5 ml Eppendorf tube.
85. Divide the MDCC-PBP into 250 µl aliquots.
CRITICAL - Ensure that the 250 µl aliquots are
wrapped in Aluminium foil to protect the MDCC
from photodegradation.
PAUSE POINT - Once collected, the MDCC-PBP
can be stored at -80 °C until required.
Determining the concentration of MDCC-PBP
86. Blank the Nanodrop 2000 probe with 2.5 µl of
desalting buffer.
87. Apply 2.5 µl of labelled PBP to the Nanodrop
2000 probe.
88. Measure absorbance of the labelled PBP at 280
nm and 430 nm in triplicate.
89. Correct for absorbance of MDCC at 280 nm
using the following formula:
Average A430 x 0.164 = α
Average A280 - α = Corrected A280 value
90. Use the corrected A280 value and to calculate the
concentration of labelled PBP.
91. Use the average A430 reading to calculate the
concentration of free MDCC.
Calculating the labelling efficiency of PBP with
MDCC
92. Calculate the labelling efficiency of PBP
according to the equation below:
𝐿𝐸 =
[𝑀𝐷𝐶𝐶]
[𝑃𝐵𝑃]
𝑥 100%
Where LE = labelling efficiency, [MDCC] =
concentration of MDCC and [PBP] = concentration
of PBP.
PAUSE POINT - Once the concentration of MDCC-
PBP and labelling efficiency have been determined,
the remaining MDCC-PBP can be stored at -80 °C
until required.
Pi titrations of MDCC-PBP
93. If necessary, rapidly defrost the MDCC-PBP
then store on ice.
94. Incubate the MDCC-PBP with 0.2 unit/ ml PNP
and 1 mM 7-MEG for 20-25 minutes at room
temperature.

8. 8
95. Exchange 200 µl of MDC-PBP into 50 mM
HEPES Buffer (see Reagent Setup), pH 8.1 using
two Zeba™ spin desalting columns (see steps 71-
74).
CRITICAL - Ensure the Eppendorf containing the
pooled eluents from the Zeba™ columns is
wrapped in Aluminium foil to protect the MDCC
from photodegradation.
96. Appropriately dilute the MDCC-PBP to 0.5 µM
in 50 mM HEPES buffer, pH 8.1 to a final volume of
2 ml in a fluorimeter cuvette.
97. Measure emission of this sample from 0-10 µM
KPi in 0.25 µM increments (4 µl of KPi from a 200
mM stock) by exciting the sample at 430 ± 2.5 nM.
An increase in fluorescence emission at 464 nm
should be observed (Figure 3).
Figure 3 Titration of MDCC-PBP with Pi Dashed line: 0 µM Pi,
0.5 µM MDCC-PBP, 50 mM HEPES, pH 8.1 black line: 3 µM
Pi, 0.5 µM MDCC-PBP, 50 mM HEPES, pH 8.1. Emission
spectra were recorded using a Horiba Fluoromax-3 fluorimeter.
Concluding Remarks
An A197C PBP variant has been cloned,
overexpressed and purified using Ni-NTA affinity
chromatography. The purified protein has been
successfully labelled with MDCC and the labelled
protein is sensitive to Pi. However, the increase in
fluorescence at 464 nm is not as high as
anticipated. This is probably because the MDCC-
PBP preparation used was contaminated with
background Pi despite steps taken to avoid this. An
improved 'Pi Mop' system that additionally uses
phosphodeoxyribomutase to sequester Pi as
ribose-5-phosphate as opposed to less
thermodynamically stable ribose-1-phosphate has
been reported.1,6
Use of this system in addition to
incubating the unlabelled PBP with this improved
'Pi Mop' prior to the labelling reaction could help
reduce Pi contamination in future work.
Acknowledgements
H.T. was supported by a summer studentship from
P3 Translational Agricultural Technologies.
References
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S., Hazlett L. T., Corrie, J. E. T. and Webb M.R.
Biochemistry 37 10370-10380 (1998)
2. Hirshberg, M., Henrick, K., Haire, L., L., Vasisht,
N., Brune M., Corrie, J.E.T. and Webb M.R.
Biochemistry 37 10381-10385 (1998)
3. Brune, M., Hunter, J. L., Corrie, J.E.T. and
Webb, M.R. Biochemistry 33 8262-8271 (1994)
4. Izui, K., Matsumura, H., Furumoto, T. and Kai,
Y. Annual Reviews in Plant Biology 55 69-84 (2004)
5. Meyer, C.P., Rustin, P. and Wedding, R. T. Plant
Physiology 86 325-328 (1988)
6. Nixon, A.E., Hunter, J. L., Bonifacio, G.,
Eccleston, J. F., and Webb, M. R. Analytical
Biochemistry 265 299-307 (1998)