pstS (A197C) in pET22b: information and sequence
Plasmid
pET22b_PstS_1
PstS gene cloned into pET22b via the two restriction sites NdeI and XhoI
Usage
The expressed protein is a single-cysteine variant of E. coli phosphate binding protein. The
gene encodes the mature protein without the N-terminal signal peptide, but with an Nterminal methionine for subsequent labeling at the cysteine with one MDCC (N-[2-(1maleimidyl)ethyl]-7-diethylaminocoumarin-3-carboxamide) per molecule. This adduct is
then used as a fluorescent phosphate biosensor, having ~7-fold fluorescence intensity
increase on binding inorganic phosphate with very high affinity.
Publications
References to usage:
Brune, M.; Hunter, J. L.; Howell, S. A.; Martin, S. R.; Hazlett, T. L.; Corrie, J. E. T.; Webb,
M. R., Mechanism of inorganic phosphate interaction with phosphate binding protein from
Escherichia coli. Biochemistry 1998, 37, 10370-10380.
Brune, M.; Hunter, J. L.; Corrie, J. E. T.; Webb, M. R., Direct, real-time measurement of
rapid inorganic phosphate release using a novel fluorescent probe and its application to
actomyosin subfragment 1 ATPase. Biochemistry 1994, 33, 8262-8271.
Webb, M. R., A fluorescent sensor to assay inorganic phosphate. In Kinetic Analysis of
Macromolecules: a Practical Approach, Johnson, K. A., Ed. Oxford University Press: Oxford,
U.K., 2003; pp 131-152.
Reference to this expression construct and expression:
Solscheid, C.; Kunzelmann, S.; Davis, C. T.; Hunter, J. L.; Nofer, A.; Webb, M. R.,
Development of a reagentless biosensor for inorganic phosphate, applicable over a wide
concentration range. Biochemistry 2015, 54, 5054-5062.
Protocol
Separate file.
Note that the expression plasmid has changed since Brune et al. (2008).
Contact
Martin Webb
The Francis Crick Institute
London
[email protected]
Expressed protein
Met
Q G
L F
Y L
S G
G K
L I
Q K
W A
D S
E
I
Q
G
T
G
S
G
Y
S
A
G
F
K
S
N
A
E
K
G
S
S
P
I
F
D
D
D
T
K
L
S
T
K
V
G
G
A
G
P
T
G
V
K
F
I
K
W
A
L
G
G
I
W
T
A
P
P
K
Y
A
V
G
D
S
A
V
I
Q
G
K
G
D
Y
F
S
T
A
A
Q
V
E
L
V
P
S
N
T
I
V
A
A
Q
T
T
D
F
I
L
I
K
R
E
T
L
P
A
A
A
V
L
E
F
D
A
N
V
K
N
P
N
I
Y
P
T
N
L
E
G
F
L
A
V
V
I
N
E
A
A
I
S
Y
D
P
P
W
I
N
H
L
A
F
G
G
K
G
A
K
P
K
G
L
L
N
Y
A
D
D
W
A
K
K
N
V
K
Q
S
A
S
S
L
V
E
G
K
V
D
D
G
P
G
Y
A
K
V
T
A
E
S
T
C
D
P
E
Y
P
L
Q
G
Y
W
E
Q
Q
L
V
N
S
A
S
Q
V
K
S
L
I
T
K
K
G
R
E
D
D
A
V
Q
T
T
A
T
E
G
V
K
N
F
E
A
G
K
K
V
W
N
A
V
W
N
L
T
R
P
L
Q
L
K
K
A
L
R
I
A
D
K
T
V
Q
G
A
G
Y
L
F
N
N
E
D
D
L
T
T
F
I
Y
G
I
G
G
K
N
D
K
Sequence
NdeI PBP A197C Stop Stop XhoI
CATATGGAAGCAAGCCTGACAGGTGCAGGTGCAACCTTCCCTGCGCCGGTGTATGCCAAATGGGCTGAC
ACTTACCAGAAAGAAACCGGTAATAAAGTTAACTACCAGGGTATCGGTTCTTCCGGTGGCGTAAAACAG
ATTATCGCTAATACCGTTGATTTTGGTGCCTCTGACGCGCCGCTGTCTGACGAAAAACTGGCTCAGGAA
GGTCTGTTCCAGTTCCCGACCGTGATTGGCGGCGTGGTGCTGGCGGTTAACATTCCAGGGCTGAAGTCT
GGCGAACTGGTGCTGGATGGTAAAACCCTCGGCGACATCTACCTGGGCAAAATCAAGAAGTGGGATGAT
GAAGCCATCGCCAAACTGAATCCGGGTCTGAAACTGCCTTCACAAAACATTGCTGTAGTACGCCGCGCA
GATGGCTCCGGGACTTCCTTCGTCTTCACCAGCTACCTGGCGAAAGTGAACGAAGAGTGGAAAAACAAC
GTTGGTACTGGCTCTACCGTAAAATGGCCGATCGGTCTGGGCGGTAAAGGTAACGACGGTATCGCCGCG
TTCGTTCAGCGTCTGCCGGGTGCAATTGGTTATGTTGAATATTGTTACGCGAAGCAGAACAACCTGGCG
TACACCAAACTGATCTCCGCTGATGGTAAACCGGTTAGTCCGACCGAAGAAAACTTCGCTAATGCAGCA
AAAGGTGCAGACTGGAGCAAAACCTTCGCTCAGGATCTGACCAACCAGAAAGGCGAAGATGCATGGCCT
ATTACCTCTACCACGTTCATTCTGATCCACAAAGATCAGAAGAAACCAGAACAAGGCACAGAAGTGCTG
AAATTCTTCGACTGGGCGTACAAAACCGGGGCTAAACAGGCGAACGACCTGGATTACGCCAGCCTGCCG
GATAGTGTAGTTGAACAGGTTCGCGCTGCGTGGAAGACCAATATTAAAGACAGTAGCGGTAAGCCGCTG
TACTAATGACTCGAG
This is cloned into pET22b via the two restriction sites NdeI and XhoI
TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGAC
CGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGC
CGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCT
CGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCG
CCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCC
TATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCT
GATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCG
GGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAG
ACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGT
CGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGT
AAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGAT
CCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGC
GGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTT
GGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGC
TGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCT
AACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGA
AGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATT
AACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGC
AGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCG
TGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACAC
GACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAA
GCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATT
TAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTT
CCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAAT
CTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAAC
TCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTA
GTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGT
GGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGC
GCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACT
GAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCC
GGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTA
TAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAG
CCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACAT
GTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGC
TCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTA
TTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCT
GATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGAC
ACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTG
TGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGC
GGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGT
TGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCT
GTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAG
AGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAAC
AACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATA
CAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGG
GCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGG
TCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAG
TAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCG
CCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAG
CGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGT
CCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCA
TAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGG
GCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCG
CTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTT
TGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACC
GCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTG
ATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCC
GCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACC
AGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTC
CAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGA
CGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACC
AGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCA
GAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCC
AGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCT
TCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATC
GCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGT
TTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTT
TCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCG
GCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGG
CGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTT
ATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAA
TGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAA
ACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCC
AGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGAT
CCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATT
TTGTTTAACTTTAAGAAGGAGATATACATATGGAAGCAAGCCTGACAGGTGCAGGTGCAACCTTCCCTG
CGCCGGTGTATGCCAAATGGGCTGACACTTACCAGAAAGAAACCGGTAATAAAGTTAACTACCAGGGTA
TCGGTTCTTCCGGTGGCGTAAAACAGATTATCGCTAATACCGTTGATTTTGGTGCCTCTGACGCGCCGC
TGTCTGACGAAAAACTGGCTCAGGAAGGTCTGTTCCAGTTCCCGACCGTGATTGGCGGCGTGGTGCTGG
CGGTTAACATTCCAGGGCTGAAGTCTGGCGAACTGGTGCTGGATGGTAAAACCCTCGGCGACATCTACC
TGGGCAAAATCAAGAAGTGGGATGATGAAGCCATCGCCAAACTGAATCCGGGTCTGAAACTGCCTTCAC
AAAACATTGCTGTAGTACGCCGCGCAGATGGCTCCGGGACTTCCTTCGTCTTCACCAGCTACCTGGCGA
AAGTGAACGAAGAGTGGAAAAACAACGTTGGTACTGGCTCTACCGTAAAATGGCCGATCGGTCTGGGCG
GTAAAGGTAACGACGGTATCGCCGCGTTCGTTCAGCGTCTGCCGGGTGCAATTGGTTATGTTGAATATT
GTTACGCGAAGCAGAACAACCTGGCGTACACCAAACTGATCTCCGCTGATGGTAAACCGGTTAGTCCGA
CCGAAGAAAACTTCGCTAATGCAGCAAAAGGTGCAGACTGGAGCAAAACCTTCGCTCAGGATCTGACCA
ACCAGAAAGGCGAAGATGCATGGCCTATTACCTCTACCACGTTCATTCTGATCCACAAAGATCAGAAGA
AACCAGAACAAGGCACAGAAGTGCTGAAATTCTTCGACTGGGCGTACAAAACCGGGGCTAAACAGGCGA
ACGACCTGGATTACGCCAGCCTGCCGGATAGTGTAGTTGAACAGGTTCGCGCTGCGTGGAAGACCAATA
TTAAAGACAGTAGCGGTAAGCCGCTGTACTAATGACTCGAGCACCACCACCACCACCACTGAGATCCGG
CTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCC
TTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT