Infrared-fluorescent proteins from Deinococcus radiodurans for whole-body imaging
Infrared-fluorescent proteins:
1, Engineered from a bacteriophytochrome DrBphP from Deinococcus radiodurans
Cloned by gene synthesis based on chromophore-binding domain (CBD),
consisting of the PAS and GAF domains, which are necessary and
sufficient for covalent incorporation of BV
2, Excitation: 684 nm; Emission 708 nm
3, Extinction coefficient > 90,000 M−1cm−1
4, Quantum yield 0.07
5, Suitable for whole-body imaging.
IFP1.0: Dimer, D207H from DrCBD
IFP1.1: Dimer, A288V, I208T, V186M from IFP1.0
IFP1.2: Dimer, M54V from IFP1.1
IFP1.3: Monomer, L311K from IFP1.2
IFP1.4: Monomer, T135I, L195M, H196Q, G119A, Y307E, L314G, V318R from IFP1.3
Fluorescent Absorption Extinct. Emission Quantum Relative Stoichiometry
protein max. (nm) coeff. max. (nm) yield brightness
(M-1 cm-1) (%)
IFP1.0 699 60,000 722 0.028 100 Dimer
IFP1.1 686 86,000 713 0.050 256 Dimer
IFP1.2 684 86,000 707 0.066 338 Dimer
IFP1.3 684 84,000 707 0.061 305 Monomer
IFP1.4 684 92,000 708 0.070 383 Monomer
Half-life of IFP1.4 in HEK293A cells is 4.03 ± 0.41 hours.
DrCBD MSRDPLPFFPPLYLGGPEITTENCEREPIHIPGSIQPHGALLTADGHSGEVLQMSLNAAT 60
DrCBD FLGQEPTVLRGQTLAALLPEQWPALQAALPPGCPDALQYRATLDWPAAGHLSLTVHRVGE 120
DrCBD LLILEFEPTEAWDSTGPHALRNAMFALESAPNLRALAEVATQTVRELTGFDRVMLYKFAP 180
DrCBD DATGEVIAEARREGLHAFLGHRFPASDIPAQARALYTRHLLRLTADTRAAAVPLDPVLNP 240
DrCBD QTNAPTPLGGAVLRATSPMHMQYLRNMGVGSSLSVSVVVGGQLWGLIACHHQTPYVLPPD 300
DrCBD LRTTLEYLGRLLSLQVQVKEA 321
IFP1.4 MARDPLPFFPPLYLGGPEITTENCEREPIHIPGSIQPHGALLTADGHSGEVLQVSLNAAT 60
IFP1.4 FLGQEPTVLRGQTLAALLPEQWPALQAALPPGCPDALQYRATLDWPAAGHLSLTVHRVAE 120
IFP1.4 LLILEFEPTEAWDSIGPHALRNAMFALESAPNLRALAEVATQTVRELTGFDRVMLYKFAP 180
IFP1.4 DATGEMIAEARREGMQAFLGHRFPASHTPAQARALYTRHLLRLTADTRAAAVPLDPVLNP 240
IFP1.4 QTNAPTPLGGAVLRATSPMHMQYLRNMGVGSSLSVSVVVGGQLWGLIVCHHQTPYVLPPD 300
IFP1.4 LRTTLEELGRKLSGQVQRKEAEFHHHHHH 329
Blast
Score = 554 bits (1427), Expect = 1e-162, Method: Compositional matrix adjust.
Identities = 307/346 (88%), Positives = 311/346 (89%), Gaps = 10/346 (2%)
DrCBD 6 MSRDPLPFFPPLYLGGPEITTENCEREPIHIPGSIQPHGALLTADGHSGEVLQMSLNAAT 65
M+RDPLPFFPPLYLGGPEITTENCEREPIHIPGSIQPHGALLTADGHSGEVLQ+SLNAAT
IFP1.4 4 MARDPLPFFPPLYLGGPEITTENCEREPIHIPGSIQPHGALLTADGHSGEVLQVSLNAAT 63
Query 66 DRCBDFLGQEPTVLRGQTLAALLPEQWPALQAALPPGCPDALQYRATLDWPAAGHLSLTV 125
FLGQEPTVLRGQTLAALLPEQWPALQAALPPGCPDALQYRATLDWPAAGHLSLTV
Sbjct 64 –IFPFLGQEPTVLRGQTLAALLPEQWPALQAALPPGCPDALQYRATLDWPAAGHLSLTV 121
Query 126 HRVGEDRCBDLLILEFEPTEAWDSTGPHALRNAMFALESAPNLRALAEVATQTVRELTGF 185
HRV E LLILEFEPTEAWDS GPHALRNAMFALESAPNLRALAEVATQTVRELTGF
Sbjct 122 HRVAE–IFPLLILEFEPTEAWDSIGPHALRNAMFALESAPNLRALAEVATQTVRELTGF 179
Query 186 DRVMLYKFAPDRCBDDATGEVIAEARREGLHAFLGHRFPASDIPAQARALYTRHLLRLTA 245
DRVMLYKFAP DATGE+IAEARREG+ AFLGHRFPAS PAQARALYTRHLLRLTA
Sbjct 180 DRVMLYKFAP–IFPDATGEMIAEARREGMQAFLGHRFPASHTPAQARALYTRHLLRLTA 237
Query 246 DTRAAAVPLDPVLNPDRCBDQTNAPTPLGGAVLRATSPMHMQYLRNMGVGSSLSVSVVVG 305
DTRAAAVPLDPVLNP QTNAPTPLGGAVLRATSPMHMQYLRNMGVGSSLSVSVVVG
Sbjct 238 DTRAAAVPLDPVLNP–IFPQTNAPTPLGGAVLRATSPMHMQYLRNMGVGSSLSVSVVVG 295
Query 306 GQLWGLIACHHQTPYVLPPDDRCBDLRTTLEYLGRLLSLQVQVKEA 351
GQLWGLI CHHQTPYVLPPD LRTTLE LGR LS QVQ KEA
Sbjct 296 GQLWGLIVCHHQTPYVLPPD–IFPLRTTLEELGRKLSGQVQRKEA 339
DNA Sequence
ATTAAG GAT CCG
GCC ACC ATGGCTCGGGACCCTCTGCCATTCTTTCCACCTCTGTACCTGGGCGGCCCTGAG 60
ATTACAACCGAGAACTGCGAGAGAGAGCCTATCCACATTCCTGGGTCCATCCAGCCACAC 120
GGGGCTCTGCTCACAGCTGACGGCCACTCCGGAGAGGTGCTCCAAGTGTCCCTGAATGCC 180
GCTACCTTCCTGGGCCAGGAGCCTACTGTGCTGCGGGGGCAGACCCTGGCTGCCCTGCTC 240
CCCGAGCAGTGGCCAGCCCTGCAGGCAGCCCTGCCCCCAGGATGTCCAGATGCCCTCCAA 300
TACAGGGCCACCCTCGACTGGCCAGCTGCTGGGCACCTCAGCCTGACTGTGCATCGGGTG 360
GCTGAACTCCTGATCCTGGAGTTCGAACCTACCGAGGCCTGGGACAGCATTGGCCCTCAC 420
GCCCTGAGGAACGCCATGTTTGCTCTGGAAAGCGCTCCAAACCTGCGGGCTCTGGCCGAA 480
GTCGCAACACAAACTGTGAGAGAACTGACTGGCTTCGATCGGGTGATGCTGTACAAATTT 540
GCCCCTGACGCCACTGGAGAGATGATTGCTGAGGCCAGACGGGAGGGCATGCAGGCTTTT 600
CTGGGCCACAGGTTTCCCGCATCCCACACCCCTGCACAAGCTAGGGCCCTCTACACAAGA 660
CACCTGCTCCGGCTGACCGCAGACACCAGGGCTGCAGCAGTGCCCCTCGACCCCGTGCTG 720
AATCCCCAGACAAATGCTCCTACACCTCTGGGCGGAGCTGTCCTCAGAGCTACATCCCCA 780
ATGCACATGCAGTACCTGAGGAATATGGGAGTGGGCTCCTCCCTGAGCGTCAGCGTCGTG 840
GTCGGCGGCCAGCTGTGGGGACTGATTGTCTGCCACCATCAGACACCCTACGTGCTGCCA 900
CCAGATCTGCGGACCACCCTGGAGGAGCTGGGGAGAAAGCTGTCCGGGCAGGTGCAGAGG 960
AAAGAAGCCGGAATGGACGAGCTGTATAAATGATCTAGA CTCGAG atcga
Oligo for gene synthesis
1 ATTAAGGATCCGGCCACCATGGCTCGGGACCCTCTGCCATTCTTTCCACCTCTGTA 56
2 CTCTCGCAGTTCTCGGTTGTAATCTCAGGGCCGCCCAGGTACAGAGGTGGAAAGAATGGC 60
3 CAACCGAGAACTGCGAGAGAGAGCCTATCCACATTCCTGGGTCCATCCAGCCACACGGGG 60
4 CACTTGGAGCACCTCTCCGGAGTGGCCGTCAGCTGTGAGCAGAGCCCCGTGTGGCTGGAT 60
5 GGAGAGGTGCTCCAAGTGTCCCTGAATGCCGCTACCTTCCTGGGCCAGGAGCCTACTGTG 60
6 CCACTGCTCGGGGAGCAGGGCAGCCAGGGTCTGCCCCCGCAGCACAGTAGGCTCCTGGCC 60
7 GCTCCCCGAGCAGTGGCCAGCCCTGCAGGCAGCCCTGCCCCCAGGATGTCCAGATGCCCT 60
8 AGGTGCCCAGCAGCTGGCCAGTCGAGGGTGGCCCTGTATTGGAGGGCATCTGGACATCCT 60
9 CAGCTGCTGGGCACCTCAGCCTGACTGTGCATCGGGTGGCTGAACTCCTGATCCTGGAGT 60
10 GCGTGAGGGCCAATGCTGTCCCAGGCCTCGGTAGGTTCGAACTCCAGGATCAGGAGTTCA 60
11 GCATTGGCCCTCACGCCCTGAGGAACGCCATGTTTGCTCTGGAAAGCGCTCCAAACCTGC 60
12 GTCAGTTCTCTCACAGTTTGTGTTGCGACTTCGGCCAGAGCCCGCAGGTTTGGAGCGCTT 60
13 CACAAACTGTGAGAGAACTGACTGGCTTCGATCGGGTGATGCTGTACAAATTTGCCCCTG 60
14 CCCTCCCGTCTGGCCTCAGCAATCATCTCTCCAGTGGCGTCAGGGGCAAATTTGTACAGC 60
15 GGCCAGACGGGAGGGCATGCAGGCTTTTCTGGGCCACAGGTTTCCCGCATCCCACACCCC 60
16 TCAGCCGGAGCAGGTGTCTTGTGTAGAGGGCCCTAGCTTGTGCAGGGGTGTGGGATGCGG 60
17 CACCTGCTCCGGCTGACCGCAGACACCAGGGCTGCAGCAGTGCCCCTCGACCCCGTGCTG 60
18 AGGACAGCTCCGCCCAGAGGTGTAGGAGCATTTGTCTGGGGATTCAGCACGGGGTCGAGG 60
19 TGGGCGGAGCTGTCCTCAGAGCTACATCCCCAATGCACATGCAGTACCTGAGGAATATGG 60
20 CGCCGACCACGACGCTGACGCTCAGGGAGGAGCCCACTCCCATATTCCTCAGGTACTGCA 60
21 GCGTCGTGGTCGGCGGCCAGCTGTGGGGACTGATTGTCTGCCACCATCAGACACCCTACG 60
22 CCCCAGCTCCTCCAGGGTGGTCCGCAGATCTGGTGGCAGCACGTAGGGTGTCTGATGGTG 60
23 CCTGGAGGAGCTGGGGAGAAAGCTGTCCGGGCAGGTGCAGAGGAAAGAAGCCGGAATGGA 60
24 TCGATCTCGAGTCTAGATCATTTATACAGCTCGTCCATTCCGGCTTCTTTCC 52
