Evrogen
 
작성일 : 15-07-03
[Evrogen] Far-red fluorescent protein Katushka2S

 

Far-red fluorescent protein Katushka2S

 

- Super bright far-red fluorescence

- high signal-to-noise ratio, fast maturation

- Recommended for whole body imaging

 

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Katushka2S is the next generation of far-red fluorescent protein TurboFP635

(Katushka) [Luker et al., 2015; Shcherbo et al., 2009; Gurskaya et al., 2011].

Katushka2S exhibits highest brightness, fastest maturation and superior

signal-to-noise ratio compared to TurboFP635 and many other available far-

red fluorescent proteins: E2-Crimson, mNeptune, mNeptune2.5, mCardinal,

TurboFP650 and NirFP [Lukeret al., 2015], which makes it the protein of

choice for visualization within living tissues. Katushka2S can also be used

together with iRFP720 [Luker et al., 2015] for dual color whole body imaging.

 

Main properties

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Katushka2S normalized excitation (thin line) and emission (thick line) spectra.

 

Characteristic

Description

  Molecular weight, kDa

  26

  Polypeptide length, aa

  235

  Fluorescence color

  far-red

  Excitation maximum, nm

  588

  Emission maximum, nm

  633

  Quantum yield

  0.44

 Extinction coefficient, M-1cm-1

  67 000

  Brightness*

  29.5

  Brightness, % of EGFP

  89

  pKa

  5.7

  Structure

  dimer

  Aggregation

  no

  Maturation rate at 37°C

  super fast

  Photostability

  high

  Cell toxicity

  not observed

  Main advantages

  super bright and fast-maturing far-red fluorescent protein;

  PH-stability

  Possible limitations

  dimer, limited applicability for fusions generation, medium photo

  stability

* Brightness is a product of extinction coefficient and quantum yield, divided by 1000.

 

Recommended filter sets and antibodies

Katushka2S can be recognized using Anti-tRFP antibody (Cat.# AB233-AB234) available from Evrogen.

Recommended Omega Optical filter sets are QMAX-Red and XF102-2. Katushka2S can also be detected

using Texas Red filter sets or similar.

For IVIS Lumina II imaging system the highest fluorescence signal for Katushka2S is observed with the

following settings:

In cell culture: "Cy5.5" (695-770 nm) channel using 570/35nm excitation

In whole body imaging: "Cy5.5" (695-770 nm) channel using 605/35nm excitation

 

Performance and use

Mammalian cells transiently transfected with Katushka2S expression vectors produce bright fluorescence in

12 hrs after transfection. No cytotoxic effects or visible protein aggregation are observed.   

Superior performance of Katushka2S in whole-body imaging was demonstrated using mouse xenograft

model. HEK293FT cells transiently transfected with plasmids encoding different far-red fluorescent proteins

were implanted into mice intramuscularly or subcutaneously. The cells were co-transfected with firefly

luciferase to normalize the transfection efficiency and total numbers of injected cells. Katushka2S produced

higher fluorescence signal and better signal-to-noise ratio than other far-red fluorescence proteins analyzed

at various excitation and emission channels.

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Whole-mouse imaging with IVIS Lumina II system (PerkinElmer).

Representative fluorescence images of nude mice injected into the gluteal muscle with HEK293FT cells

transiently expressing mNeptune, E2-Crimson, Katushka2S, TurboFP635, TurboFP650 and NirFP captured

with indicated excitation (35-nm bandwidth centered at the indicated wavelength) and emission (DsRed 575-

650nm and Cy5.5 695-770nm) filter combination. Pseudocolor scale bar: radiant efficiency (photons/s)/

(µW/cm2). Note that scale bar differ for images with different excitation and emission filters.

Images from [Luker et al., 2015]

 

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Whole-mouse imaging with IVIS Lumina II system (PerkinElmer).

(A) Representative fluorescence images of mice injected in backs with HEK293FT cells transiently

expressing Katushka2S (K2S), TurboFP650 (650), mCardinal (Car) and mNeptune2.5(N2.5). captured with

indicated excitation (35-nm bandwidth centered at the indicated wavelength) and emission (640/20 and

660/20)

filter combination. Pseudocolor scale bar: radiant efficiency (photons/s/cm2/sr)/(µW/cm2).

(B) Graphs display mean values ±SEM for fluorescence radiant efficiency normalized to luciferase photon

flux for each implant (n=4 per condition) for 570-nm (left) and 605-nm (right) excitation and listed emission

filters. Fluorescent proteins are depicted by the following colors: Katushka2S (brown), TurboFP650 (pink),

mCardinal (blue) and mNeptune2.5 (green).

Images from [Luker et al., 2015]

 

Katushka2S was also compared with one of the best phytochrome photoreceptors iRFP720. Katushka2S

produced brighter fluorescence intensity at its optimum conditions as compared with the best output from

iRFP720. At the same time, both proteins showed comparable signal-to-noise ratios at the optimum

wavelengths for excitation and emission since autofluorescence from mouse tissue was lower in the

channel optimal for iRFP720.

 

Katushka2S was also compared with one of the best phytochrome photoreceptors iRFP720. Katushka2S

produced brighter fluorescence intensity at its optimum conditions as compared with the best output from

iRFP720. At the same time, both proteins showed comparable signal-to-noise ratios at the optimum

wavelengths for excitation and emission since autofluorescence from mouse tissue was lower in the

channel optimal for iRFP720.

 

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Fluorescence signals acquired with IVIS Spectrum system (PerkinElmer).

Fluorescence signals normalized to corresponding firefly luciferase luminescence in mice injected in backs

with HEK293FT cells transiently expressing Katushka2S or iRFP70.

(A) Pairwise comparisons of the signals from Katushka2S (brown) and iRFP720 (violet) at different

excitation/emission wavelengths. * p<0.0005

(B) Signal-to-noise ratios at optimal excitation and emission wavelengths for each protein.

 

pKatushka2S-C vector

 

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Vector description

pKatushka2S-C is a mammalian expression vector encoding far-red fluorescent protein Katushka2S.

The vector allows generation of fusions to the Katushka2S C-terminus and expression of Katushka2S fusions

or Katushka2S alone in eukaryotic (mammalian) cells.

 

Katushka2S codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al.,

1996]. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated

upstream of the Katushka2S coding sequence [Kozak, 1987]. Multiple cloning site (MCS) is located between

Katushka2S coding sequence and SV40 polyadenylation signal (SV40 polyA).

 

The vector backbone contains immediate early promoter of cytomegalovirus (PCMV IE) for protein

expression, SV40 origin for replication in mammalian cells expressing SV40 T-antigen, pUC origin of

replication for propagation in E. coli, and f1 origin for single-stranded DNA production. SV40 polyadenylation

signals (SV40 poly A) direct proper processing of the 3'-end of the reporter mRNA.

 

SV40 early promoter (PSV40) provides neomycin resistance gene (Neor) expression to select stably

transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene

expression (Kanr) in E. coli. Kanr/Neorgene is linked with herpes simplex virus (HSV) thymidine kinase

(TK) polyadenylation signals.

 

 

pKatushka2S-N vector

 

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Vector description

pKatushka2S-N is a mammalian expression vector encoding far-red fluorescent protein Katushka2S.

The vector allows generation of fusions to the Katushka2S N-terminus and expression of Katushka2S fusions

or Katushka2S alone in eukaryotic (mammalian) cells.

 

Katushka2S codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al.,

1996]. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated

upstream of the Katushka2S coding sequence [Kozak, 1987]. Multiple cloning site (MCS) is located between

Katushka2S coding sequence and SV40 polyadenylation signal (SV40 polyA).

 

 

The vector backbone contains immediate early promoter of cytomegalovirus (PCMV IE) for protein

expression, SV40 origin for replication in mammalian cells expressing SV40 T-antigen, pUC origin of

replication for propagation in E. coli, and f1 origin for single-stranded DNA production. SV40 polyadenylation

signals (SV40 poly A) direct proper processing of the 3'-end of the reporter mRNA.

 

SV40 early promoter (PSV40) provides neomycin resistance gene (Neor) expression to select stably

transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene

expression (Kanr) in E. coli. Kanr/Neorgene is linked with herpes simplex virus (HSV) thymidine kinase

(TK) polyadenylation signals.

 

 

 Vector type :

  Mammalian expression vector

 Reporter :

  Katushka2S

 Reporter codon usage :

  Mammalian

 Promoter for TagGFP2 :

  PCMV IE

 Host cells :

  Mammalian

 Selection :

  Prokaryotic – kanamycin

  Eukaryotic – neomycin (G418)

 Replication :

  Prokaryotic – pUC ori

  Eukaryotic – SV40 ori

 Use :

  Katushka2S expression in mammalian cells;

 generation of fusions to the Katushka2S C,N-terminus

* The vector sequence has been compiled using the information from sequence databases, published

   literature, and other sources, together with partial sequences obtained by Evrogen. This vector has not

   been completely sequenced.

 

Generation of Katushka2S fusion proteins

A localization signal or a gene of interest can be cloned into MCS of the vector. It will be expressed as a

fusion to the Katushka2S C,N-terminus when inserted in the same reading frame as Katushka2S and no in-

frame stop codons are present. Katushka2S-tagged fusions retain fluorescent properties of the native protein

allowing fusion localization in vivo. Unmodified vector will express Katushka2S when transfectedinto

eukaryotic (mammalian) cells.

 

Note: The plasmid DNA was isolated from dam+-methylated E. coli. Therefore some restriction sites are

blocked by methylation. If you wish to digest the vector using such sites you will need to transform the vector

into a dam- host and make fresh DNA.

 

Expression in mammalian cells

pKatushka2S-C,N vector can be transfected into mammalian cells by any known transfection method. CMV

promoter provides strong, constitutive expression of Katushka2S or its fusions in eukaryotic cells. If required,

stable transformants can be selected using G418 [Gorman, 1985].

 

Propagation in E. coli

Suitable host strains for propagation in E. coli include DH5alpha, HB101, XL1-Blue, and other general purpose

strains. Plasmid incompatibility group is pMB1/ColE1. The vector confers resistance to kanamycin

(30 μg/ml) to E. coli hosts. Copy number in E. coli is about 500.

 

 

pKatushka2S-B vector

 

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 Vector type :

  bacterial expression vector

 Reporter :

  Katushka2S

 Reporter codon usage :

  Mammalian

 Promoter for Katushka2S :

  T5 promoter/lac operator

 Host cells :

  prokaryotic

 Selection :

  ampicillin

 Replication :

  ColE1 ori

 Use :

  Source of the Katushka2S coding sequence;

  Katushka2S expression in bacterial cells

* The vector sequence has been compiled using the information from sequence databases, published

   literature, and other sources, together with partial sequences obtained by Evrogen. This vector has not

   been completely sequenced.

 

Vector description

pKatushka2S-B is a prokaryotic expression vector encoding far-red fluorescent protein Katushka2S (see

reporter description). Reporter codon usage is optimized for high expression in mammalian cells

(humanized) [Haas et al., 1996].

 

The vector is primarily intended as a source of Katushka2S coding sequence. Flanking restriction sites are

convenient for excision of Katushka2S sequence and its further insertion into other expression vectors of

choice. Alternatively, Katushka2S coding sequence can be amplified by PCR.

 

Note: The plasmid DNA was isolated from dam+-methylated E. coli. Therefore some restriction sites are

blocked by methylation. If you wish to digest the vector using such sites you will need to transform the vector

into a dam- host and make fresh DNA.

 

The vector can be also used for Katushka2S expression in prokaryotes under the control of T5 promoter/lac

operator. The vector backbone contains ColE1 origin of replication and ampicillin resistance gene for

propagation and selection in E. coli.

 

 

Antibody against katushka2S(Anti-tRFP antibody)

Rabbit polyclonal antibody against TurboRFP, TurboFP602, TurboFP635, Katushka2S, TurboFP650, NirFP,

TagBFP, TagRFP, FusionRed, TagFP635, mKate2 and PA-TagRFP.

DATA SHEET (PDF)

 

 

Ordering informations

Catalog No.

Product Name

Size

 FP761 (pdf)

pKatushka2S-C vector

20 μg

FP762 (pdf)

pKatushka2S-N vector

20 μg

FP763 (pdf)

pKatushka2S-B vector

20 μg

 

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