Methods and protein design to increase the efficiency and contrast of photo convertible fluorescent proteins

Tech ID:

We disclose two fluorescent protein technologies, which we refer to as “Janus” and “Ignis,” respectively. Both

are green-to-red photoconvertible fluorescent proteins (PC-FPs) of the EosFP family, generated through sitedirected

mutagenesis of the mEos4b protein coding sequence at amino acid positions 41 and 70 (Janus) and

positions 41, 70 and 197 (Ignis) relative to the start codon ATG. Methionine at position 41 has been mutated to

isoleucine (Met41Ile) and valine at position 70 has been mutated to threonine (Val70Thr) in both proteins.

Ignis fluorescent protein contains the additional mutation, isoleucine at position 197 to methionine (Ile197Met).

The genotype of Janus is therefore mEos4b-Met41Ile-Val70Thr, and Ignis is mEos4b-Met41Ile-Val70Thr-

Ile197Met. Ignis is Janus with an additional Ile197Met mutation.

Janus Fluorescent Protein

Janus was derived from the pure monomer, mEos4b, without modification of surface residues that confer

monomeric character. As expected, it behaves as a monomer with indistinguishable performance to

documented monomeric PC-FPs, mEos3.2 and mEos4b, in challenging fusion constructs (membrane-localized

fusion proteins CTLA4 and N-myristoylated DmrB) in our experiments.

The excitation and emission spectra of green-state Janus are blue-shifted relative to mEos4b like mEos4b-

V70T. This improves its excitation under commonly-employed 488nm laser illumination, compensating for its

lower peak excitation coefficient. (mEos4b is only about 55% maximally excited at this wavelength whereas

Janus is nearer its excitation maximum). As such Janus is bright in green channel confocal applications.

However, the Janus red state retains longer-wavelength spectral peaks, with excitation maximum at 571 nm and

emission maximum at 585 nm. This is one of the largest red Stokes-shifts among available PC-FPs. The

excitation spectrum in the red form also permits convenient and efficient excitation under both 561 and 568 nm

laser illumination commonly employed in confocal and super-resolution experiments. We note this as superior

to both mKikGR and Dendra2, which have red excitation peaks considerably above and below 561 and 568 nm.

Relative to commonly-used PC-FPs in the EosFP family, Janus displays exceptional photoconversion contrast.

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Daniel Rafferty
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(210) 562-4038
Brian Stoveken
James Lechleiter
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