A kind of far red fluorescent protein, fusion protein, isolated nucleic acid, carrier and application
A red fluorescent protein and fusion protein technology, applied in fusion protein, isolated nucleic acid, far-red fluorescent protein, carrier and application fields, can solve the problems of weak sensitivity, low protein extinction coefficient and quantum yield, and poor imaging effect
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Embodiment 1
[0050] The far-red fluorescent protein provided in this example has an amino acid sequence as shown in SEQ ID NO: 2, which has the following amino acid mutation sites relative to the fluorescent protein mMaroon1 (SEQ ID NO: 1): S128A, R157Y, F194A, D223T, Deletion of P225G and the 226-234 amino acid fragment.
[0051] That is, the amino acid sequence of the far-red fluorescent protein in this embodiment is mutated from serine at position 128 of mMaroon1 to alanine, arginine at position 157 is mutated to tyrosine, and phenylalanine at position 194 is mutated to alanine acid and through the mutation of the above amino acid sites and the deletion of the 226-234 amino acid fragment.
[0052] Amino acid site mutations at the above positions and the deletion of amino acid fragments at positions 226-234 changed the dimer properties of the original fluorescent protein mMaroon1, so that the far red fluorescent protein (SEQ ID NO: 2) of this embodiment has Good monomer performance, eas...
Embodiment 2
[0054] On the basis of the far-red fluorescent protein (SEQ ID NO: 2) of Example 1, further site-directed mutations were carried out to improve its maturation rate.
[0055] The far-red fluorescent protein provided in this example has an amino acid sequence as shown in SEQ ID NO: 3, and it not only has the following amino acid mutations relative to the fluorescent protein mMaroon1 (its amino acid sequence is as SEQ ID NO: 1): S128A, R157Y, F194A , D223T, P225G, and the deletion of the 226-234th amino acid fragment; it also has the following amino acid mutation sites: E2R, E3L, N8E, H10T, T38K, C114V, H214Y, V216H, Y221H, C222T, and L224V; that is, the second position Mutation of glutamic acid to arginine, glutamic acid at position 3 to leucine, asparagine at position 8 to glutamic acid, histidine at position 10 to threonine, position 38 Threonine at position 114 is mutated to lysine, cysteine at position 114 is mutated to valine, histidine at position 214 is mutated to tyros...
Embodiment 3
[0058] On the basis of the far-red fluorescent protein (SEQ ID NO: 2) of Example 1, it was further subjected to site-directed mutation to increase its brightness.
[0059] The far-red fluorescent protein provided in this example has an amino acid sequence as shown in SEQ ID NO: 4. Compared with the fluorescent protein mMaroon1, it not only has the following amino acid mutations: S128A, R157Y, F194A, D223T, P225G, and amino acids 226-234 The deletion of the fragment; it also has the following amino acid mutation sites: V46I, Q134R, L138R, V165E, L170Y and E175V, that is, the valine at position 46 of mMaroon1 is mutated to isoleucine, and the glutamine at position 134 is mutated to Arginine, leucine at position 138 is mutated to arginine, valine at position 165 is mutated to glutamic acid, leucine at position 170 is mutated to tyrosine, glutamic acid at position 175 Mutated to valine.
[0060] Mutations at these sites can make the electrostatic interaction between the amino aci...
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