CN116478264B - Recombinant chromoprotein, preparation method and application thereof - Google Patents

Recombinant chromoprotein, preparation method and application thereof Download PDF

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CN116478264B
CN116478264B CN202310732760.0A CN202310732760A CN116478264B CN 116478264 B CN116478264 B CN 116478264B CN 202310732760 A CN202310732760 A CN 202310732760A CN 116478264 B CN116478264 B CN 116478264B
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chromoprotein
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CN116478264A (en
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杨熹
钱莉春
赵碧玉
吴昕
郭文雅
姜伟
黎建军
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Suzhou Zuo Xuanxing Biotechnology Co ltd
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Abstract

The invention relates to recombinant chromoproteins, a preparation method and application thereof. The recombinant chromoprotein is obtained by replacing a sequence of a predetermined length at the N-terminus of the target chromoprotein with MASSE and replacing a sequence of a predetermined length at the C-terminus thereof with GA. The amino acid sequence of the recombinant chromoprotein is one of SEQ ID NO.1 to 5. The recombinant chromoprotein has no toxicity to escherichia coli, and has colors superior to fwYellow and mRFP 1; can be applied to screening of gene clones and also can be applied to induction of metabolic inducers to indicate the growth state of escherichia coli.

Description

Recombinant chromoprotein, preparation method and application thereof
Technical Field
The invention relates to a recombinant chromoprotein, a preparation method and application thereof, and belongs to the technical field of biology.
Background
The Chromoprotein family (Chromoprotein), the fluorescent protein family (Fluorescent Protein), and engineered mutants thereof, are widely used as reporter genes for gene expression. Most of them are derived from coelenterates, coral and jellyfish, containing homologous tri-amino acid chromophores/fluorophores, of which the best studied is of GFP and its derivatives from Aequorea victoria, and DsRed and its derivatives from Discosoma sp. In natural environment, compared with fluorescent protein, the chromoprotein can strongly absorb visible light, has unique advantages in specific scenes, such as no instrument detection and analysis, light ultraviolet damage, and background fluorescence avoidance, and can be used for quenching of Fluorescence Resonance Energy Transfer (FRET) and photoacoustic imaging. Chromoproteins have been used for labeling, teaching and biosensors in cloned organisms.
Most of the currently used chromoproteins are still natural amino acid sequences, often have insufficiently vivid colors compared to fluorescent proteins that have been deeply studied and engineered, and are often toxic to E.coli, which can significantly interfere with the growth of E.coli. GFP and its derivatives from Aequorea victoria, and DsRed and its derivatives from Discosoma sp, while vivid in color, are mostly so toxic to e. This greatly limits the options and range of applications of chromoproteins. There are only two chromoproteins known to date that do not interfere with E.coli growth, namely mRFP1 and fwYellow. In addition, currently found chromoproteins are in red and blue color, and also yellow, but there are few suitable green proteins. Therefore, there is a need to find or develop more chromoproteins that are not toxic to E.coli and have more colors to better expand the applications of chromoproteins.
Disclosure of Invention
The main purpose of the invention is as follows: the problems existing in the prior art are overcome, and the recombinant chromoprotein is provided, has no toxicity to escherichia coli, obvious color development and practical value; also provides a preparation method and application thereof.
The technical scheme for solving the technical problems is as follows:
a recombinant chromoprotein obtained by replacing a sequence of a predetermined length at the N-terminus of a target chromoprotein with MASSE and replacing a sequence of a predetermined length at the C-terminus with GA.
Preferably, the amino acid sequence of the recombinant chromoprotein is one of SEQ ID NO.1 to 5.
Preferably, the target chromoprotein is mScarlet-I, the sequence of the preset length of the N end of the target chromoprotein is MVSKGE, the sequence of the preset length of the C end of the target chromoprotein is GGMDELYK, and the amino acid sequence of the recombinant chromoprotein obtained after replacement is SEQ ID NO.1; the recombinant chromoprotein is named as CP-Pink, and shows magenta color under white light and red fluorescence under blue light;
or the target chromoprotein is dToma, the sequence of the preset length of the N end of the target chromoprotein is MVSKGE, the sequence of the preset length of the C end of the target chromoprotein is YGMDELYK, and the amino acid sequence of the recombinant chromoprotein obtained after replacement is SEQ ID NO.2; the recombinant chromoprotein is named as CP-Orange, and shows Orange red under white light and Orange fluorescence under blue light;
or, the target chromoprotein is mCherry, the sequence of the preset length of the N end of the target chromoprotein is MVSKGEEDNM, the sequence of the preset length of the C end of the target chromoprotein is GGMDELYK, and the amino acid sequence of the recombinant chromoprotein obtained after replacement is SEQ ID NO.3; the recombinant chromoprotein is named as CP-Voilet, and is purple under white light and dark red fluorescence under blue light;
or the target chromoprotein is mhYFP, the sequence of the preset length of the N end of the target chromoprotein is MVSKGE, the sequence of the preset length of the C end of the target chromoprotein is MNELYK, and the amino acid sequence of the recombinant chromoprotein obtained after replacement is SEQ ID NO.4; the recombinant chromoprotein is named as CP-Yellow, and is Yellow under white light and Yellow fluorescent under blue light;
or the target chromoprotein is sfGFP, the sequence of the preset length of the N end of the target chromoprotein is MSKGE, the sequence of the preset length of the C end of the target chromoprotein is GMDELYK, and the amino acid sequence of the recombinant chromoprotein obtained after replacement is SEQ ID NO.5; the recombinant chromoprotein is named as CP-Green, and is Green under white light and Green fluorescent under blue light.
Each of the above-mentioned target chromoproteins is strongly toxic itself, and its continuous expression leads to death of E.coli, whereas the recombinant chromoproteins are not toxic to E.coli and have a color superior to fwYellow and mRFP 1; experiments prove that the recombinant chromoprotein can be applied to screening of gene cloning, has quicker color development and high accuracy compared with blue and white spot screening, and is not limited by escherichia coli strains; it can also be applied to sensing metabolic inducers in the medium to indicate the growth state of E.coli.
The invention also provides:
a method for preparing recombinant chromoprotein, comprising: and replacing the sequence with the preset length at the N end of the target chromoprotein with MASSE, and replacing the sequence with the preset length at the C end of the target chromoprotein with GA, thereby obtaining the recombinant chromoprotein.
Preferably, the amino acid sequence of the recombinant chromoprotein is one of SEQ ID NO.1 to 5.
Preferably, the specific process of the preparation method is as follows:
replacing the N-terminal MVSKGE of the target chromoprotein mScarle-I with MASSE, replacing the C-terminal GGMDELYK with GA, and the obtained recombinant chromoprotein is named as CP-Pink, and the amino acid sequence of the recombinant chromoprotein is SEQ ID NO.1; the recombinant chromoprotein shows magenta color under white light and red fluorescence under blue light;
alternatively, the N-terminal MVSKGE of the target chromoprotein dTopith is replaced by MASSE, the C-terminal YGMDELYK is replaced by GA, the obtained recombinant chromoprotein is named as CP-Orange, and the amino acid sequence of the recombinant chromoprotein is SEQ ID NO.2; the recombinant chromoprotein shows orange red under white light and shows orange fluorescence under blue light;
alternatively, the N-terminal MVSKGEEDNM of the target chromoprotein mCherry is replaced by MASSE, the C-terminal GGMDELYK is replaced by GA, the obtained recombinant chromoprotein is named CP-Voilet, and the amino acid sequence of the recombinant chromoprotein is SEQ ID NO.3; the recombinant chromoprotein is purple under white light and shows dark red fluorescence under blue light;
alternatively, the N-terminal MVSKGE of the target chromoprotein mhYFP is replaced by MASSE, the C-terminal MNELYK is replaced by GA, the obtained recombinant chromoprotein is named as CP-Yellow, and the amino acid sequence of the recombinant chromoprotein is SEQ ID NO.4; the recombinant chromoprotein is yellow under white light and yellow fluorescent under blue light;
alternatively, the N-terminal MSKGE of the target chromoprotein sfGFP is replaced by MASSE, the C-terminal GMDELYK is replaced by GA, the obtained recombinant chromoprotein is named as CP-Green, and the amino acid sequence of the recombinant chromoprotein is SEQ ID NO.5; the recombinant chromoprotein is green under white light and green fluorescent under blue light.
The invention also provides:
the use of a recombinant chromoprotein as described hereinbefore for screening gene clones.
The use of a recombinant chromoprotein as hereinbefore described for sensing a metabolic inducer in a culture medium.
Nucleic acid encoding a recombinant chromoprotein as described hereinbefore.
A vector comprising a nucleic acid as hereinbefore described.
Compared with the prior art, the invention obtains the series of recombinant chromoproteins by modifying the N-terminal sequence and the C-terminal sequence of the existing chromoproteins, has no toxicity to escherichia coli, and has colors superior to fwYellow and mRFP 1; can be applied to screening of gene clones and also can be applied to induction of metabolic inducers to indicate the growth state of escherichia coli.
Drawings
FIG. 1 is a plasmid map of pLevo-Amp-BluntGreen in example 2 of the present invention.
Detailed Description
The invention is described in further detail below with reference to the accompanying drawings in combination with embodiments. The invention is not limited to the examples given.
Example 1
This example is to construct a chromoprotein plasmid and screen for chromoproteins.
The plasmid pLevo-Kan-Red is constructed by using the vector pLevo existing in the applicant, and the sequence of the plasmid pLevo-Kan-Red is as follows, SEQ ID NO.6:
wherein, the italic sequence part is mRFP1, the sequence part in brackets is kanamycin resistance gene, and the underlined sequence part is pUC origin.
The backbone sequence was amplified using PCR, the amplification primers were:
cttcggaggaagccatttagtatttctc（SEQ ID NO.7）
ggtgcttaataatgagcttcaaataaaacg（SEQ ID NO.8）
then, the 1.8kb fragment was cut by electrophoresis and recovered using a monatin bio-gel recovery kit (MI 17101).
The following PCR products were purchased from general organisms, and the amino acid sequences corresponding to these fragments were replaced with MASSE at the N-terminal and GA at the C-terminal based on the reported sequences. And (3) injection: fwYellow is the original sequence.
>E2-Crimson，SEQ ID NO.9：
ctaaatggcttcctccgaaaacgtgattaaaccgtttatgcgctttaaagttcacatggaaggcagcgtgaacggccatgaatttgaaattgaaggcgtgggcgaaggcaaaccgtatgaaggcacgcagaccgcgaaactgcaagtgaccaaaggcggcccgctgccgtttgcgtgggatattctgagcccgcagtttttttatggcagcaaagcgtatattaaacatccggcggatattccggattatctgaaacagagctttccggaaggctttaaatgggaacgcgtgatgaactttgaagatggcggcgtggtgaccgtgacccaagatagcagcctgcaagatggcaccctgatttatcatgtgaaatttattggcgtgaactttccgagcgatggcccggtgatgcagaaaaaaaccctgggctgggaaccgagcaccgaacgcaactatccgcgcgatggcgtgctgaaaggcgaaaaccacatggcgctgaaactgaaaggcggtggccattatctgtgcgaatttaaaagcatttatatggcgaaaaagccggtgaaactgccgggctatcattatgtggattataaactggatattacgagccataacgaagattataccgtggtggaacagtatgaacgcgcggaagcgcgccatagcaccggtgcttaataatgagcttc
>CP-Pink，SEQ ID NO.10：
ctaaatggcttcctccgaagcggtgattaaagaatttatgcgctttaaagttcacatggaaggcagcatgaacggccatgaatttgaaattgaaggcgaaggcgaaggccgcccgtatgaaggcacgcagaccgcgaaactgaaagtgaccaaaggcggcccgctgccgtttagctgggatattctgagcccgcagtttatgtatggcagccgcgcgtttattaaacatccggcggatattccggattattataaacagagctttccggaaggctttaaatgggagcgcgtgatgaactttgaagatggcggcgcggtgaccgtgacccaagatacgagcctggaagatggcaccctgatttataaagtgaaactgcgcggcaccaactttccgccggatggcccggtgatgcagaaaaaaacgatgggctgggaagcgagcaccgaacgcctgtatccggaagatggcgtgctgaaaggcgatattaaaatggcgctgcgcctgaaagatggtggccgctatctggcggattttaaaaccacctataaagcgaaaaagccggtgcagatgccgggcgcgtataacgtggatcgcaaactggatattacgagccataacgaagattataccgtggtggaacagtatgaacgcagcgaaggtcgtcatagcaccggtgcttaataatgagcttc
>mNeptune，SEQ ID NO.11：
ctaaatggcttcctccgaagaactgattaaagaaaacatgcataccaaactgtatatggaaggcaccgtgaacaaccatcattttaaatgcacccatgaaggcgaaggcaaaccgtatgaaggcacgcagaccaaccgcattaaagtggtggaaggcggcccgctgccgtttgcgtttgatattctggcgacctgctttatgtatggcagcaaaacctttattaaccatacccaaggcattccggatttttttaaacagagctttccggaaggctttacctgggagcgcgtgaccacctatgaagatggcggcgtgctgaccgtgacccaagatacgagcctgcaagatggctgcctgatttataacgtgaaactgcgcggcgtgaactttccgagcaacggcccggtgatgcagaaaaaaaccctgggctgggaagcgagcaccgaaaccctgtatccggcggatggcggcctggaaggccgctgcgatatggcgctgaaactggtgggcggtggccatctgcattgcaacctgaaaaccacctatcgcagcaaaaaaccggcgaaaaacctgaaaatgccgggcgtgtattttgtggatcgccgcctggaacgcattaaagaagcggataacgaaacctatgtggaacagcatgaagtggcggtggcgcgctattgcgatctgccgagcaaactgggccataaactgaacggtgcttaataatgagcttc
>fwYellow，SEQ ID NO.12：
ctaaatggcttcctccgaaggcgcaaaactgttcgagaaagaaatcccatatatcactgagctggaaggtgacgttgaaggtatgaagtttatcatcaagggtgaaggtaccggtgacgcgagcgtcggtaaagtggatgctcagttcatttgtaccacgggcgacgttccggttccgtggagcacgctggtcaccacgctgacgtatggtgctcagtgctttgccaagtatccgcgccacattgcggatttcttcaaaagctgcatgccggaaggttacgtccaagagcgcaccatcacctttgagggtgatggcgtgttcaagacccgtgcggaagtcacctttgaaaatggcagcgtgtacaaccgtgtaaaactgaacggccagggtttcaagaaggacggccacgtgctgggcaaaaatctggagtttaactttacccctcattgtttgtacatttggggtgaccaagcgaatcatggcctgaagtctgcgttcaaaatcatgcatgagatcaccggctccaaagaggatttcattgttgccgatcacacccaaatgaataccccgattggtggtggtccggtgcacgtgccggagtaccaccacattacgtatcatgttaccctgtctaaagacgtcaccgatcaccgtgaccatttgaacattgttgaggtgatcaaggcagttgacctggaaacgtaccgtggtgcttaataatgagcttc
>CP-Yellow，SEQ ID NO.13：
ctaaatggcttcctccgaagaactgtttaccggcgtggtgccgattctggtggaactggatggcgatgtgaacggccataaatttagcgtgcgcggcgaaggcgaaggcgatgcgaccaacggcaaactgaccctgaaactgattagcaccaccggcaaactgccggtgccgtggccgaccctggtgaccaccctgggctatggcctgatggtgtttgcgcgctatccggatcacatgaaacagcatgatttttttaaaagcgcgatgccggaaggctatgtgcaagaacgcaccattagctttgaagatgatggctattataaaacccgcgcggaagtgaaatttgaaggcgataccctggtgaaccgcattgtgctgaaaggcattgattttaaagaagatggcaacattctgggccataaactggaatataactttaacccgcataacgtgtatattaccgcggataaacagaaaaacggcattaaagcgaactttaaaattcgccataacgtggaagatggcggcgtgcaactggcggatcattatcagcagaacaccccgattggcgatggcccggtgctgatgccggataaccattatctgagctatcagagcaaactgagcaaagatccgaacgaaaaacgcgatcacatggtgctgaaagagcgcgtgaccgcggcgggcattacccatgatggtgcttaataatgagcttc
>mNeonGreen，SEQ ID NO.14：
gagaaatactaaatggcttcctccctcccagcgacacatgagttacacatctttggctccatcaacggtgtggactttgacatggtgggtcagggcaccggcaatccaaatgatggttatgaggagttaaacctgaagtccaccaagggtgacctccagttctccccctggattctggtccctcatatcgggtatggcttccatcagtacctgccctaccctgacgggatgtcgcctttccaggccgccatggtagatggctccggataccaagtccatcgcacaatgcagtttgaagatggtgcctcccttactgttaactaccgctacacctacgagggaagccacatcaaaggagaggcccaggtgaaggggactggtttccctgctgacggtcctgtgatgaccaactcgctgaccgctgcggactggtgcaggtcgaagaagacttaccccaacgacaaaaccatcatcagtacctttaagtggagttacaccactggaaatggcaagcgctaccggagcactgcgcggaccacctacacctttgccaagccaatggcggctaactatctgaagaaccagccgatgtacgtgttccgtaagacggagctcaagcactccaagaccgagctcaacttcaaggagtggcaaaaggcctttaccgatgtgatgggtgcttaataatgagcttc
>StayGold，SEQ ID NO.15：
gagaaatactaaatggcttcctcccccttcaagtttcaactgaagggcaccatcaacggcaagagcttcaccgtggagggcgagggcgagggcaacagccacgagggcagccacaagggcaagtacgtctgcacaagcggcaagctgcccatgagctgggccgccctgggcacatccttcggctacggcatgaagtactacaccaagtaccctagcggcctgaagaactggttccacgaggtgatgcccgagggcttcacctacgacagacacattcagtacaagggcgacggcagcatccacgccaagcatcagcacttcatgaagaacggcacctaccacaacatcgtggagttcaccggccaagacttcaaggagaacagccccgtgctgaccggcgacatgaacgtgagcctgcccaacgaggtgcagcacatccctagagacgacggcgtggagtgccccgtgaccctgctgtaccccctgctgagcgacaagagcaagtgcgtggaggcccatcagaacaccatctgcaagcccctgcacaatcagcccgcccccgacgtgccctaccactggatcagaaagcagtacacacagagcaaggacgacaccgaggagcgggaccacatctgtcagagcgaaaccctggaggcccacctcggtgcttaataatgagcttc
>CP-Orange，SEQ ID NO.16：
ctaaatggcttcctccgaagaagtgattaaagaatttatgcgctttaaagtgcgcatggaaggcagcatgaacggccatgaatttgaaattgaaggcgaaggtgagggccgcccgtacgaaggcacgcagaccgcgaaactgaaagtgaccaaaggcggcccgctgccgtttgcgtgggatattctgagcccgcagtttatgtatggcagcaaagcgtatgtgaaacatccggcggatattccggattataaaaaactgagctttccggaaggctttaaatgggagcgcgtgatgaactttgaagatggcggcctggtgaccgtgacccaagatagcagcctgcaagatggcaccctgatttataaagtgaaaatgcgcggcaccaactttccgccggatggcccggtgatgcagaaaaaaacgatgggctgggaagcgagcaccgaacgcctgtatccgcgcgatggcgtgctgaaaggcgaaattcatcaagcgctgaaactgaaggatggcggccattatctggtggaatttaaaaccatttatatggcgaaaaagccggtgcaactgccgggctattactatgtggataccaaactggatattacgagccataacgaagattataccattgtggaacagtatgaacgcagcgaaggccgccatcatctgtttctgggtgcttaataatgagcttc
>CP-Voilet，SEQ ID NO.17：
ctaaatggcttcctccgaagcgattattaaagaatttatgcgctttaaagttcacatggaaggcagcgtgaacggccatgaatttgaaattgaaggcgaaggcgaaggccgcccgtatgaaggcacgcagaccgcgaaactgaaagtgaccaaaggcggcccgctgccgtttgcgtgggatattctgagcccgcagtttatgtatggcagcaaagcgtatgtgaaacatccggcggatattccggattatctgaaactgagctttccggaaggctttaaatgggagcgcgtgatgaactttgaagatggcggcgtggtgaccgtgacccaagatagcagcctgcaagatggcgaatttatttataaagtgaaactgcgcggcaccaactttccgagcgatggcccggtgatgcagaaaaaaacgatgggctgggaagcgagcagcgaacgcatgtatccggaagatggcgcgctgaaaggcgaaattaaacagcgcctgaaactgaaagatggcggccattatgatgcggaagtgaaaaccacctataaagcgaaaaagccggtgcaactgccgggcgcgtataacgtgaacattaaactggatattacgagccataacgaagattataccattgtggaacagtatgaacgcgcggaaggccgccatagcaccggtgcttaataatgagcttc
>CP-Green，SEQ ID NO.18：
ctaaatggcttcctccgaagaactgtttaccggcgtggtgccgattctggtggaactggatggcgatgtgaacggccataaatttagcgtgcgcggcgaaggcgaaggcgatgcgaccaacggcaaactgaccctgaaatttatttgcaccaccggcaaactgccggtgccgtggccgaccctggtgaccaccctgacctatggcgtgcagtgctttagccgctatccggatcacatgaaacgccatgatttttttaaaagcgcgatgccggaaggctatgtgcaagaacgcaccattagctttaaagatgatggcacctataaaacccgcgcggaagtgaaatttgaaggcgataccctggtgaaccgcattgaactgaaaggcattgattttaaagaagatggcaacattctgggccataaactggaatataactttaacagccataacgtgtatattaccgcggataaacagaaaaacggcattaaagcgaactttaaaattcgccataacgtggaagatggcagcgtgcaactggcggatcattatcagcagaacaccccgattggcgatggcccggtgctgctgccggataaccattatctgagcacgcagagcgtgctgagcaaagatccgaacgaaaaacgcgatcacatggtgctgctggaatttgtgaccgcggcgggcattacccatggtgcttaataatgagcttc
>shadowR，SEQ ID NO.19：
ctaaatggcttcctccgaagattatattaaagaatttatgcgctataaagtgcatatggaaggcagcgtgaacggccatgaatttgaagtggaaggcgaaggcgaaggccgcccgtatgaaggcacgcagaccgcgaaactgaaagtgaccaaaggcggcccgctgccgtttgcgtgggatattctgagcccgcagtgtcagtatggcagcattccgtttaccaaatatccggaagatattccggattatgtgaaacagagctttccggaaggctttacctgggaacgcgaaatgaactttgaagatggcggcaaagtgaccgtgagcaacgatagcagcctgcaagatggcgaatttatttataaagtgaaactgcgcggcgaaaactttccgccgaacggcccggtgatgcagaaaaaaaccgaaggctgggaaccgaacagcgaacgcctgaccccgcatgatggcgcgctgaaaggcaacaaccgcatgagcctgaaactgaaagatggcggccattatcgcgtggaatttaaaaccacctataaagcgaaaaagccggtgaaaatgccgggccatcattgggtggatcgcaaactggatgtgaccaaccataacgaagattatacgagcgtggaacagtatgaacgcagcgaagcgcgcaaaagcaccggtgcttaataatgagcttc
The above-mentioned each chromoprotein fragment was recombined with the plasmid backbone fragment (simultaneously using the original vector pLevo-Kan-Red containing mRFP1 as a control) using a monatin single fragment seamless cloning kit (MC 40201), transformed into NEB 10. Beta. Competent, plated on kanamycin resistance plates, and grown at 37℃for 18 hours.
The specific results are as follows:
(1) The fwYellow growth rate was consistent with mRFP1 (i.e., original vector pLevo-Kan-Red), both of which are known to be chromoproteins that do not affect E.coli growth; under the experimental conditions, fwYellow, mRFP1 can grow normally and develop color normally, and can be used as a reference standard to measure the performance of other chromoproteins.
(2) E2-Crismon and shadow R have normal growth speed, but have very weak color development and light purple color, and are not obvious even if the E2-Crismon and shadow R are continuously placed at 4 ℃ for 48 hours; staygold does not develop color at all; the mNapptone grows extremely slowly; the mNanGreen showed yellow color, but grew slowly. Therefore, these chromoproteins have poor practicality and do not meet the screening conditions.
(3) The growth rates of the five colored proteins of CP-Orange, CP-Voilet, CP-GFP, CP-Yellow, and CP-Pink are consistent with fwYellow, mRFP, which indicates that the growth of the Escherichia coli is not affected.
Under white light, CP-Orange shows Orange red, CP-Voilet shows purple, CP-Pink shows magenta, CP-Yellow shows Yellow, and CP-Green shows Green; except for CP-Yellow, the color is much more pronounced than fwYellow.
Under blue light, CP-Orange shows Orange fluorescence, CP-Voilet shows dark red fluorescence, CP-Pink shows red fluorescence, CP-Yellow shows Yellow fluorescence, CP-Green shows Green fluorescence; except for CP-Voilet, fluorescence was stronger than mRFP 1.
Therefore, these five proteins can be used as chromoproteins that work well in E.coli, meeting the screening conditions.
The original proteins of these five chromoproteins and the modified specific conditions of this example are as follows:
1. the original protein of CP-Pink is mScarlet-I, wherein the amino acid sequence of mScarlet-I is SEQ ID NO.20:
MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTGGMDELYK
the amino acid sequence of CP-Pink is SEQ ID NO.1:
MASSEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTGA
note that: the italic sequence portions at both ends of each sequence are specific sequences before and after substitution of the N-terminal or the C-terminal, as follows.
2. The original protein of CP-Orange is dTommao, wherein, the amino acid sequence of dTomoto is SEQ ID NO.21:
MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRHHLFLYGMDELYK
the amino acid sequence of CP-Orange is SEQ ID NO.2:
MASSEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRHHLFLGA
3. the original protein of CP-Voilet is mCherry, wherein the amino acid sequence of mCherry is SEQ ID NO.22:
MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK
the amino acid sequence of CP-Voilet is SEQ ID NO.3:
MASSEAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGA
4. the original protein of CP-Yellow is mhYFP, wherein the amino acid sequence of mhYFP is SEQ ID NO.23:
MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKLISTTGKLPVPWPTLVTTLGYGLMVFARYPDHMKQHDFFKSAMPEGYVQERTISFEDDGYYKTRAEVKFEGDTLVNRIVLKGIDFKEDGNILGHKLEYNFNPHNVYITADKQKNGIKANFKIRHNVEDGGVQLADHYQQNTPIGDGPVLMPDNHYLSYQSKLSKDPNEKRDHMVLKERVTAAGITHDMNELYK
the amino acid sequence of CP-Yellow is SEQ ID NO.4:
MASSEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKLISTTGKLPVPWPTLVTTLGYGLMVFARYPDHMKQHDFFKSAMPEGYVQERTISFEDDGYYKTRAEVKFEGDTLVNRIVLKGIDFKEDGNILGHKLEYNFNPHNVYITADKQKNGIKANFKIRHNVEDGGVQLADHYQQNTPIGDGPVLMPDNHYLSYQSKLSKDPNEKRDHMVLKERVTAAGITHDGA
5. the original protein of CP-Green is sfGFP, wherein the amino acid sequence of sfGFP is SEQ ID NO.24:
MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK
the amino acid sequence of CP-Green is SEQ ID NO.5:
MASSEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGA
example 2
This example is a test to determine whether the chromoprotein obtained from the screening of example 1 can be used in the screening of blunt-ended clones.
Constructing a plasmid pLevo-Amp-Red, wherein the sequence of the plasmid pLevo-Amp-Red is SEQ ID NO.25:
wherein the sequence part in the middle brackets is ampicillin resistance gene, and the underlined sequence part is pUC origin.
The pLevo-Amp-Red was digested with EcoRV, followed by electrophoresis, and the 1.7 k-sized fragment was recovered by digestion.
The CP-Green gene vector and the CP-Pink gene vector of example 1 were amplified separately using the following primers:
gccttttgctcataagttgacagcagatatcagctgaggtaccctgctaactactagag（SEQ ID NO.26）
cctgacctctaaggatgagagcgttcaccgac（SEQ ID NO.27）
the amplified products are 870bp fragments, and are recombined with EcoRV digestion products of pLevo-Amp-Red respectively by using a monad fragment seamless cloning kit (MC 40201), then NEB10 beta competence is converted, and an Amp plate is coated to obtain pLevo-Amp-BluntGreen and pLevo-Amp-BluntRed. As an example, a plasmid map of pLevo-Amp-BluntGreen is shown in FIG. 1. The 13bp position of the plasmid is EcoRV restriction enzyme site, and the 18bp position is PvuII restriction enzyme site.
The sequence of pLevo-Amp-BluntGreen is SEQ ID NO.28:
gttgacagcagatatcagctgaggtaccctgctaactactagagaaagaggagaaatactaaatggcttcctccgaagaactgtttaccggcgtggtgccgattctggtggaactggatggcgatgtgaacggccataaatttagcgtgcgcggcgaaggcgaaggcgatgcgaccaacggcaaactgaccctgaaatttatttgcaccaccggcaaactgccggtgccgtggccgaccctggtgaccaccctgacctatggcgtgcagtgctttagccgctatccggatcacatgaaacgccatgatttttttaaaagcgcgatgccggaaggctatgtgcaagaacgcaccattagctttaaagatgatggcacctataaaacccgcgcggaagtgaaatttgaaggcgataccctggtgaaccgcattgaactgaaaggcattgattttaaagaagatggcaacattctgggccataaactggaatataactttaacagccataacgtgtatattaccgcggataaacagaaaaacggcattaaagcgaactttaaaattcgccataacgtggaagatggcagcgtgcaactggcggatcattatcagcagaacaccccgattggcgatggcccggtgctgctgccggataaccattatctgagcacgcagagcgtgctgagcaaagatccgaacgaaaaacgcgatcacatggtgctgctggaatttgtgaccgcggcgggcattacccatggtgcttaataatgagcttcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcatccttagaggtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctgatgagacaataaccctgataaatgcttcaataatatcgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcccgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgaatactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaactatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgaaaactattaactggcgaactacttactctagcttcccggcaacaactaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcgcttccggctggctggtttattgctgataaatctggagccggtgagcgtggttctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacaccacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaaggattgaggatctaggtgaagatcctttttgcgcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcataa
the sequence of pLevo-Amp-BluntRed is SEQ ID NO.29:
gttgacagcagatatcagctgaggtaccctgctaactactagagaaagaggagaaatactaaatggcttcctccgaagcggtgattaaagaatttatgcgctttaaagttcacatggaaggcagcatgaacggccatgaatttgaaattgaaggcgaaggcgaaggccgcccgtatgaaggcacgcagaccgcgaaactgaaagtgaccaaaggcggcccgctgccgtttagctgggatattctgagcccgcagtttatgtatggcagccgcgcgtttattaaacatccggcggatattccggattattataaacagagctttccggaaggctttaaatgggagcgcgtgatgaactttgaagatggcggcgcggtgaccgtgacccaagatacgagcctggaagatggcaccctgatttataaagtgaaactgcgcggcaccaactttccgccggatggcccggtgatgcagaaaaaaacgatgggctgggaagcgagcaccgaacgcctgtatccggaagatggcgtgctgaaaggcgatattaaaatggcgctgcgcctgaaagatggtggccgctatctggcggattttaaaaccacctataaagcgaaaaagccggtgcagatgccgggcgcgtataacgtggatcgcaaactggatattacgagccataacgaagattataccgtggtggaacagtatgaacgcagcgaaggtcgtcatagcaccggtgcttaataatgagcttcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcatccttagaggtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctgatgagacaataaccctgataaatgcttcaataatatcgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcccgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgaatactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaactatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgaaaactattaactggcgaactacttactctagcttcccggcaacaactaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcgcttccggctggctggtttattgctgataaatctggagccggtgagcgtggttctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacaccacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaaggattgaggatctaggtgaagatcctttttgcgcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcataa
pUC57, pLevo-Amp-BluntGreen and pLevo-Amp-BluntRed were digested with EcoRV, respectively, followed by electrophoresis and gel cutting recovery.
A fragment of the following sequence (SEQ ID No. 30) was synthesized in the universal biosynthesis of the Anhui:
acgcgtgccaccatgggagtcaaagttctgtttgccctgatctgcatcgctgtggccgaggccaagcccaccgagaacaacgaagacttcaacatcgtggccgtggccagcaacttcgcgaccacggatctcgatgctgaccgcgggaagttgcccggcaagaagctgccgctggaggtgctcaaagagttggaagccaatgcccggaaagctggctgcaccaggggctgtctgatctgcctgtcccacatcaagtgcacgcccaagatgaagaagttcatcccaggacgctgccacacctacgaaggcgacaaagagtccgcacagggcggcataggcgaggcgatcgtcgacattcctgagattcctgggttcaaggacttggagcccttggagcagttcatcgcacaggtcgatctgtgtgtggactgcacaactggctgcctcaaagggcttgccaacgtgcagtgttctgacctgctcaagaagtggctgccgcaacgctgtgcgacctttgccagcaagatccagggccaggtggacaagatcaagggggccggtggtgactaacaggcct
ligation of the above fragments with recovered pUC57, pLevo-Amp-BluntGreen, pLevo-Amp-BluntRed, respectively, was performed using monatin rapid T4 DNA ligase (MC 00102), DH 5. Alpha. Competence was transformed, and Amp-resistant plates were applied, wherein the corresponding plates of pUC57 required addition of IPTG and X-gal for 14 hours at 37 ℃; at this time, it can be seen that pLevo-Amp-BluntGreen and pLevo-Amp-BluntRed already have a more pronounced color, and that if they are in blue light, the fluorescence is very pronounced and pUC57 shows a very light blue color.
16 white spots were picked each and the number of positive clones was examined, with the following results:
pUC57:12
pLevo-Amp-BluntGreen:14
pLevo-Amp-BluntRed:15
Note that: the results obtained by the screening of example 1 were substantially identical to those obtained by the above experimental procedure, and specific experimental data thereof will not be described here.
As can be seen from this, the chromoprotein obtained by the screening of example 1 can be used in place of the blue-white screening for screening of gene clones.
Example 3
This example is based on example 2 and tests for the screening of chromoproteins for limitation by competent strains.
Stbl3 (lacZ complete) and JM108 (complete no lacZ sequence) were transformed with the ligation product of example 2, respectively, and ampicillin plates were coated, with IPTG and X-gal added to the pUC corresponding plates. The results of the growth at 37℃for 16 hours were as follows:
note that: the results obtained by the screening of example 1 were substantially identical to those obtained by the above experimental procedure, and specific experimental data thereof will not be described here.
As can be seen, the chromoprotein obtained in the screening of example 1 was not limited by the strain when used in the screening of gene clones.
Example 4
This example is a test of whether the chromoprotein obtained by the screening of example 1 can be used to detect small amounts of metabolic inducer in the medium.
pET-28a vector was digested with BamHI and HindIII. The CP-Green gene vector and the CP-Pink gene vector of example 1 were amplified separately using the following primers:
cagcaaatgggtcgcggcatggcttcctccgaag（SEQ ID NO.31）
cgagtgcggccgcaagcgaagctcattattaagcacc（SEQ ID NO.32）
the amplified products are all 750bp fragments, and are recombined with BamHI and HindIII enzyme digestion products of pET-28a respectively by using a monatin single-fragment seamless cloning kit (MC 40201), then NEB10 beta competence is converted, and an Amp plate is coated to obtain pET-CP-Green and pET-CP-Pink. These two plasmids were transformed into NEB T7 Express competent, spread on Amp plates, and spotted on LB medium for shaking. Four hours after addition of IPTG at different concentrations, the colour was observed. The results were as follows:
IPTG concentration 0 40μM 0.1mM 1mM
ER2566 (control) Primary color Primary color Primary color Primary color
pET-CP-Green Primary color Green colour Green colour Green colour
pET-CP-Pink Primary color Red color Red color Red color
Note that: the results obtained by the screening of example 1 were substantially identical to those obtained by the above experimental procedure, and specific experimental data thereof will not be described here.
From this, it can be seen that the chromoprotein obtained by the screening of example 1 can be used to detect low concentrations of metabolic inducer.
In practical studies, the applicant research team found that many of the MVSKGEE sequences at the N-terminus and GMDELYK sequences at the C-terminus of the existing chromoproteins might be key factors responsible for their toxicity to e.coli, even related proteins derived from mRFP1 remain highly toxic to e.coli. However, mRFP1 itself is not toxic, and has a MASSEE at the N-terminus and an STGA at the C-terminus. Applicant research team produced the following ideas: the MVSKGEE sequence at the N end of the existing chromoprotein is replaced by MASSE, the GMDELYK sequence at the C end of the existing chromoprotein is replaced by GA, and whether the existing chromoprotein can remove toxicity to escherichia coli or not is judged, so that the chromoprotein which is nontoxic to escherichia coli is obtained.
Through a great deal of repeated practical research, the applicant research team obtains:
1. the N-terminal MVSKGE of mScarlet-I (mRFP 1 derived protein) is replaced by MASSE, and the C-terminal GGMDELYK is replaced by GA, so that the chromoprotein CP-Pink is obtained, the color is magenta under white light, and red fluorescence is blue light.
2. The N-terminal MVSKGE of dTotato (mRFP 1 derived protein) is replaced by MASSE, and the C-terminal YGMDELYK is replaced by GA, so that the chromoprotein CP-Orange is obtained, orange red is displayed under white light, and Orange fluorescence is displayed under blue light.
3. The N-terminal MVSKGEEDNM of mCherry (mRFP 1 derived protein) is replaced by MASSE, and the C-terminal GGMDELYK is replaced by GA, so that the chromoprotein CP-Voilet is obtained, and the chromoprotein CP-Voilet is purple under white light and shows dark red fluorescence under blue light.
4. The N-terminal MVSKGE of mhYFP (GFP derived protein) is replaced by MASSE, and the C-terminal MNELYK is replaced by GA, so that the color protein CP-Yellow is obtained, and the color protein CP-Yellow is Yellow under white light and Yellow fluorescence is blue light.
5. The N-terminal MSKGE of sfGFP (GFP derived protein) is replaced by MASSE, and the C-terminal GMDELYK is replaced by GA, so as to obtain the chromoprotein CP-Green, which is Green under white light and Green fluorescence under blue light.
All five recombinant chromoproteins are not toxic to E.coli and have a color superior to fwYellow and mRFP1 (their original proteins are strongly toxic themselves, and their continued expression can lead to E.coli death); can be applied to screening of gene clone, has quicker color development and high accuracy compared with blue and white spot screening, and is not limited by escherichia coli strains; the method can also be used for detecting low-concentration metabolic inducer, plays a role in induction, and can indicate the growth state of the escherichia coli.
In addition to the embodiments described above, other embodiments of the invention are possible. All technical schemes formed by equivalent substitution or equivalent transformation fall within the protection scope of the invention.

Claims (6)

1. A recombinant chromoprotein obtained by replacing a sequence of a predetermined length at the N-terminus of a target chromoprotein with MASSE and replacing a sequence of a predetermined length at the C-terminus with GA;
the amino acid sequence of the recombinant chromoprotein is one of SEQ ID NO.1 to 5;
the target chromoprotein is mScarlet-I, the sequence of the preset length of the N end of the target chromoprotein is MVSKGE, the sequence of the preset length of the C end of the target chromoprotein is GGMDELYK, and the amino acid sequence of the recombinant chromoprotein obtained after replacement is SEQ ID NO.1; the recombinant chromoprotein is named as CP-Pink, and shows magenta color under white light and red fluorescence under blue light;
or the target chromoprotein is dToma, the sequence of the preset length of the N end of the target chromoprotein is MVSKGE, the sequence of the preset length of the C end of the target chromoprotein is YGMDELYK, and the amino acid sequence of the recombinant chromoprotein obtained after replacement is SEQ ID NO.2; the recombinant chromoprotein is named as CP-Orange, and shows Orange red under white light and Orange fluorescence under blue light;
or, the target chromoprotein is mCherry, the sequence of the preset length of the N end of the target chromoprotein is MVSKGEEDNM, the sequence of the preset length of the C end of the target chromoprotein is GGMDELYK, and the amino acid sequence of the recombinant chromoprotein obtained after replacement is SEQ ID NO.3; the recombinant chromoprotein is named as CP-Voilet, and is purple under white light and dark red fluorescence under blue light;
or the target chromoprotein is mhYFP, the sequence of the preset length of the N end of the target chromoprotein is MVSKGE, the sequence of the preset length of the C end of the target chromoprotein is MNELYK, and the amino acid sequence of the recombinant chromoprotein obtained after replacement is SEQ ID NO.4; the recombinant chromoprotein is named as CP-Yellow, and is Yellow under white light and Yellow fluorescent under blue light;
or the target chromoprotein is sfGFP, the sequence of the preset length of the N end of the target chromoprotein is MSKGE, the sequence of the preset length of the C end of the target chromoprotein is GMDELYK, and the amino acid sequence of the recombinant chromoprotein obtained after replacement is SEQ ID NO.5; the recombinant chromoprotein is named as CP-Green, and is Green under white light and Green fluorescent under blue light.
2. A method for preparing recombinant chromoprotein, comprising: replacing a sequence with a preset length at the N end of the target chromoprotein with MASSE, and replacing a sequence with a preset length at the C end of the target chromoprotein with GA, so as to obtain recombinant chromoprotein;
the amino acid sequence of the recombinant chromoprotein is one of SEQ ID NO.1 to 5;
the preparation method comprises the following specific processes:
replacing the N-terminal MVSKGE of the target chromoprotein mScarle-I with MASSE, replacing the C-terminal GGMDELYK with GA, and the obtained recombinant chromoprotein is named as CP-Pink, and the amino acid sequence of the recombinant chromoprotein is SEQ ID NO.1; the recombinant chromoprotein shows magenta color under white light and red fluorescence under blue light;
alternatively, the N-terminal MVSKGE of the target chromoprotein dTopith is replaced by MASSE, the C-terminal YGMDELYK is replaced by GA, the obtained recombinant chromoprotein is named as CP-Orange, and the amino acid sequence of the recombinant chromoprotein is SEQ ID NO.2; the recombinant chromoprotein shows orange red under white light and shows orange fluorescence under blue light;
alternatively, the N-terminal MVSKGEEDNM of the target chromoprotein mCherry is replaced by MASSE, the C-terminal GGMDELYK is replaced by GA, the obtained recombinant chromoprotein is named CP-Voilet, and the amino acid sequence of the recombinant chromoprotein is SEQ ID NO.3; the recombinant chromoprotein is purple under white light and shows dark red fluorescence under blue light;
alternatively, the N-terminal MVSKGE of the target chromoprotein mhYFP is replaced by MASSE, the C-terminal MNELYK is replaced by GA, the obtained recombinant chromoprotein is named as CP-Yellow, and the amino acid sequence of the recombinant chromoprotein is SEQ ID NO.4; the recombinant chromoprotein is yellow under white light and yellow fluorescent under blue light;
alternatively, the N-terminal MSKGE of the target chromoprotein sfGFP is replaced by MASSE, the C-terminal GMDELYK is replaced by GA, the obtained recombinant chromoprotein is named as CP-Green, and the amino acid sequence of the recombinant chromoprotein is SEQ ID NO.5; the recombinant chromoprotein is green under white light and green fluorescent under blue light.
3. Use of the recombinant chromoprotein of claim 1 for screening gene clones.
4. Use of the recombinant chromoprotein of claim 1 for sensing a metabolic inducer in a culture medium.
5. A nucleic acid encoding the recombinant chromoprotein of claim 1.
6. A vector comprising the nucleic acid of claim 5.
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