CN114057893B - Myristoylation polypeptide for encoding mitochondrial localization and preparation method and application thereof - Google Patents

Myristoylation polypeptide for encoding mitochondrial localization and preparation method and application thereof Download PDF

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CN114057893B
CN114057893B CN202111307669.1A CN202111307669A CN114057893B CN 114057893 B CN114057893 B CN 114057893B CN 202111307669 A CN202111307669 A CN 202111307669A CN 114057893 B CN114057893 B CN 114057893B
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CN114057893A (en
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周亮
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Abstract

The invention provides a polypeptide for encoding mitochondrial localization myristoylation and a preparation method and application thereof, belonging to the technical field of molecular genetics. The polypeptide sequence has an amino acid sequence represented by an M-N formula, wherein M is an amino acid sequence shown in SEQ ID No.1 and N is an amino acid sequence shown in SEQ ID No. 2. The invention includes the use of polypeptides, polypeptide compositions, nucleic acids, polypeptide conjugates having the sequence set forth in SEQ ID No.3 for expressing myristoylated mitochondrial localization protein. The amino terminal of the sequence of the invention is provided with a myristoylation conserved sequence which can be myristoylated and then inserted into a mitochondrial cell membrane.

Description

Myristoylation polypeptide for encoding mitochondrial localization and preparation method and application thereof
Technical Field
The invention relates to the technical field of molecular genetics, in particular to a myristoylation polypeptide for encoding mitochondrial localization and a preparation method and application thereof.
Background
The present invention is in the field of molecular genetics. Mitochondrial function studies rely on mitochondrial co-localization of marker genes and associated fluorescent dyes. However, it is difficult to introduce a protein of interest for fatty acid modification into mitochondria.
Mitochondrial function was observed and studied, relying on marker genes and fluorescent dyes for mitochondrial localization. Meanwhile, the target gene and the mitochondrial peptide are fused, so that the target protein coded by the target gene can be introduced into mitochondria. These methods make it simple and straightforward to study mitochondrial function.
Introduction of fatty acid modified proteins of interest (i.e., myristoylation) into mitochondria is a difficulty of the related art. The existing mitochondrial peptide cannot lead exogenous protein modified by fatty acid into mitochondria.
Disclosure of Invention
To solve the above technical problems, the present invention provides a polypeptide encoding mitochondrially localized myristoylation and a method for preparing the same.
A polypeptide encoding a mitochondrially localized myristoylation, said polypeptide having an amino acid sequence represented by the formula M-N, wherein M is the amino acid sequence shown in SEQ ID No.1 and N is the amino acid sequence shown in SEQ ID No.2, or an amino acid sequence having at least 70% homology to the amino acid sequence represented by the formula M-N, and the amino acid terminus of said amino acid sequence is myristoylated.
Furthermore, the amino acid sequence of the polypeptide is shown as SEQ ID No. 3.
A polypeptide composition, which comprises the polypeptide with the amino acid sequence shown as SEQ ID No. 3.
A nucleic acid comprising a sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID No. 3.
A polypeptide conjugate comprising a polypeptide portion, wherein the polypeptide portion comprises said polypeptide.
Further, the polypeptide conjugate further comprises a coupling moiety, wherein the coupling moiety is linked to the polypeptide moiety.
Further, the coupling moiety is selected from at least one of hemocyanin, ovalbumin and bovine serum albumin.
The polypeptide, the polypeptide composition, the nucleic acid and the polypeptide conjugate are applied to the preparation of a fluorescent probe for targeted subcellular organelle fluorescence imaging.
The polypeptide, the polypeptide composition, the nucleic acid and the application of the polypeptide conjugate in the expression of mitochondrial localization protein.
Further, the subcellular organelles include mitochondria.
Compared with the prior art, the technical scheme of the invention has the following advantages:
the amino terminal of the sequence of the invention is provided with a myristoylation conserved sequence which can be myristoylated and then inserted into a mitochondrial cell membrane. Meanwhile, the sequence at the carboxyl terminal of the sequence is derived from the C0 region of the NMDA receptor GluN1 subunit, and the C0 region can be combined with Calmodulin, so that the combination with mitochondria is promoted. Therefore, the sequence obtained in the invention can target and locate mitochondria.
Drawings
In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the present disclosure taken in conjunction with the accompanying drawings.
FIG. 1 is a fluorescence map of localized expression of myristoylated mitochondrial localization polypeptide sequences in example 2 of the present invention;
FIG. 2 is a fluorescence plot of myristoylated mitotic-localized GFP fusion plasmid (myr-Mito-GFP) and a commercial mitotic-localized plasmid (Mito-RFP) of example 3 of the present invention.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Example 1
Myristoylation mitochondrial localization polypeptide sequence screening and validation:
(1) The myristoylation conserved sequence is derived from the protein MARCKS front-end sequence: MGAQFSK.
The corresponding nucleotides are: ATGGGTGCCCAGTTCTCCAAG.
(2) The PCR primers for the GluN 1C 0 region with the myristoylation conserved sequence were customized as follows:
GluN1:5'-3' (with myristoylation conserved sequence): TCGGCTAGCACCATGGTGCCCAGTTCCCAAGATCGCCTACAAGCGACACAAGGATGCC; 3'-5': AGCTGTCGACCTGCAGGTTTCTTCCTCCACCGTTCAC.
(3) The GluN1 plasmid was used as a template for conventional PCR. (PCR kit available from TaKaRa Co., ltd.) system. The PCR system (25. Mu.L) was as follows (unit:. Mu.L):
10×Buffer:2.5,
dNTP:2,
Taq:0.5；
primer:2；
vector:300ng。
(4) Performing PCR, wherein the conditions of PCR are as follows:
(4.1) one cycle, 30 seconds at 95 ℃;
(4.2) 30 cycles of 95 ℃ for 30 seconds, 58 ℃ for 30 seconds, and 72 ℃ for 2 minutes;
(4.3) one cycle, 10 minutes at 72 ℃.
(5) The gel was subjected to agarose gel electrophoresis, and the gel was recovered and digested with NEB fast-cutting enzymes Nhe I and Sal I for 20 minutes. And recovering the product after enzyme digestion by using the nucleic acid gel.
(6) The pEGFP-N3 vector is cut by NEB fast cutting enzymes Nhe I and Sal I for 1-3 hours, and the cut vector is recovered through nucleic acid gel.
(7) The ligation was carried out at 16 ℃ for 1-2 hours using ligase Solution I from TaKaRa Limited (see description for details).
(8) The ligation products were added to competent cell DH 5. Alpha. (all-gold organism), incubated first for 20 minutes on ice, followed by heat shock at 42 ℃ for 1 minute, and finally placed on ice for 2-5 minutes, and the sequencing was correct after plating.
Example 2
Expression of myristoylated mitochondrial localization polypeptide sequence (myr-mito):
HEK293 cells were first seeded in 24-well plates, and then after 24 hours, the GFP fusion plasmid with the myristoylated mitochondrial localization sequence (myr-mito) was transfected into HEK293 cells using lipo2000 transfection reagent at a plasmid amount of 300ng per well, and its localization was observed 24 hours after transfection with a fluorescence microscope (scale: 20 μm). It can be seen that the mitochondrial localization sequence with myristoylation of the present invention is capable of expression in HEK293 cells and exhibits mitochondrial localization.
Example 3
Localization of myristoylated mitochondrial localization polypeptide sequence (myr-mito):
HEK293 cells were first seeded in 24-well plates, and then after 24 hours, GFP fusion plasmids of myristoylated mitochondrial localization sequence (myr-Mito) and either commercial mitochondrial localization plasmids (Mito-RFP) or endoplasmic reticulum localization plasmids (ER-RFP) were transfected into HEK293 cells using lipo2000 transfection reagents in an amount of 300ng of plasmid per well, the localization of which was observed by fluorescence microscopy 24 hours after transfection (scale: 20 μm). It can be seen that the localization sequence of mitochondria with myristoylation in the present invention is able to localize to mitochondria, not to the endoplasmic reticulum.
SEQ ID No.1：Met GlyAla Gln Phe Ser Lys；
SEQ ID No.2：Ile Ala Tyr Lys Arg His Lys Asp Ala Arg Arg Lys Gln Met Gln LeuAla PheAlaAlaValAsnVal TrpArg Lys Asn Leu Gln；
SEQ ID No.3：Met Gly Ala Gln Phe Ser Lys Ile Ala Tyr Lys Arg His Lys Asp AlaArgArg Lys Gln Met Gln LeuAla Phe AlaAla Val Asn Val Trp Arg Lys Asn Leu Gln。
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.
Figure BDA0003340716620000061
Figure BDA0003340716620000071
SEQUENCE LISTING
<110> Suzhou university
<120> a polypeptide encoding mitochondrially localized myristoylation, a process for its preparation and its use
<130> 3
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 7
<212> PRT
<213> (Artificial sequence)
<400> 1
Met Gly Ala Gln Phe Ser Lys
1 5
<210> 2
<211> 29
<212> PRT
<213> (Artificial sequence)
<400> 2
Ile Ala Tyr Lys Arg His Lys Asp Ala Arg Arg Lys Gln Met Gln Leu
1 5 10 15
Ala Phe Ala Ala Val Asn Val Trp Arg Lys Asn Leu Gln
20 25
<210> 3
<211> 36
<212> PRT
<213> (Artificial sequence)
<400> 3
Met Gly Ala Gln Phe Ser Lys Ile Ala Tyr Lys Arg His Lys Asp Ala
1 5 10 15
Arg Arg Lys Gln Met Gln Leu Ala Phe Ala Ala Val Asn Val Trp Arg
20 25 30
Lys Asn Leu Gln
35

Claims (7)

1. A polypeptide encoding a mitochondrially localized myristoylation, comprising: the amino acid sequence of the polypeptide is shown as SEQ ID No. 3.
2. A polypeptide composition, characterized by: the polypeptide composition comprises the polypeptide with the amino acid sequence shown as SEQ ID No.3 in claim 1.
3. A nucleic acid, wherein: the nucleic acid is a coding sequence of polypeptide with an amino acid sequence shown as SEQ ID No. 3.
4. A polypeptide conjugate, comprising: the polypeptide conjugate comprises a polypeptide portion, wherein the polypeptide portion is the polypeptide of claim 1.
5. The polypeptide conjugate of claim 4, wherein: the polypeptide conjugate further comprises a coupling moiety linked to the polypeptide moiety.
6. The polypeptide conjugate of claim 4, wherein: the coupling part is selected from at least one of hemocyanin, ovalbumin and bovine serum albumin.
7. Use of the polypeptide of claim 1, the polypeptide composition of claim 2, the nucleic acid of claim 3, the polypeptide conjugate of any one of claims 4-6 for the preparation of a fluorescent probe for targeted subcellular organelle fluorescence imaging; the subcellular organelles are mitochondria.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998040477A1 (en) * 1997-03-14 1998-09-17 The Regents Of The University Of California Fluorescent protein sensors for detection of analytes
CN107446027A (en) * 2017-09-05 2017-12-08 苏州大学 With the transdermal small peptide and its application for appraising and deciding capability
CN109575116A (en) * 2018-11-09 2019-04-05 广东海洋大学 A kind of mitochondria positioning leads peptide and its discovery method and application
WO2020101740A1 (en) * 2018-11-16 2020-05-22 Codiak Biosciences, Inc. Engineered extracellular vesicles and uses thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180126003A1 (en) * 2016-05-04 2018-05-10 Curevac Ag New targets for rna therapeutics

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998040477A1 (en) * 1997-03-14 1998-09-17 The Regents Of The University Of California Fluorescent protein sensors for detection of analytes
CN107446027A (en) * 2017-09-05 2017-12-08 苏州大学 With the transdermal small peptide and its application for appraising and deciding capability
CN109575116A (en) * 2018-11-09 2019-04-05 广东海洋大学 A kind of mitochondria positioning leads peptide and its discovery method and application
WO2020101740A1 (en) * 2018-11-16 2020-05-22 Codiak Biosciences, Inc. Engineered extracellular vesicles and uses thereof

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