CN110511273B - Preparation method and application of cell-penetrating polypeptide - Google Patents
Preparation method and application of cell-penetrating polypeptide Download PDFInfo
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Abstract
The invention discloses a preparation method of cell-penetrating polypeptide, which comprises the following steps: altering a portion of the amino acid residues in the angular homeodomain (HDAntp) amino acid sequence such that its overall net positive charge to protein mass ratio is greater than 1.6. The cell-penetrating polypeptide obtained by the preparation method can efficiently penetrate through membranes to enter cytoplasm of various mammalian cells. The preparation method of the invention can lead the polypeptide or the protein to have the membrane penetration performance by improving the total net positive charge on the basis of not influencing the functions of the original protein or the polypeptide, thereby having great methodological value and significance for the transmembrane transport of the exogenous drug protein which needs to play a specific function in cells.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to a preparation method and application of efficient cell-penetrating polypeptide.
Background
The cell membrane is a barrier for the entry and exit of substances into and out of the cell and is composed of lipid bilayers, and many biological macromolecules such as polypeptides, proteins and DNA cannot freely cross the cell membrane to carry out substance and information transfer. In recent years, some protein domains having a cell membrane penetration ability, such as trans-Transcription Activator (TAT) of Human immunodeficiency virus (HIV-1), drosophila homeodomain (HDAntp), and herpes simplex virus type I VP22 transcription factor, have not only a cell penetration ability but also a function of mediating macromolecules such as heterologous proteins, oligonucleotides, and liposomes to directly penetrate through a cell membrane into a cell, and are called Protein Transduction Domains (PTDs). Further studies have shown that mediating the membrane-penetrating function of protein transduction domains are cell-penetrating polypeptides (CPPs) located within the PTD and generally consisting of around 20 amino acid residues, e.g. amino acids 47-57 of TAT protein play a key role in the membrane-penetrating effect; the 43-58 residue fragment of HDAntp is the main functional element mediating the membrane penetration of HDAntp and is therefore named Penetratin. Cell-penetrating polypeptides CPPs can penetrate cell membranes without damaging cell membranes, and are gradually the focus of research attention and increasingly expanded short peptide families, including MPG, PEP-I, TAT, oligoarginine, oligolysine and the like.
A large number of researches show that substances carried by the CPPs can be proteins (green fluorescent protein, RNase, galactosidase and the like), polypeptides (less than 100 amino acid residues), DNA, chemical small molecule drugs, oligonucleotides, antisense nucleic acids, peptide nucleic acids, nanoparticles, fluorescein, organic molecules, adenovirus vectors, imaging substances, liposomes, iron particles and the like. CPPs can carry various biological macromolecules by means of chemical combination or gene fusion, and the connection mode of the CPPs and the drugs comprises non-covalent connection and covalent connection.
However, none of these transmembrane polypeptides is high in transmembrane efficiency. Currently, common methods for introducing biological macromolecules into cells include a liposome-mediated method, a viral vector-mediated method, an electroporation method, a microinjection method and the like, but these methods have the disadvantages of low transport efficiency, high cytotoxicity, poor safety, poor targeting specificity and the like, and greatly limit the large-scale application of the methods. Therefore, the development of efficient and safe transmembrane transport technology for biological macromolecules is an important challenge.
Disclosure of Invention
In order to solve the above technical problems, the inventors conducted extensive research to increase the number of positively charged amino acid residues in the HDAntp sequence such that the total net positive charge to protein mass Ratio (Ratio of positive charges per kDa) is higher than 1.6, and unexpectedly found that the modified HDAntp, i.e., HDSup +, can efficiently penetrate the membrane into the cytoplasm of various mammalian cells, thereby completing the present invention.
The first aspect of the invention provides a preparation method of a high-efficiency cell-penetrating polypeptide, which comprises the following steps:
altering a portion of the amino acid residues in the angular homeodomain (HDAntp) amino acid sequence such that its overall net positive charge to protein mass ratio is greater than 1.6.
Further, the HDAntp has an amino acid sequence shown in SEQ ID NO. 1.
In some embodiments of the invention, said altering a portion of the amino acid residues in the HDAntp amino acid sequence comprises changing a portion of the amino acid residues to positively charged amino acid residues. Among them, the positively charged amino acid residues include lysine (K), arginine (R) and histidine (H), and preferably, the positively charged amino acids include K and R.
In some embodiments of the present invention, the changing of a part of the amino acid residues in the HDAntp amino acid sequence refers to changing leucine (L) at position 14 to lysine (K), alanine (a) at positions 35 and 37 to arginine (R), cysteine (C) at position 39 to serine (S) to avoid disulfide bond formation of the cysteine residue to generate polypeptide dimer, and asparagine (N) at position 60 to arginine (R) in SEQ ID No.1, thereby obtaining the amino acid sequence shown in SEQ ID No. 2.
In a second aspect, the present invention provides a high efficiency cell-penetrating polypeptide obtained by altering a portion of the amino acid residues in the HDAntp amino acid sequence such that the total net positive charge to protein mass ratio is greater than 1.6.
Further, the HDAntp has an amino acid sequence shown in SEQ ID NO. 1.
In some embodiments of the invention, said altering a portion of the amino acid residues in the HDAntp amino acid sequence comprises changing a portion of the amino acid residues to positively charged amino acid residues. Among them, the positively charged amino acid residues include lysine (K), arginine (R) and histidine (H), and preferably, the positively charged amino acids include K and R.
In some embodiments of the invention, the cell-penetrating polypeptide has the amino acid sequence shown in SEQ ID No.2, which is obtained by changing leucine (L) at position 14 to lysine (K), alanine (a) at positions 35 and 37 to arginine (R), cysteine (C) at position 39 to serine (S), and asparagine (N) at position 60 to arginine (R) in SEQ ID No. 1.
In a third aspect, the invention provides a gene encoding a cell-penetrating polypeptide according to the second aspect of the invention. The nucleotide sequence encoding the corresponding amino acid sequence may be plural in number according to the codon encoding rule, but the functions thereof are identical.
In a fourth aspect, the present invention provides a vector comprising a gene according to the third aspect of the invention.
In some embodiments of the invention, the vector is pET/RosettaTM2(DE3)pLysS。
In a fifth aspect, the present invention provides a cell comprising a gene according to the third aspect of the present invention or a vector according to the fourth aspect of the present invention.
Further, the cell is a prokaryotic cell, and further, the cell is an Escherichia coli cell.
In a sixth aspect, the invention provides the use of a cell-penetrating polypeptide according to the second aspect of the invention or a gene according to the third aspect or a vector according to the fourth aspect or a cell according to the fifth aspect of the invention in the manufacture of a medicament for use in a cell.
In some embodiments of the invention, the agent comprises a polypeptide. In some preferred embodiments of the invention, the polypeptide is a protein, and in some more preferred embodiments of the invention, the polypeptide is an antibody.
In some embodiments of the invention, the N-terminus or C-terminus of the amino acid sequence of the polypeptide is fused to the cell penetrating polypeptide.
The invention has the advantages of
Compared with the prior art, the invention has the following effective effects:
according to the preparation method, the total net positive charge and protein mass ratio is higher than 1.6 by increasing the number of positively charged amino acid residues in the HDAntp sequence, so that the modified HDSup + of the HDAntp is obtained. The preparation method of the invention can be used for obtaining cytoplasm which can efficiently penetrate through membranes and enter various mammalian cells.
The cell-penetrating polypeptide has a penetrating efficiency higher than that of Penetratin, and can reach 5 times at most.
The preparation method of the invention can enable the polypeptide or the protein to have the membrane penetrating performance by improving the total net positive charge on the basis of not influencing the functions of the original protein or the original polypeptide. This has significant methodological value and significance for transmembrane transport of exogenous pharmaceutical proteins that are required to perform specific functions within the cell.
Drawings
FIG. 1 shows the Antennapdia homeodomain (HDAntp) and the modified HDAntp (HDSup +) with naturally increased charge. (A) The amino acid sequences of HDAntp and HDSup +. (B) HDAntp and HDSup + specifically bind to the D4 enhancer probe; where pmol represents the loading of the complex of HDAntp or HDSup + and D4 probe.
FIG. 2 shows a comparison of the transport efficiency of HDSup + and Penetratin in Hela cells. (A) Confocal fluorescence microscopy of live Hela cells. Cells were incubated for 1 hour at 37 ℃ in 2.5. mu.M extracellular protein containing HDSup +, Pentratin and mCherry, respectively. The fluorescence signal is shown by the flame color of ImageJ, the maximum projection z is superimposed into 20 confocal sections, and the scale bar represents 10 μm. (B) The amount of cellular uptake at various concentrations was determined by Fluorescence Activated Cell Sorting (FACS). Data are mean ± SD of triplicate experiments.
Figure 3 shows a comparison of HDSup + and Penetratin in terms of transport efficiency in SHSY5Y cells and NuMuMG cells.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.
Examples
The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
The experimental procedures used in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
The mammalian cell lines Hela, SHSY5Y and NMuMG used were cultured in DMEM (Dulbecco's Modified Eagle's Medium) with high W/Glutamax-I (Invitrogen) containing 10% heat-inactivated fetal bovine serum (FBS, Invitrogen) (37 ℃, 5% CO)2)。
Example 1 preparation of HDSup +
As shown in fig. 1A, by increasing the number of positively charged amino acid residues in the HDAntp sequence (SEQ ID No.1), leucine (L) at position 14 in SEQ ID No.1 was changed to lysine (K), alanine (a) at positions 35 and 37 was changed to arginine (R), cysteine (C) at position 39 was changed to serine (S), and asparagine (N) at position 60 was changed to arginine (R), thereby obtaining an engineered HDAntp, HDSup +, having the amino acid sequence shown in SEQ ID No. 2. HDSup + Total Net Positive Charge and protein Mass Ratio (Ratio of positive charges per kDa) is higher than 1.6.
Example 2 recombinant protein preparation
To be able to compare the efficiency of the transfer of HDSup + and its transduction domain, Penetratin, we fused them to the mCherry fluorescent protein using the same linker and orientation. The amino acid sequence of the mCherry fluorescent protein is as follows (SEQ ID NO. 3):
MHHHHHHHHHHIEGRVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKG
penetrain (DSLEFIASKLA, SEQ ID NO.4) and mCherry are fused and expressed to obtain a recombinant expression protein mCherry-Penetrain, and the sequence is as follows (SEQ ID NO. 5):
MHHHHHHHHHHIEGRVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKGGDSLEFIASKLA
the HDAntp and the mCherry are fused and expressed to obtain a recombinant expression protein mCherry-HDAntp, and the sequence is as follows (SEQ ID NO. 6):
MHHHHHHHHHHIEGRVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKGGRKRGRQTYTRYQTLELEKEFHFNRYLTRRRRIEIAHALCLTERQIKIWFQNRRMKWKKEN
the HDSup + and the mCherry are fused and expressed to obtain a recombinant expression protein mCherry-HDSu +, the sequence of the recombinant expression protein mCherry-HDSu + is as follows (SEQ ID NO. 7):
MHHHHHHHHHHIEGRVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKGGRKRGRQTYTRYQTKELEKEFHFNRYLTRRRRIEIRHRLSLTERQIKIWFQNRRMKWKKER
the recombinant proteins are expressed by an escherichia coli expression system: pET/RosettaTM2(DE3) pLysS for fusion expression.
Example 2 electrophoretic mobility Shift assay
To determine whether a recombinant protein can retain its ability to specifically bind to DNA, the inventors used the [ α -32P ] dATP labeled D4 probe for Electrophoretic Mobility Shift Analysis (EMSA). D4 consists of 62 base pair DNA, corresponding to a fragment within the invertebrate enhancer in drosophila, which is bound by endogenous Antp and other co-transcription factors during leg development. The specific operation is as follows:
based on the sequence of the drosophila-derived D4 enhancer, probes were designed for Electrophoretic Mobility Shift Analysis (EMSA) as follows:
d4 forward probe sequence: (SEQ ID NO.8)
5’-AGTTTACCATTAAATTCCCATTTAGGCTGTCAATCATTTGCGCT-3’
D4 reverse probe sequence: (SEQ ID NO.9)
5’-AAGCCGCCAAGAAAAATTAGCGCAAATGATTGACAGCCTAAATGGG-3’
The probe sequence was then labeled with [ alpha-32P ] dATP using the Klenow fragment of DNA pol I. 10ng of annealed oligonucleotide was used for each reaction. Unincorporated labels were removed using an illustra MicroSpin G-25 column (GE). Poly (dI. dC) (Sigma) was used to reduce non-specific binding. The resulting solution was analyzed by non-denaturing electrophoresis using 6% acrylamide gel and developed with X-ray film.
The result is shown in FIG. 1B, the recombinant HDAntp and HDSup + protein can both specifically bind to the 62bp D4 probe, and the protein/DNA complex formed by the HDSup + protein and the probe is in positive correlation with the added protein quality, which indicates that both the recombinant HDAntp and HDSup + can maintain the specific binding ability with the complementary DNA sequence.
Example 3 comparison of HDSup + and penetratin efficiency
1. Live cell imaging
Since the fixation of cells leads to an artificial redistribution of CPPs, the inventors monitored cellular uptake of recombinant proteins in living cells. Cells were seeded onto 8-well μ -slide (ibidi) containing 220 μ L of medium (1 × 10 per well)4One). After 22 hours, the cells were washed once with PBS and incubated for 30 minutes in serum-free DMEM, or pre-treated with the corresponding drugs and then incubated for 60 minutes in each protein solution. After incubation, cells were washed three times with PBS containing 20U/mL heparin to remove membrane bound proteins and imaged in pre-warmed growth medium. Cells were imaged on a heating stage using a 60 × or 100 × lens in a Leica SP5 MP confocal microscope and images were processed using ImageJ software.
2. Flow cytometry
Cells were seeded into 24-well plates at 6X 10 per well4And (4) cells. After 22 hours, cells were incubated as described above for live cell imaging, then trypsinized, resuspended in 200 μ l growth medium and placed on ice. Cells were analyzed for mCherry internalization on a LSRII flow cytometer (BD Biosciences) (561 nm). Cells were gated to obtain viable cells and at least 10,000 viable cells per treatment were analyzed. Data were analyzed using FlowJo software (Tree Star, Inc.).
As a result, HDSup + fusion proteins were found to signal more strongly intracellularly than the Penetratin fusion proteins (fig. 2A), while mCherry alone gave hardly any detectable signal under the same microscope setup.
To further quantify the transmembrane efficiency of each protein, the inventors applied Fluorescence Assisted Cell Sorting (FACS) to Hela cells by an additional trypsinization step prior to FACS analysis, under the same treatment. The forward and lateral scatter gate settings of FACS exclude signals from dead cells, which typically produce strong false signals. At an extracellular protein concentration of 2.5 μ M, HDSup + was able to deliver mCherry with up to 5-fold higher efficiency than pennetratin into Hela cells (fig. 2B). And the same difference was observed as low as 0.5 μ M and as high as 5 μ M, although the difference between the latter HDSup + and Penetratin was reduced to 4-fold (fig. 2B). Overall, HDSup + showed a greater capacity to penetrate Hela cells than Penetratin.
To rule out the possibility that the difference is specific to Hela cells, the inventors used other cell lines to compare the transmembrane efficiency between HDSup + and Penetratin. In both SHSY5Y and NuMuMG cell lines, higher transmembrane efficiency of HDSup + was observed compared to the permeatin penetretin (fig. 3). It was shown that the difference in transmembrane efficiency between HDSup + and Penetratin was not affected by the cell type.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
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Arg Gln Thr Tyr Thr Arg Tyr Gln Thr Lys Glu Leu Glu Lys Glu Phe
260 265 270
His Phe Asn Arg Tyr Leu Thr Arg Arg Arg Arg Ile Glu Ile Arg His
275 280 285
Arg Leu Ser Leu Thr Glu Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg
290 295 300
Arg Met Lys Trp Lys Lys Glu Arg
305 310
<210> 8
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
agtttaccat taaattccca tttaggctgt caatcatttg cgct 44
<210> 9
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
aagccgccaa gaaaaattag cgcaaatgat tgacagccta aatggg 46
Claims (5)
1. A cell-penetrating polypeptide, which is obtained by changing partial amino acid residues in an HDAntp amino acid sequence so that the ratio of the total net positive charge to the protein mass is higher than 1.6, and which is the amino acid sequence shown in SEQ ID NO. 2.
2. A gene encoding the cell-penetrating polypeptide of claim 1, wherein said gene is capable of encoding said cell-penetrating polypeptide.
3. Use of a cell-penetrating polypeptide according to claim 1 in the preparation of a medicament intended to function intracellularly.
4. The use of claim 3, wherein the drug comprises a polypeptide.
5. The use of claim 4, wherein the N-terminus or C-terminus of the amino acid sequence of the polypeptide is fused to the cell penetrating polypeptide.
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