CN110172467B - Construction of orthogonal aminoacyl-tRNA synthetase/tRNA system by chimeric design method - Google Patents
Construction of orthogonal aminoacyl-tRNA synthetase/tRNA system by chimeric design method Download PDFInfo
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Abstract
An orthogonal aminoacyl-tRNA synthetase/tRNA system constructed by a chimeric design method belongs to the technical field of chemical biology. It is characterized by the broad orthogonality of the transplanted pyrrollysyl-tRNA synthetase/tRNA pair in eukaryotic and prokaryotic cells. The system of the invention transplants eukaryotic and prokaryotic promiscuous orthogonality of the pyrrollysyl-tRNA synthetase/tRNA system through reasonable chimeric design, including several chimeric tRNA synthetase/tRNA systems, including but not limited to histidine, phenylalanine, alanine and serine systems, which not only have high efficiency, but also have pyrrollysyl-tRNA synthetase/tRNAPylThe flexibility of the system can be applied to prokaryotes and eukaryotes. The methods can be applied without doubt to the orthogonal engineering of other aminoacyl-tRNA synthetase/tRNA pairs.
Description
Technical Field
The invention belongs to the technical field of chemical biology, and particularly relates to an orthogonal aminoacyl-tRNA synthetase/tRNA system constructed by a chimeric design method.
Background
Proteins are the main substances that function in the body, and 20 amino acids encoded by 61 codons are synthesized in the ribosome. Although the 20 amino acids endow the protein with the characteristics of participating in various physiological and biochemical activities, only few active groups such as sulfydryl, hydroxyl and the like in the 20 amino acids can be chemically controlled. In order to better study the physiological function of protein, the genetic code expansion technology of other unnatural amino acids with active groups can be introduced. The genetic code expansion technology utilizes an orthogonalized aminoacyl tRNA synthetase/tRNA system to recognize unnatural amino acids with different functions and decode unassigned codons (stop codons, 4-linked codons, etc.) so as to realize the site-specific insertion of the unnatural amino acid. This system has achieved up to now the insertion of more than 150 unnatural amino acids with different reactive groups, performing different functions, such as bio-tracking and imaging, regulation of protein function in vivo, studying post-translational modifications, proteomic analysis and biotherapeutics.
In the genetic codon amplification technique,the core is the need for an orthogonalized aminoacyl tRNA synthetase/tRNA, which does not recognize each other with the endogenous aminoacyl tRNA synthetase and tRNA in the cell, and which does not affect the normal physiological activity in the cell to achieve the insertion of a specific unnatural amino acid. The orthogonal pairs of aminoacyl tRNA and tRNA used so far are mainly 4, but only the pyrrolyl-tRNA synthetase (PylRS)/tRNA synthetase derived from Methanococcus methanolica mazei or Methanosacina barkeriCUAOrthogonal pairs can be universally applied in bacteria, eukaryotic cells and individuals.
Pyrrolosyl-tRNA synthetase (PylRS)/tRNACUAOrthogonal system although about 100 unnatural amino acids have been successfully introduced into bacteria and eukaryotic cells, it suffers from several problems: 1, the activity to some unnatural amino acids is very low, which results in high experimental cost and low signal-to-noise ratio; 2, these introduced unnatural amino acids are derivatives of lysine and phenylalanine, and are difficult to introduce for amino acids with other chemical structures. We have evolved to introduce novel unnatural amino acids, although we can take advantage of other 20 aminoacyltrna synthetases that occur naturally. However, there are problems with this approach: 1, directed evolution of the anticodon-binding domain of aminoacyl tRNA synthetase is required for other aminoacyl tRNA synthetase/tRNA systems to recognize unassigned codons (stop codon, rare codon, and 4-linked codon); 2, the system has no universal orthogonality, the source and the bacteria can be used in eukaryotic cells, and the source and the bacteria can be used in eukaryotic organisms, so that a plurality of limitations are added to the application of the system.
In order to overcome the above problems, researchers have devised different approaches to coping with them. To increase Pyrrolosyl-tRNA synthetase (PylRS)/tRNACUAOrthogonal system activity, Diter Soll and David R Liu construct Pyrrolyl-tRNA synthetase of two species chimera Mm and Mb, and the chimeric protein is modified by PACE (phage assisted continuous evolution) method to improve activity. From the tRNA point of view, there have also been some researchers who have performed corresponding mutagenesis of the anticodon loop, anticodon arm, and T loop, respectively, to enhance the insertion of an unnatural amino group into this systemEfficiency of the acid. For the problem that other aminoacyl tRNA synthetase/tRNA systems are not universally orthogonal, researchers can change the Escherichia coli to replace TrpRS/tRNA in the Escherichia coli genome with TrpRS/tRNA in yeast by changing the Escherichia coli, so that the Escherichia coli TrpRS/tRNA system can be orthogonal in the changed Escherichia coli strain and eukaryotes, and screening of unnatural amino acids is facilitated. The above solutions, while overcoming to some extent the problems in the field of genetic codon expansion, do not fundamentally solve the problem of non-extensive orthogonality of natural amino acid aminoacyl-tRNA synthetases.
Disclosure of Invention
In view of the problems of the prior art, the present invention provides a technical solution for constructing an orthogonal aminoacyl-tRNA synthetase/tRNA system by a chimeric design method, which is not limited to four systems of a chimeric histidyl-tRNA synthetase/tRNA pair (chHisRS/chHisT), a chimeric phenylalanyl-tRNA synthetase/tRNA pair (chHmPheRS/chHmPheT), a chimeric seryl-tRNA synthetase/tRNA pair (chSerRS/chSerT), and a chimeric alanyl-tRNA synthetase/tRNA pair (chAlaRS/chAlaT), the gene sequence of the chimeric histidyl-tRNA synthetase is shown in SEQ ID No.3, the gene sequence of the chimeric phenylalanyl-tRNA synthetase is shown in SEQ ID No.4, the gene sequence of the chimeric seryl-tRNA synthetase is shown as SEQ ID No.5, the gene sequence of the chimeric alanyl-tRNA synthetase is shown in SEQ ID No. 6. More specifically, we do not exemplify a specific mosaic system, but all mosaic systems are constructed in accordance with the following method. Meanwhile, in the present invention, the E.coli expression system uses pNEG and pBK vectors, and the animal cell expression system uses pcDNA3.1 vector as an example, but is not limited to these three vectors. Maps of these three vectors are shown in figure 1.
In the construction of the orthogonal aminoacyl tRNA synthetase/tRNA/system by chimeric design method, we provide specific method to construct and optimize chimeric system, which includes the following steps:
s1: constructing a chimeric aminoacyl-tRNA synthetase/tRNA vector;
s2: the activity of the chimeric aminoacyl-tRNA synthetase/tRNA was evaluated in escherichia coli using GFP amber suppression efficiency;
s3: confirmation of amino acid insertions, including GFP expression, identification of LC-MS and LC-MS/MS;
s4: testing amber inhibition efficiency of the chimeric system in a mammalian cell expression system;
s5: for the low-efficiency chimeric system, the acceptor arm of the chimeric tRNA is selected to construct a mutant library for screening.
The chimeric aminoacyl-tRNA synthetase/tRNA vector is constructed by the following steps:
s6: constructing a chimeric tRNA vector;
s7: construction of chimeric aminoacyl-tRNA synthetase vectors.
The chimeric tRNA vector is constructed by the following steps:
s8: designing and synthesizing chimeric tRNA;
s9: the reporter gene GFP190TAG-His6 is synthesized, and the sequence is shown as SEQ ID No. 1;
s10: chimeric tRNA and GFP190TAG-His6 were ligated into pNEG vector under the control of glns and pBAD promoters, respectively.
The chimeric aminoacyl-tRNA synthetase is constructed by the following steps:
s11: selection of a tRNA binding domain from a pyrrolysinyl-tRNA synthetase, selection of an aminoacyl-tRNA synthetase catalytic domain in a chimeric aminoacyl-tRNA synthetase; separately ligated to pBK vectors. The complete sequence of the tRNA binding domain of the pyrrollysyl-tRNA synthetase is shown in SEQ ID No. 2;
s12: linker peptides of varying lengths and types are synthesized and loaded between two moieties in a chimeric aminoacyl-tRNA synthetase. The kind of linker peptide: GS-rich, helix, P-rich, the sequence is shown in Table 3-1.
S13: the synthetic glns promoter, pBAD promoter, oxb20 promoter, trp promoter were inserted into pBK vector to control the expression of chimeric aminoacyltRNA synthetase, and the sequences are shown in Table 4-1.
Also provided is a method for assessing the activity of a chimeric aminoacyl-tRNA synthetase/tRNA in E.coli using GFP amber suppression efficiency, as follows:
s14: the pNEG vector carrying the GFP190TAG-His6 gene and the chimeric tRNA and the pBK vector carrying the chimeric aminoacyl-tRNA synthetase are co-transformed into DH10B competent cells;
s15: selecting a monoclonal antibody to be cloned in a liquid culture medium, and inducing gene expression by arabinose;
s17: lysing the expressed cells;
s18: the fluorescence of GFP in cell supernatants after lysis was measured and the GFP amber inhibition efficiency of the chimeric system was calculated (fig. 4).
For the constructed chimeric aminoacyl-tRNA synthetase/tRNA system, we also provide a method for identifying the correct insertion of an amino acid, as follows:
s19: culturing Escherichia coli in S14 in large scale, and allowing arabinose to induce protein expression;
s20: collecting the expressed escherichia coli, carrying out ultrasonic disruption, and carrying out affinity chromatography purification;
s21: western-blotting, LC-MS and LC-MS/MS identification of the purified protein.
The chimeric system which can be orthogonally used in Escherichia coli, the analysis of the efficiency of transfer into mammalian cells and the confirmation of orthogonality are as follows:
s22: transferring the chimeric aminoacyl tRNA synthetase gene and the chimeric tNRA gene into a pcDNA3.1 vector;
s23: the plasmid of S22 and pEGFP-EGFP-191TAG-Histag transfect HEK 293T cells together;
s24: GFP fluorescence and Western-blotting measure the efficiency of the chimeric system in mammalian cells.
For the initial construction of chimeric systems with low activity and orthogonality, we provide a screening system based on a library of chimeric tRNA receptor arm regions, as follows:
s25: constructing a chimeric tRNA library and cloning the library into a pNEG vector;
s26: preparing a DH10B competent cell containing the chimeric aminoacyl-tRNA synthetase plasmid;
s27: electrotransformation of the library into prepared competent cells and plating of plates containing arabinose;
s28: screening clones with fluorescence;
s29: confirming the orthogonality of the chimeric tRNA;
s30: sequencing the chimeric tRNA.
The chimeric system inherits the flexibility of the pyrrollysyl-tRNA synthetase/tRNA system, can realize the efficient site-specific introduction of amino acid by using codons UAG and UAA and UGA codons, and is concretely as follows:
s31: constructing vectors of a UAA system and a UGA system;
s32: the efficiency of amino acid introduction was compared for the three systems.
The system of the invention transplants eukaryotic and prokaryotic promiscuous orthogonality of the pyrrollysyl-tRNA synthetase/tRNA system through reasonable chimeric design, including several chimeric tRNA synthetase/tRNA systems, including but not limited to histidine, phenylalanine, alanine and serine systems, which not only have high efficiency, but also have pyrrollysyl-tRNA synthetase/tRNAPylThe flexibility of the system can be applied to prokaryotes and eukaryotes. The methods can be applied without doubt to the orthogonal engineering of other aminoacyl-tRNA synthetase/tRNA pairs.
Drawings
FIG. 1 is a map of a plasmid used in the present invention, in which A: pBK plasmid map, B: pNEG plasmid map, C: pcDNA3.1 plasmid map, D: pEGFP-EGFP-191 TAG-Histagg plasmid map.
FIG. 2 is a schematic diagram of the construction of an orthogonal aminoacyl-tRNA synthetase/tRNA system by chimeric design, in which A: Pyrrolysinyl-tRNA synthetases/RNAsPylA structure of an orthogonal pair (left) and a structure of a traditional aminoacyl-tRNA synthetase/tRNA pair (right), a chimeric aminoacyl-tRNA synthetase (chRS) in which the tRNA binding domain of the pyrrollysyl-tRNA synthetase is fused to the catalytic domain of the traditional aminoacyl-tRNA synthetase, and the chimeric tRNA (cht) is generated by replacing the pylT acceptor arm with a specific tRNA acceptor arm (middle); b: amino acid structure in this study.
FIG. 3 is a diagram showing the construction and efficiency measurement of chimeric histidine tRNA, in which A: a clover structure having histidine tRNA of G-1 structure and pyrrolysine tRNA; b: the clover structure of the chimera of chHisT consists of the sequence of pyl T and the sequence of his T; c: the chimera structure of chHisRS-1 is composed of fusion of pylRS NTD (1-149aa) and hisRS CD (1-326 aa); d: the GFP fluorescence intensity method is used for detecting amber inhibition efficiency.
As shown in fig. 4 for the construction of chimeric histidyl-tRNA synthetases and the corresponding efficiency and specificity assays, a: the chHisRS chimera consists of a pylRS sequence and a hisRS sequence, and the pylRS NTD mutation carries an IPYE mutation; b: analyzing amber inhibition efficiency of the chHisRSS by a GFP (green fluorescent protein) report method; c: the amber suppression efficiency of pylRSs at 2mM Boc-L-lysine was analyzed by GFP fluorescence at 22 ℃ and 30 ℃ with the noted activity of pylRS at 30 ℃ being higher than at 22 ℃ and similar to that of chHisRS at 22 ℃ or 30 ℃; d: mass spectrometry confirmed the introduction of histidine at a specific site of GFP by the chimeric histidyl-tRNA synthetase/tRNA; e: analyzing amber suppression efficiency of the chHisRS under different promoter conditions through a GFP signal and a non-denaturing polyacrylamide gel; f: and superposing the histidyl-tRNA synthetase/tRNA complex with a pyrrolysinyl-tRNA synthetase/tRNA complex structure by taking tRNA as an overlapped object.
FIG. 5 is a graph depicting the activity and orthogonality of a chimeric histidyl-tRNA synthetase/tRNA system in cells, where A: the amber suppression efficiency in mammalian cells was determined by western testing for the expression level of full-length GFP. In this experiment, HEK 293T cells were co-transfected with two plasmids: one plasmid carries chimeric aminoacyl tRNA synthetase and chimeric tRNA gene, the tRNA is divided into terminal CCA sequence and non, the other plasmid carries GFP-190TAG-His6A gene. Then by anti-His6The antibodies were analyzed either in a western analysis (A) or by fluorescence microscopy (B) on transfected cells. A plasmid not carrying the chimeric aaRS gene was used as a negative control. C: the amber inhibitory activity of chHisRS was analyzed with the GFP reporter under different tRNA conditions.
Another system of chimerization, as constructed using the chimerization design in FIG. 6, wherein A: the amber suppressing activity of the resulting chimeric aminoacyl-tRNA synthetases on the corresponding chimeric tRNAs was analyzed with GFP as a reporter gene. In this figure, EC stands for E.coli, H for human, H-MT for human mitochondria; b: analyzing amber inhibitory activity of chPheRSs by GFP reporter gene; c: mass spectrometry confirmed the introduction of phenylalanine at a specific site of GFP by the chimeric phenylalanyl-tRNA synthetase/tRNA; d: analysis of amber inhibitory activity of chAlaRSs with GFP reporter gene E: mass spectrometry confirmed the introduction of alanine at a specific site of GFP by the chimeric alanyl-tRNA synthetase/tRNA.
FIG. 7 is a graph of the molecular evolution of chimeric seryl tRNA, in which A: the general two-step approach to chimeric tRNA receptor arm evolution is a combination of tRNA randomization and tRNA selection; b: GFP reporter assay amber suppression efficiency C of chSerTs under chSerRS conditions: the mass spectrum analysis of serine for GFP fidelity.
FIG. 8 shows a clover structure of tRNA of the invention, in which A: the structures of pylT and hisT in the invention, pylT has U25C mutation to improve pyrrolidine efficiency, and hisT has G-1The structure of (1); b: a chimeric construct of Histidyl-tRNA-1 to-6, comprising pylT and hisT; c: coli and CUA anticodon in humans; d: other chimeric structures of tRNAs of the invention, chPheT, chAlaT, chAlaT (G-U), have an unusual G-U pair mutated to an AU pair; e: the chimeric serine tRNA was named chSerT-2, which had high amber suppression efficiency from original-1 to-2, -3 after selection.
FIG. 9 is a schematic representation of the chimeric histidine system, in which A: purified histidine-inserted GFP proteins were analyzed by coomassie blue staining; b: mass spectrometry confirmed the insertion of histidine in GFP; c: LC-MS/MS analysis confirmed the insertion of histidine in GFP; d: non-denaturing polyacrylamide gel electrophoresis analysis of the insertion of histidine in the GFP protein; e: amber suppression efficiency of the chimeric histidyl-tRNA/tRNA system at different histidine concentrations at 22 ℃ or 30 ℃.
FIG. 10 shows the structure of a chimeric histidyl-tRNA synthetase. Protein complexes were fitted by tRNA overlap.
FIG. 11 is a graph of the optimization of chimeric histidine tRNA synthetases, in which A: an overview of chHisRS of different length linker peptides; b: GFP report method and native gel fluorescence analysis of different lengths of chHisRS amber inhibitory activity; c: the GFP reporter assay analyzed the amber inhibitory activity of chHisRS carrying different types of linker peptides, with GS representing a Gly and Ser rich linker peptide, Pro representing a linker peptide rich in Pro, and helix representing a linker peptide with a helical structure.
As shown in FIG. 12, the suppression efficiency of the nonsense code of the chimeric histidine system was analyzed for the suppression efficiency of the GFP gene carrying three stop codons (ochre, opal and amber codon).
As figure 13 is a test of the orthogonality of the chimeric histidine system in mammalian cells, where a & B, in figure5, shows a complete Western blot gel, regions in the main text are highlighted by dashed boxes, D: the inhibitory activity of chHisRS on endogenous hisTs amber was analyzed by the GFP assay.
FIG. 14 is a schematic diagram of a chimeric phenylalanyl-tRNA synthetase/tRNA system, wherein A: analyzing amber inhibition activity of chPheRSs by a GFP method; b: complete Western blot gel in Figure5B, highlighted by the dashed box in Figure 5B; c: purified GFP protein was analyzed by coomassie blue staining; d: mass spectrometry raw data confirmed the introduction of phenylalanine at a specific site of GFP by the chimeric phenylalanyl-tRNA synthetase/tRNA; e: amber suppression efficiency of phenylalanine chimeric system at 22 ℃ or 30 ℃.
FIG. 15 is a schematic diagram of a chimeric alanyl-tRNA synthetase/tRNA system, in which A: the structure of the alanine-tRNA synthetase (chAlaRS) contains a pylRS sequence and an alaRS sequence, the tRNA binding domain of pylRS carries an IPYE mutation; b: analyzing the amber inhibition activity of the chAlaRSs and chAlaT (G-U) by a GFP method; c: the GFP method was used to analyze the amber-suppressing activity of the chAlaRSs and chAlaT (A-U). D: the complete Western blot gel is shown in Figure 5D, with regions in the main text highlighted by dashed boxes; e: purified GFP protein was analyzed by coomassie blue staining; f: g mass spectrometry confirmed the original data for the introduction of alanine at a specific site of GFP by the chimeric alanyl-tRNA synthetase/tRNA.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments, which are illustrative of the present invention and are not limited to the following embodiments.
The invention is further described below with reference to chimeric histidine tRNA synthetase/tRNA systems and molecular modifications of chimeric serine tRNA's.
Example 1: construction of chimeric histidine tRNA
In the present invention, the chimeric tRNA and the reporter gene GFP190TAG-His6 are constructed on the same plasmid and controlled by lpp promoter and pBAD promoter, respectively. The specific construction method is as follows:
(1) construction of GFP190TAG-His6 into pNEG vector
The sequence of GFP190TAG-His6 is shown in SEQ ID No.1, primers pNEG-GFP-F and pNEG-GFP-R are designed, the existing plasmid containing the gene is used as a template for amplification, and pNEG-GFP-v-F and pNEG-GFP-v-R are designed and synthesized at the same time, and the vector is amplified by using the previous pNEG as the template. Recovering agarose gel, assembling by Gibson, transforming DH10B competent cells, selecting monoclonal sequencing to obtain plasmid pNEG-GFP190TAG-His 6;
(2) chimeric histidine tRNA was designed and cloned into pNEG-GFP190TAG-His6 vector
In order to obtain the optimal chimeric histidine tRNA, we designed chimeric histidine tRNA with different regions of the acceptor arm region of the histidine tRNA grafted, named chHisT-1, 2, 3, 4, 5 and 6, and see FIGS. 3 and 8 for model and detailed sequence. Corresponding primers were designed and cloned into pNEG-GFP190TAG-His6 vector constructed in the previous step. The corresponding primers are shown in Table 1-1.
TABLE 1-1 primers required for construction of chimeric tRNA
| Name | Sequence |
| GFP190TAG-F | TTTAAGAAGGAGATATACATATGGGTAAAGGAGAAGAACTTTTC |
| GFP190TAG-R | CCGCAAGGAATGGTGCATGCTCAATGGTGATGGTGATGATGGGGCCC |
| ChHisT-1-F | ATCCGTTCAGCCGGGTTAGATTCCCGGGGTTTCCCCCACTGCAGATCCTTAGCGAAAGC |
| ChHisT-1-R | TTAGAGTCCATTCGATCTACATGATCAGGTTTCCCGAATTCAGCGTTACAAGTATTACAC |
| ChHisT-2-F | ATCCGTTCAGCCGGGTTAGATTCCCGGGGTTTACCCCACTGCAGATCCTTAGCGAAAGC |
| ChHisT-2-R | TTAGAGTCCATTCGATCTACATGATCAGGTTTACCGAATTCAGCGTTACAAGTATTACAC |
| ChHisT-3-F | ATCCGTTCAGCCGGGTTAGATTCCCGGGGTCCACCCCACTGCAGATCCTTAGCGAAAGC |
| ChHisT-3-R | TTAGAGTCCATTCGATCTACATGATCAGGTCCACCGAATTCAGCGTTACAAGTATTACAC |
| ChHisT-4-F | TTAGAGTCCGTTCGATCTACATGATCATAGCCACCGAATTCAGCGTTACAAGTATTACAC |
| ChHisT-4-R | ATCCGTTCAGCCGGGTTAGATTCCCGGTAGCCACCCCACTGCAGATCCTTAGCGAAAGC |
| ChHisT-5-F | GAATTCAGCGTTACAAGTATTACACA |
| ChHisT-5-R | GTGGCTATGATCATGTAGATCGAACGG |
| ChHisT-6-F | AAGAGTCCGTTCGATCTACATG |
| ChHisT-6-R | AATCCGTTCAGCCGGGTTAGATTC |
Example 2: construction of chimeric histidyl-tRNA synthetases
The chimeric histidyl-tRNA synthetase is composed of two parts, one is the tRNA binding domain of the pyrrollysyl-tRNA synthetase and the other is the catalytic domain of the histidyl-tRNA synthetase, which are designed as shown in FIGS. 2 and 3C.
The construction of chimeric histidyl-tRNA synthetases involves the selection of the tRNA binding domain of the pyrrolysinyl-tRNA synthetase, the selection of the catalytic domain of the histidyl-tRNA synthetase, and the selection of the manner in which the two are fused.
(1) Analysis and selection of the tRNA binding Domain of Pyrrolysyl-tRNA synthetase
In the present invention, it is necessary to clone the tRNA binding domain of the pyrrollysyl-tRNA synthetase into the pBK vector, and the tRNA binding domain can be divided into two subdomains, wherein the N-terminal 1-149 recognizes the variable region and T-loop of the corresponding tRNA, and the D-stem region of the corresponding tRNA is recognized by 185-240, and the mutation of V31I, T56P, H62Y and A100E (IPYE for short) can improve the activity of the system. Therefore, we selected four tRNA binding domain portions, N149+185-240(N240), N149-IPYE, N149+185-240-IPYE, and designed primers to construct into pBK vectors (FIG. 4A).
(2) Selection of catalytic domains of histidyl-tRNA synthetases
The invention then analyzes the structure of histidyl-tRNA synthetases (FIGS. 3F, 4A, and 10), selecting the N-terminal 326 amino acids (1-326) as part of the catalytic domain of the chimeric design.
(3) Construction of chimeric histidyl-tRNA synthetases with different fusion modes
Primers were designed to amplify histidyl-tRNA synthetases 1-326, linked to the N-or C-terminus of the pyrrollysyl-tRNA synthetase tRNA binding domain, to construct chHis-1, 2, 3, 4 and 5, and cloned into a pBK vector. The corresponding primers are shown in Table 2-1.
TABLE 2-1 construction of chimeric histidyl-tRNA synthetase primer List
| Name | Sequence |
| Pyl-N149-F | TACGCTTTGAGGAATCCCATATGGATAAGAAGCCGCTGGATG |
| Pyl-N149-R | GTCAGAGCTGGGGCGCTTGCAGAGGTCGGAACCGGAGAGTTC |
| Pyl-N205-R | GCCGCCGCTTCCGCCACCCTCGCGTTCCTCAGC |
| ChHisRS-1-F | GTGGCGGAAGCGGCGGCATGGCAAAAAACATTCAAGC |
| ChHisRS-1-R | TAGCGTTTGAAACTGCAGTTACGGATTAACGGCCTGTACTAACAATACAAGACGTTC |
| ChHisRS-3-F | CGCTTTGAGGAATCCCATATGGCAAAAAACATTCAAGCC |
| ChHisRS-3-R | TTCCGCCGCCGCTTCCGCCACCTATATCGACAACAGGATCGGCT |
Example 3: optimization of chimeric histidyl-tRNA synthetase linker peptides
In the construction of chimeric proteins, the linker peptide between the two chimeric fragments was critical to the activity of the chimeric molecule, and in the present invention we tested the length of the linker peptide and the type of linker peptide (FIG. 11).
(1) Optimization of linker peptide length
The length (GS-type) of the flexible linker peptides, 0, 6, 12, 18 and 24 amino acids, were designed and named chHisRS4-2, -3, -4, -5 and-6, respectively, and the nucleotide sequences of the corresponding linker peptides are shown in Table 3-1. Meanwhile, 15 amino acids at the N end of the histidine-tRNA synthetase are deleted on the basis of 0 amino acid connecting peptide, the name is chHisRS4-1, a designed primer is constructed on a carrier through a Q5 site directed mutagenesis kit (NEB), and the corresponding primer is shown in a table 3-2.
(2) Construction of chimeric histidyl-tRNA synthetases of different linker peptide species
In addition to the flexible linker peptide (GS-type), proline rich linker peptide (APAPAPAPAPAPAPAPAPAP) and alpha helix type linker peptide (AEAAAAAAKA) were selected in the present invention, and the corresponding nucleotide sequences are shown in Table 3-1. Primers were designed and constructed on the vector by Q5 site directed mutagenesis kit (NEB), and the corresponding primers are shown in Table 3-2.
TABLE 3-1 nucleotide sequences of different linker peptides
TABLE 3-2 primers used for linker peptide construction
| Name | Sequence |
| 12aa-GS-linker-F | TCTGGTGGCATGGCAAAAAACATTCAAGCC |
| 18aa-GS-linker-F | TCTGGTGGCGGTGGCGGAAGCGGCGGTATGGCAAAAAACATTCAAGCC |
| 24aa-GS-linker-R | ACCGCCACCAGACCCACCGCCGCCGCCGCTTCCGCCACCCTCGCGTTCCTCAG |
| 24aa-GS-linker-F | GGCGGAAGCGGCGGTGGCGGTGGCAGCGGGGGTATGGCAAAAAACATTCAAGCC |
| P-rich-linker-R | GAGCAGGGGCCGGCGCTGGCGCGGGGGCCTCGCGTTCCTCAGCGTAGAT |
| P-rich-linker-F | CAGCGCCTGCCCCGGCACCCGCCCCCATGGCAAAAAACATTCAAGCCA |
| α-helix-linker-F | GCTGCAAAGGAAGCTGCAGCGAAGGCTATGGCAAAAAACATTCAAGCCA |
| α-helix-linker-R | GGCCTCTTTTGCAGCCGCTTCGGCCTCGCGTTCCTCAGCGTAGA |
Example 4: optimization of control of chimeric histidyl-tRNA synthetase promoters
In E.coli expression systems, the promoter has a large influence on the enzyme activity. To obtain optimal amino acid insertion efficiency, glns promoter, pBAD promoter, oxb20 promoter, trp promoter were selected, and the nucleotide sequences of the promoters are shown in Table 4-1. The corresponding primers are shown in Table 4-2.
TABLE 4-1 nucleotide sequences of different promoters
TABLE 4-2 primers required for promoter construction
| Name | Sequence |
| pBAD-F | TTTGCTGAGTTGAAGGTTATGACAACTTGACGGCTACAT |
| pBAD-R | CCAGCGGCTTCTTATCCATGGTTAATTCCTCCTGTTAGC |
| Trp-F | GTGTGGAAGCGGTCGCTTTCATAAGGAGGTCGCAAATGGATAAGAAGCCGCTGGATG |
| Trp-R | ATTAAACTAGTTCGATGATTAATTGTCAACAGCCCGAGGATCCTTCAACTCAGCA |
| Oxb20-F | TCCCGCTTATAAAAGCTGTTGTGACCGCTTGCTCTAGCCAGC |
| Oxb20-R | TATAACGGCGGCCGGGTAATACCGGATAGTCAATATGTTCTG |
Example 5: evaluating the activity of the different types of chimeric histidyl-tRNA synthetases/tRNA constructed by utilizing GFP amber suppression efficiency in escherichia coli to obtain the optimal chimeric histidyl-tRNA synthetase/tRNA system
In this example, the chimeric histidyl-tRNA synthetase plasmid and chimeric histidine tRNA plasmid constructed in the above example were co-transformed into DH10B competent cells. The transformed cells were cultured in 2XYT medium at 37 ℃ for 1 hour with shaking, plated on LB agar plates containing 50. mu.g/ml kanamycin (kan) and 100. mu.g/ml Ampicillin (AMP), and cultured at 37 ℃ for 12 hours, while using as a negative control cells transformed with pNEG plasmid alone carrying GFP-190(TAG) and a chimeric tRNA. 3 spots were picked from each plate and cultured in 2XYT medium at 37 ℃ with shaking until OD600 became 0.8, arabinose was added to a final concentration of 0.2%, and protein expression was induced at 22 ℃ for 14 hours. After expression was complete, 1ml of the cell culture was centrifuged, the medium removed and lysed with 150. mu.l of BugBuster protein extraction reagent (Millipore). After the cleavage was completed, the cells were centrifuged at 12000rpm for 1min, and 100. mu.l of the supernatant was applied to a 96-well plate (COSTAR). The GFP signal of the supernatant was recorded and normalized using Bio Tek Synergy NEO 2. The measured data were statistically processed to find the average and error. Through the above tests, we have concluded the following:
(1) chimeric histidine tRNA requires an intact transplantation of histidine tRNA acceptor arms (fig. 3);
(2) the tRNA binding domain of the pyrrolysinyl-tRNA synthetase is better at the N-terminus than at the C-terminus of the chimeric protein (FIGS. 4A and B);
(3) the more complete the tRNA binding domain of the pyrrollysyl-tRNA synthetase, the higher the activity of the chimeric molecule, and the introduction of the iphe mutation can significantly improve the activity of the chimeric molecule (fig. 4A and B);
(4) the kind of linker peptide does not greatly affect the activity, and 18 amino acid linker peptides are preferred (FIGS. 11B and C);
(5) oxb20 promoter is best used in E.coli expression systems (FIG. 4E).
Example 6: confirmation of insertion of histidine into specific site of GFP protein
We obtained chimeric histidyl-tRNA synthetase/tRNA pairs and also required verification of the accuracy of their amino acid introduction, and required purification of the protein, confirmed by LC-MS and LC-MS/MS.
In this example, for the expression and purification of proteins, DH10B cells cultured overnight were inoculated into 100ml of fresh LB medium at an inoculum size of 1:100 and supplemented with the required antibiotics, and then grown until OD600 reached 0.8. L-arabinose was added to the final concentration of 0.2%, to induce expression of GFP (22 ℃, 220rpm, 14 h). The cells after induction were centrifuged at 4000rpm at 4 ℃ for 5 minutes, and the resulting cell pellet was resuspended in precooled NTA-0 buffer (25mM Tris, 250mM NaCl, pH 8.0) and sonicated. The lysate was centrifuged at 12000rpm at 4 ℃ for 60 minutes, and the resulting supernatant was subjected to nickel affinity chromatography chelate chromatography equilibrated in advance with NTA-0 buffer, followed by washing with 6 volumes of NTA-0 buffer containing 50mM imidazole. Finally the protein was eluted with NTA-0 buffer supplemented with 500mM imidazole. The purified proteins were analyzed by SDS-PAGE and LC-MS.
For LC-MS analysis, the purified proteins were analyzed on an LCQ Deca XP MAX mass spectrometer (Thermo Fisher Science) equipped with an electrospray ionization (ESI) source and agilent 1200 HPLC. The separation and desalting were carried out on an Agilent 300SB-C18 column (300X 2.1, 150mm, 5 μm). Mobile phase a was set to 0.1% formic acid in water and mobile phase B0.1% formic acid in acetonitrile and set at a flow rate of 0.200 ml/min. The data was analyzed using XCalbur-Quar browser software. In the UniDec software (version 2.6.8, Oxford university), mass spectrum deconvolution was performed using the core Bayesian deconvolution algorithm. The theoretical molecular weight of the protein was predicted using the ExPASY computer pI/Mw tool (https:// web. ExPASy. org/computer _ pI /).
In the LC-MS/MS analysis, protein bands were excised from the gel and digested with trypsin overnight. The digest was loaded onto a Q exact Orbitrap (Thermo Fisher) mass spectrometer incorporating Proxeon Easy-nLC II HPLC (Thermo Fisher Science) and Proxeon nanospray sources. MS/MS spectra were searched using the MASCOT engine (Matrix Science, London, UK; version 2.2) and further processed with pLabel software (version 2.4, University of Florida Herbarium). The results of the treatment showed that histidine was inserted into the 190 position of GFP, as shown in FIG. 4D and FIG. 9.
Example 7: molecular evolution of chimeric serine tRNAs
The chimeric seryl-tRNA synthetase/tRNA system constructed by the above example is less active, and the acceptor arm region of the chimeric serine tRNA is then modified, specifically as follows:
s25: constructing a chimeric tRNA library and cloning the library into a pNEG vector;
s26: preparing a DH10B competent cell containing the chimeric aminoacyl-tRNA synthetase plasmid;
s27: electrotransformation of the library into prepared competent cells and plating of plates containing arabinose;
s28: screening clones with fluorescence;
s29: confirming the orthogonality of the chimeric tRNA;
s30: sequencing the chimeric tRNA;
more specifically, for establishing the chSerT library, G2: C71, U3: A70, G4: C69, A5: U68, G6: C67 and G7: C666 base pairs are selected, primers chSerT-lib-F and chSerT-lib-R PCR random library fragments are designed, primers chSerT-v-F and chSerT-v-R are simultaneously designed to amplify pNEG vectors, and the vectors and the fragments are assembled by Gibson after being recovered by a gel recovery kit. The assembled library plasmids were transformed into DH10B competent cells containing pBK-oxb20-chSerRS by electroporation. The transformed cells were added to 0.9ml of SOC medium and thawed at 37 ℃ for 1 hour, and then plated on LB agar plates containing 50. mu.g/ml kanamycin, 100. mu.g/ml ampicillin and 0.2% L-arabinose. After 12h incubation at 37 ℃ followed by 48h incubation at 22 ℃ clones with fluorescence were picked from the plates using Azure bio.c400 on Cy2 channel and plated in 96 well plates. The cells were incubated at 37 ℃ and 220rpm for 10 hours and then induced with 0.2% arabinose for 22 hours. OD600 and GFP fluorescence (λ ex-490/10 nm, λ em-510/10 nm) were recorded with Bio Tek Synergy NEO 2. The cells with the highest GFP/OD600 ratio were selected and inoculated into 2XYT medium containing 100. mu.g/ml ampicillin, and plasmids were extracted using a plasmid minikit. The extracted DNA was digested with BglII restriction enzyme, and the pBK-oxb20-chSerRS plasmid was removed and transformed into E.coli DH10B competent cells. The pNEG plasmid containing the chSerT variant was extracted and sequenced. A comparison of the tRNA screened and the corresponding activities is shown in FIG. 7. Specific primers are shown in the following table.
TABLE 7-1 construction of primer List for chimeric serine tRNA library
| Name | Sequence |
| chSerT-lib-F | TTCGCTAAGGATCTGCAGTGGCGGNNNNNCCGGGAATCTAACCCG |
| chSerT-lib-R | ATACTTGTAACGCTGAATTCGGNNNNNTGATCATGTAGATCGAAC |
| chSerT-v-F | GAATTCAGCGTTACAAGTATTACACAAAGTTTTTTATG |
| chSerT-v-R | CCACTGCAGATCCTTAGCGAAAGCTAAGGATTTTTTTTAAG |
Example 8: assay for amber suppression efficiency of chimeric histidyl-tRNA synthetase/tRNA systems in mammalian cells
(1) Construction of chimeric histidyl-tRNA synthetase/tRNA mammalian expression vectors
Primers were designed to amplify chimeric histidyl-tRNA synthetase and chimeric histidine tRNA, cloned into pcdna3.1 vector, and maps of the empty vectors under the control of CMV and U6 promoters, respectively, are shown in fig. 1. The chimeric histidine tRNA retained CCA and not both of the acceptor arms, respectively. The primers are shown in Table 8-1.
(2) Chimeric system transfection of HEK 293T cells
The plasmid constructed above and pEGFP-EGFP190TAG-His are mixed by PEI reagent6Were co-transfected into HEK 293T cells in a ratio of 1:1 (G: G).
(3) Cell fluorescence and WB analysis transfection and inhibition efficiency
48h after transfection, fluorescence imaging analysis was performed using GE DV Elite Applied Precision DeltaVision system followed by Western blot analysis. The efficiency of CCA-terminated chimeric histidine tRNA was significantly higher than none (fig. 5 and 13).
The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by using the contents of the present specification and the accompanying drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Sequence listing
<110> Zhejiang university
<120> construction of an orthogonal aminoacyl-tRNA synthetase/tRNA System Using chimeric design approach
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atgggtaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60
gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga 120
aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt 180
gtcactactt tctcttatgg tgttcaatgc ttttcccgtt atccggatca catgaaacgg 240
catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaacgcac tatatctttc 300
aaagatgacg ggaactacaa gacgcgtgct gaagtcaagt ttgaaggtga tacccttgtt 360
aatcgtatcg agttaaaagg tattgatttt aaagaagatg gaaacattct cggacacaaa 420
ctcgagtaca actataactc acacaacgta tacatcacgg cagacaaaca aaagaatgga 480
atcaaagcta acttcaaaat tcgccacaac attgaagatg gatccgttca actagcagac 540
cattatcaac aaaatactcc aattggctag ggccctgtcc ttttaccaga caaccattac 600
ctgtcgacac aatctgccct ttcgaaagat cccaacgaaa agcgtgacca catggtcctt 660
cttgagtttg taactgctgc tgggattaca catggcatgg atgaactcta caaagggccc 720
catcatcacc atcaccattg 740
<210> 2
<211> 615
<212> DNA
<213> Artificial sequence (synthetic sequence)
<400> 2
atggataaga agccgctgga tgttctgatc tctgcgaccg gtctgtggat gtcccgtacc 60
ggcacgctgc acaagatcaa gcactatgag atttctcgtt ctaaaatcta catcgaaatg 120
gcgtgtggtg accatctggt tgtgaacaac tctcgttctt gtcgtcccgc acgtgcattc 180
cgttatcata aataccgtaa aacctgcaaa cgttgtcgtg tttctgacga agatatcaac 240
aacttcctga cccgttctac cgaaggcaaa acctctgtta aagttaaagt tgtttctgag 300
ccgaaagtga aaaaagcgat gccgaaatct gtttctcgtg cgccgaaacc gctggaaaat 360
ccggtttctg cgaaagcgtc taccgacacc tctcgttctg ttccgtctcc ggcgaaatct 420
accccgaact ctccggttcc gacctctgca agcgccccag ctctgactaa atcccagacg 480
gaccgtctgg aggtgctgct gaacccaaag gatgaaatct ctctgaacag cggcaagcct 540
ttccgtgagc tggaaagcga gctgctgtct cgtcgtaaaa aggatctgca acagatctac 600
gctgaggaac gcgag 615
<210> 3
<211> 1635
<212> DNA
<213> Escherichia coli (E
<400> 3
atggataaga agccgctgga tgttctgatc tctgcgaccg gtctgtggat gtcccgtacc 60
ggcacgctgc acaagatcaa gcactatgag atttctcgtt ctaaaatcta catcgaaatg 120
gcgtgtggtg accatctggt tgtgaacaac tctcgttctt gtcgtcccgc acgtgcattc 180
cgttatcata aataccgtaa aacctgcaaa cgttgtcgtg tttctgacga agatatcaac 240
aacttcctga cccgttctac cgaaggcaaa acctctgtta aagttaaagt tgtttctgag 300
ccgaaagtga aaaaagcgat gccgaaatct gtttctcgtg cgccgaaacc gctggaaaat 360
ccggtttctg cgaaagcgtc taccgacacc tctcgttctg ttccgtctcc ggcgaaatct 420
accccgaact ctccggttcc gacctctgca agcgccccag ctctgactaa atcccagacg 480
gaccgtctgg aggtgctgct gaacccaaag gatgaaatct ctctgaacag cggcaagcct 540
ttccgtgagc tggaaagcga gctgctgtct cgtcgtaaaa aggatctgca acagatctac 600
gctgaggaac gcgagggtgg cggaagcggc ggcggcggtg ggtctggtgg cggtggcgga 660
agcggcggta tggcaaaaaa cattcaagcc attcgcggca tgaacgatta cctgcctggc 720
gaaacggcca tctggcagcg cattgaaggc acactgaaaa acgtgctcgg cagctacggt 780
tacagtgaaa tccgcttgcc gattgtagag cagaccccgc tattcaaacg tgcgattggt 840
gaagtcaccg acgtggttga aaaagagatg tacacctttg aggatcgcaa tggcgacagc 900
ctgactctgc gccctgaagg gacggcgggc tgtgtacgcg ccggcatcga gcatggtctt 960
ctgtacaatc aggaacagcg tctgtggtat atcgggccga tgttccgtca cgagcgtccg 1020
cagaaagggc gttatcgtca gttccatcag ttgggctgcg aagttttcgg tctgcaaggt 1080
ccggatatcg acgctgaact gattatgctc accgcccgct ggtggcgcgc gctgggtatt 1140
tccgaacacg taactcttga gctgaattct atcggttcgc tggaagcacg cgccaattac 1200
cgcgatgcgc tggtggcatt ccttgagcag cataaagaaa agctggacga agactgcaaa 1260
cgccgcatgt acactaaccc gctgcgcgtg ctggattcaa aaaatccgga agtgcaggcg 1320
cttctcaacg acgctccggc attaggtgat tatctggacg aggaatctcg tgagcatttt 1380
gccggtctgt gcaaactgct tgagagcgcg gggatcgctt acaccgtaaa ccagcgtctg 1440
gtgcgtggtc tggattacta taaccgtacc gttttcgagt gggtgactaa cagtctcggc 1500
tcccagggca ccgtgtgtgc aggcggtcgt tatgacggtc ttgtggaaca actgggcggt 1560
cgtgcaacac cggctgtcgg ttttgcgatg ggcctcgaac gtcttgtatt gttagtacag 1620
gccgttaatc cgtaa 1635
<210> 4
<211> 1665
<212> DNA
<213> human (H.sapiens)
<400> 4
atggataaga agccgctgga tgttctgatc tctgcgaccg gtctgtggat gtcccgtacc 60
ggcacgctgc acaagatcaa gcactatgag atttctcgtt ctaaaatcta catcgaaatg 120
gcgtgtggtg accatctggt tgtgaacaac tctcgttctt gtcgtcccgc acgtgcattc 180
cgttatcata aataccgtaa atgcaaacgt tgtcgtgttt ctgacgaaga tatcaacaac 240
ttcctgaccc gttctaccga aggcaaaacc tctgttaaag ttaaagttgt ttctgagccg 300
aaagtgaaaa aagcgatgcc gaaatctgtt tctcgtgcgc cgaaaccgct ggaaaatccg 360
gtttctgcga aagcgtctac cgacacctct cgttctgttc cgtctccggc gaaatctacc 420
ccgaactctc cggttccgac ctctgcaagc gccccagctc tgactaaatc ccagacggac 480
cgtctggagg tgctgctgaa cccaaaggat gaaatctctc tgaacagcgg caagcctttc 540
cgtgagctgg aaagcgagct gctgtctcgt cgtaaaaagg atctgcaaca gatctacgct 600
gaggaacgcg agggtggcgg aagcggcggc ggaagcggtg gcggaagtgg tggcggaagc 660
ggcggcggaa gccaggcctg gggatcgagg cctcctgcag cagagtgtgc cacccaaaga 720
gctccaggca gtgtggtgga gctgctgggc aaatcctacc ctcaggacga ccacagcaac 780
ctcacccgga aggtcctcac cagagttggc aggaacctgc acaaccagca gcatcaccct 840
ctgtggctga tcaaggagag ggtgaaggag cacttctaca agcagtatgt gggccgcttt 900
gggaccccgt tgttctcggt ctacgacaac ctttctccag tggtcacgac ctggcagaac 960
tttgacagcc tgctcatccc agctgatcac cccagcagga agaaggggga caactattac 1020
ctgaatcgga ctcacatgct gagagcgcac acgtctgcac accagtggga cttgctgcac 1080
gcgggactgg atgccttcct ggtggtgggt gatgtctaca ggcgtgacca gatcgactcc 1140
cagcactacc ctattttcca ccagctggag gccgtgcggc tcttctccaa gcatgagtta 1200
tttgctggta taaaggatgg agaaagcctg cagctctttg aacaaagttc tcgctctgcg 1260
cataaacaag agacacacac catggaggcc gtgaagcttg tagagtttga tcttaagcaa 1320
acgcttacca ggctcatggc acatcttttt ggagatgagc tggagataag atgggtagac 1380
tgctacttcc cttttacaca tccttccttt gagatggaga tcaactttca tggagaatgg 1440
ctggaagttc ttggctgcgg ggtgatggaa caacaactgg tcaattcagc tggtgctcaa 1500
gaccgaatcg gctgggcttt tggcctagga ttagaaaggc tagccatgat cctctacgac 1560
atccctgata tccgtctctt ctggtgtgag gacgagcgct tcctgaagca gttctgtgta 1620
tccaacatta atcagaaggt gaagtttcag cctcttagca aataa 1665
<210> 5
<211> 1623
<212> DNA
<213> Escherichia coli (E
<400> 5
atggataaga agccgctgga tgttctgatc tctgcgaccg gtctgtggat gtcccgtacc 60
ggcacgctgc acaagatcaa gcactatgag atttctcgtt ctaaaatcta catcgaaatg 120
gcgtgtggtg accatctggt tgtgaacaac tctcgttctt gtcgtcccgc acgtgcattc 180
cgttatcata aataccgtaa aacctgcaaa cgttgtcgtg tttctgacga agatatcaac 240
aacttcctga cccgttctac cgaaggcaaa acctctgtta aagttaaagt tgtttctgag 300
ccgaaagtga aaaaagcgat gccgaaatct gtttctcgtg cgccgaaacc gctggaaaat 360
ccggtttctg cgaaagcgtc taccgacacc tctcgttctg ttccgtctcc ggcgaaatct 420
accccgaact ctccggttcc gacctctgca agcgccccag ctctgactaa atcccagacg 480
gaccgtctgg aggtgctgct gaacccaaag gatgaaatct ctctgaacag cggcaagcct 540
ttccgtgagc tggaaagcga gctgctgtct cgtcgtaaaa aggatctgca acagatctac 600
gctgaggaac gcgagggtgg cggaagcggc ggcaccatcc ctaacctgcc tgcagatgaa 660
gtgccggtag gtaaagacga aaatgacaac gttgaagtca gccgctgggg taccccgcgt 720
gagtttgact ttgaagttcg tgaccacgtg acgctgggtg aaatgcactc tggcctcgac 780
tttgcagctg cagttaagct gactggttcc cgctttgtgg taatgaaagg gcagattgct 840
cgcatgcacc gcgcactgtc gcagtttatg ctggatctgc ataccgaaca gcatggctac 900
agtgagaact atgttccgta cctggttaac caggacacgc tgtacggtac gggtcaactg 960
ccgaaatttg ctggcgatct gttccatact cgtccgctgg aagaagaagc agacaccagt 1020
aactatgcgc tgatcccaac ggcagaagtt ccgctgacta acctggtgcg cggtgaaatc 1080
atcgatgaag atgatctgcc aattaagatg accgcccaca ccccatgctt ccgttctgaa 1140
gccggttcat atggtcgtga cacccgtggt ctgatccgta tgcaccagtt cgacaaagtt 1200
gaaatggtgc agatcgtgcg cccagaagac tcaatggcgg cgctggaaga gatgactggt 1260
catgcagaaa aagtcctgca gttgctgggc ctgccgtacc gtaaaatcat cctttgcact 1320
ggcgacatgg gctttggcgc ttgcaaaact tacgacctgg aagtatggat cccggcacag 1380
aacacctacc gtgagatctc tagctgctcc aacgtttggg atttccaggc acgtcgtatg 1440
caggcacgtt gccgcagcaa gtcggacaag aaaacccgtc tggttcatac cctgaacggt 1500
tctggtctgg ctgttggtcg tacgctggtt gcagtaatgg aaaactatca gcaggctgat 1560
ggtcgtattg aagtaccaga agttctgcgt ccgtatatga acggactgga atatattggc 1620
taa 1623
<210> 6
<211> 1671
<212> DNA
<213> Escherichia coli (E
<400> 6
atgagcaaga gcaccgctga gatccgtcag gcgtttctcg actttttcca tagtaaggga 60
catcaggtag ttgccagcag ctccctggta ccccataacg acccaacttt gttgtttacc 120
aacgccggga tgaaccagtt caaggatgtg ttccttgggc tcgacaagcg taattattcc 180
cgcgctacca cttcccaacg ctgcgtgcgt gcgggtggta aacacaacga cctggaaaac 240
gtcggttaca ccgcgcgtca ccataccttc ttcgaaatgc tgggcaactt cagcttcggc 300
gactatttca aacacgatgc cattcagttt gcatgggaac tgctgaccag cgaaaaatgg 360
tttgccctgc cgaaagagcg tctgtgggtt accgtctatg aaagcgacga cgaagcctac 420
gaaatctggg aaaaagaagt agggatcccg cgcgaacgta ttattcgcat cggcgataac 480
aaaggtgcgc catacgcatc tgacaacttc tggcagatgg gtgacactgg tccgtgcggc 540
ccgtgcaccg aaatcttcta cgatcacggc gaccacattt gggggggccc tccgggaagc 600
ccggaagaag acggcgaccg ctacattgag atctggaaca tcgtcttcat gcagttcaac 660
cgccaggccg atggcacgat ggaaccgctg ccgaagccgt ctgtagatac cctgatgggt 720
ctggagcgta ttgctgcggt gctgcaacac gttaactcta actatgacat cgacctgttc 780
cgcacgttga tccaggcggt agcgaaagtc actggcgcga ccgatctgag caataaatcg 840
ctgcgcgtaa tcgctgacca cattcgttct tgtgcgttcc tgatcgcgga tggcgtaatg 900
ccgtccaatg aaaaccgtgg ttatgtactg cgtcgtatca ttcgtcgcgc agtgcgtcac 960
ggtaatatgc tcggcgcgaa agaaaccttc ttctacaaac tggttggtcc gctgatcgac 1020
gttatgggct ctgcgggtga agacctgaaa cgccagcagg cgcaggttga gcaggtgctg 1080
aagactgaag aagagcagtt tgctcgtact ctggagcgcg gtctggcgtt gctggatgaa 1140
gagctggcag gtggcggaag cggcggcgga agcggtggcg gaagcggcgg cggaagcggt 1200
ggcggaagtg gtggcggaag catggataag aagccgctgg atgttctgat ctctgcgacc 1260
ggtctgtgga tgtcccgtac cggcacgctg cacaagatca agcactatga gatttctcgt 1320
tctaaaatct acatcgaaat ggcgtgtggt gaccatctgg ttgtgaacaa ctctcgttct 1380
tgtcgtcccg cacgtgcatt ccgttatcat aaataccgta aaacctgcaa acgttgtcgt 1440
gtttctgacg aagatatcaa caacttcctg acccgttcta ccgaaggcaa aacctctgtt 1500
aaagttaaag ttgtttctga gccgaaagtg aaaaaagcga tgccgaaatc tgtttctcgt 1560
gcgccgaaac cgctggaaaa tccggtttct gcgaaagcgt ctaccgacac ctctcgttct 1620
gttccgtctc cggcgaaatc taccccgaac tctccggttc cgacctctta a 1671
Claims (9)
1. An orthogonal aminoacyl-tRNA synthetase/tRNA system is constructed by a chimeric design method, which is characterized in that the broad-spectrum orthogonal orthogonality of a pyrrolysinyl-tRNA synthetase/tRNA pair and the substrate diversity of the traditional aminoacyl-tRNA synthetase are organically integrated through the idea of chimeric design to construct a broad-spectrum orthogonal chimeric aminoacyl-tRNA synthetase/tRNA system, the orthogonal chimera system is a chimeric histidyl-tRNA synthetase/tRNA pair, a chimeric phenylalanyl-tRNA synthetase/tRNA pair, a chimeric seryl-tRNA synthetase/tRNA pair or a chimeric alanyl-tRNA synthetase/tRNA pair, the gene sequence of the chimeric histidyl-tRNA synthetase is shown as SEQ ID No.3, the gene sequence of the chimeric phenylalanyl-tRNA synthetase is shown as SEQ ID No.4, the gene sequence of the chimeric seryl-tRNA synthetase is shown as SEQ ID No.5, and the gene sequence of the chimeric alanyl-tRNA synthetase is shown as SEQ ID No. 6.
2. The method of claim 1, wherein said orthogonal aminoacyl-tRNA synthetase/tRNA system chimerism is achieved by:
s1: constructing a chimeric aminoacyl-tRNA synthetase/tRNA vector;
s2: the activity of the chimeric aminoacyl-tRNA synthetase/tRNA was evaluated in escherichia coli using GFP amber suppression efficiency;
s3: confirmation of amino acid insertions, including GFP expression, LC-MS and LC-MS/MS identification;
s4: testing amber inhibition efficiency of the chimeric system in a mammalian cell expression system;
s5: the acceptor arm of the chimeric tRNA was selected and a library of mutations was constructed and screened.
3. The method of claim 2, wherein the chimeric aminoacyl-tRNA synthetase/tRNA/system of S1 is configured by a method comprising:
s6: constructing a chimeric tRNA vector;
s7: construction of chimeric aminoacyl-tRNA synthetase vectors.
4. The method of claim 3, wherein the chimeric tRNA vector construct of S6 comprises:
s8: designing and synthesizing chimeric tRNA;
s9: the synthesis of reporter gene GFP190TAG-His 6;
s10: the chimeric tRNA and GFP190TAG-His6 were ligated into pNEG vectors, respectively atglnsAndpBADunder the control of a promoter.
5. The method of claim 3, wherein the chimeric aminoacyl-tRNA synthetase/tRNA/system as S7 is constructed by the chimeric design method, wherein the construction of the chimeric aminoacyl-tRNA synthetase comprises:
s11: selecting a pyrro-lysyl-tRNA synthetase tRNA binding domain and a histidyl-tRNA synthetase catalytic domain, and respectively connecting the binding domain and the histidyl-tRNA synthetase catalytic domain to a pBK vector;
s12: synthesizing connecting peptides of different lengths and types and loading the connecting peptides between two parts of a tRNA binding domain of the pyrrollysyl-tRNA synthetase and a catalytic domain of the histidyl-tRNA synthetase of the chimeric aminoacyl-tRNA synthetase, connecting the peptide species: GS-rich, helix, P-rich;
s13: synthesis ofglnsA promoter,pBADA promoter,oxb20A promoter,trpA promoter and inserted into a pBK vector to control the expression of the chimeric aminoacyltRNA synthetase.
6. The method of claim 2, wherein the step of assessing the activity of the chimeric aminoacyl-tRNA synthetase/tRNA in e.coli using GFP amber suppression efficiency in S2 comprises:
s14: the pNEG vector carrying the GFP190TAG-His6 gene and the chimeric tRNA and the pBK vector carrying the chimeric aminoacyl-tRNA synthetase are co-transformed into DH10B competent cells;
s15: selecting a monoclonal antibody to be cloned in a liquid culture medium, and inducing gene expression by arabinose;
s17: lysing the expressed cells;
s18: the fluorescence of GFP in cell supernatants after lysis was measured and the GFP amber inhibition efficiency of the chimeric system was calculated.
7. The method of claim 2, wherein the amino acid insertion of S3 is confirmed by constructing an orthogonal aminoacyl-tRNA synthetase/tRNA system according to the chimeric design method, comprising:
s19: culturing Escherichia coli in S14 in large scale, and allowing arabinose to induce protein expression;
s20: collecting the expressed escherichia coli, carrying out ultrasonic disruption, and carrying out affinity chromatography purification;
s21: the expression level of full-length GFP is detected by Western-blotting, and the purified protein is identified by LC-MS and LC-MS/MS.
8. The method of claim 2, wherein the chimeric system of S4 is tested for efficiency in mammalian cells by constructing an orthogonal aminoacyl-tRNA synthetase/tRNA system according to the chimeric design method comprising:
s22: transferring the chimeric aminoacyl tRNA synthetase gene and the chimeric tNRA gene into a pcDNA3.1 vector;
s23: the vector obtained by S22 and pEGFP-EGFP-191TAG-Histag transfect HEK 293T cells together;
s24: GFP fluorescence and Western-blotting measure the efficiency of the chimeric system in mammalian cells.
9. The method of claim 2, wherein the library screening system for chimeric tRNA receptor arm regions of S5 comprises:
s25: constructing a chimeric tRNA library and cloning the library into a pNEG vector;
s26: preparing a DH10B competent cell containing the chimeric aminoacyl-tRNA synthetase plasmid;
s27: electrotransformation of the library into prepared competent cells and plating of plates containing arabinose;
s28: screening clones with fluorescence;
s29: confirming the orthogonality of the chimeric tRNA;
s30: sequencing the chimeric tRNA.
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| CN114134141B (en) * | 2021-11-03 | 2024-01-30 | 杭州嵌化合生医药科技有限公司 | Chimeric phenylalanine translation system with introduced unnatural amino acid and construction method thereof |
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| CN114657156A (en) * | 2022-03-11 | 2022-06-24 | 南京中医药大学 | Pyrrolysine aminoacyl-tRNA synthetase mutant and screening system and application thereof |
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