KR101582655B1 - Methionyl tRNA synthase mutants for photoactive methionine mimetics labelled protein biosynthesis - Google Patents

Abstract

The present invention relates to a methionyl tRNA synthase (MRS) mutant for biosynthesis of a photoactive methionine marker protein. More specifically, the present invention relates to a methionyl tRNA synthase (MRS) mutant having an alanine at position 12 from the N-terminal of the amino acid sequence of wild- The MRS mutant in which the tyrosine at the 260th position is substituted with phenylalanine, the isoleucine valine at the 297th position, and the histidine leucine at the 301st position is substituted with leucine serine at the i-th position, effectively biosynthes the target protein with pM By confirming, the MRS variant can be usefully used for biosynthesis of a target protein to which pM is labeled.

Description

{Methionyl tRNA synthase mutants for photoactive methionine mimetics labeled protein biosynthesis for biosynthesis of photoactive methionine mimetic proteins}

The present invention relates to a mutant of methionyl tRNA synthase (MRS) for introducing photomethionine (pM) into a target protein in vivo.

Covalent bond formation by photoactivity between biopolymers plays an important role in structure and function research by providing important information for many intermolecular bonds. This method can be used for protein-protein conjugation (Suchanek et al., Nat Methods, 2, 261-7, 2005; Chou et al., Chem Sci, 2, 480-83) (Pingoud et al., Mol Biosyst, 1, 135-41, 2005), and the like.

To introduce a photoactive group into a protein, a photoactive group such as diazirine, benzophonone, and azide group bonded to an N-hydroxysuccinimide group is reacted with a lysine of a protein, In this case, the hydrophilic amino acid may be changed into a hydrophobic state to selectively interfere with the binding between the molecule and the protein, and may cause non-specific binding. In order to overcome such disadvantages, Jung introduced a cysteine residue at a specific position of the protein and introduced a photoactive group into the protein by introducing cysteine into a substance that combines a photoactive group and a maleimide group (Jung et. al., Anal Chem, 81, 936-42, 2009).

Studies have also been conducted to introduce non-ionic amino acids into proteins in vivo protein biosynthetic steps (Davis and Chin, Nat Reviews Mol Cell Biol, 13, 168-182, 2012). Schulz's team at the Scripps Institute in the US conducted a study to introduce specific amino acid mimetics to specific protein positions using the amino acid tRNA _CUA synthase and tRNA _CUA pair, which recognizes the amber stop codon TAG Thus, various amino acid mimics have been successfully introduced at the protein biosynthetic stage (Xie and Schulz, Nat Reviews Mol Cell Biol, 7, 775-82, 2006). Professor Tirrell's team at CALTECH in the US produced a mutant of methionyl tRNA synthase (MRS) to study the introduction of non-ionic amino acids. In particular, mutations were induced at Leu13, Tyr260 and His301 positions known as the binding sites of methionine in MRS (Crepin et al., J Mol Biol, 332, 59-72, 2003), and azidonorleucine Of the dihydrofolate reductase (Tanrikulu et al., Proc Nat Acad Sci USA, 106, 15285-90, 2009).

Studies have also been carried out to introduce non-ionic amino acids having a photoactivity into proteins in vivo protein biosynthetic steps. Tippmann et al. Used the amino acid tRNA _CUA synthetase / tRNA _CUA system to synthesize 4 '- [3- (trifluoromethyl) -3H-diazin-3-yl] -1- phenylalanine (4' - [3- ) -3H-diazin-3-yl] -1-phenylalanine, TfmdPhe) was successfully introduced into Z-mail protein in E. coli (Tippmann et al., ChemBioChem, 8, 2210-14, 2007). In addition, pyrrolysyl (Pyl) tRNA _CUA synthetase / PyltRNA _CUA system was used to successfully introduce diazyl group-introduced lysine in E. coli and animal cells into the target protein, (Chou et al., Chem Sci, 2, 480-83). However, these residues have bulky or long carbon chains, which have disadvantages in elaborate covalent bonding with the target material. These problems are caused by the formation of photolucine (pL; L-2-amino-4,4-azi-pentanoic acid) or photomethionine (pM; L- azi-hexanoic acid). Suchanek et al. (Nat Methods, 2, 261-7, 2005) reported that biosynthesis of proteins by adding pM or pL photoactive amino acid mimic as an amino acid nutrient to medium lacking methionine or leucine when cultured animal cells, The photoactive amino acid mimetics were used for protein binding analysis. However, the inventors of the present invention found that protein expression was very low as a result of expressing the protein by adding pM to the minimal medium without methionine in E. coli B834, a methionine auxotroph E. coli strain.

Accordingly, the present inventors have made efforts to enhance the introduction of pM into the biosynthetic protein, and have succeeded in producing a mutant of E. coli methionyl tRNA synthetase (MRS) capable of promoting the expression of a target protein into which pM has been introduced in E. coli. In the MRS of the present invention, in addition to the position known as the binding site of the methionine residue of the existing E. coli MRS, substitution of glycine (Gly) and valine (Val) for Ala12 and Ile297 residues, respectively, The present invention has been completed.

It is an object of the present invention to provide a mutant of methionyl tRNA synthase (MRS) for introducing photomethionine (pM) into a target protein in vivo.

In order to achieve the above object,

In the amino acid sequence of methionyl tRNA synthase (MRS), alanine (Ala) at the 12th position from the N-terminus is substituted with glycine, leucine at the thirteenth position is serine, Wherein the tyrosine at the 260th position is substituted with phenylalanine, the isoleucine at the 297th position is substituted with valine, and the histidine at position 301 is substituted with leucine. To provide MRS variants for the introduction of photomethionine (pM).

The present invention also provides a method for producing MRS in which the alanine at the 12th position from the N-terminus is substituted with glycine, the leucine at the thirteenth position is serine, the tyrosine at the 260th position is substituted with phenylalanine, Wherein the isoleucine is substituted with valine and the histidine at position 301 is replaced with leucine, to prepare a MMS variant for introduction of pM into a target protein.

The present invention also provides a reagent composition for introducing pM to a target protein comprising an MRS variant.

In addition,

1) preparing an expression vector comprising an expression vector comprising a polynucleotide encoding the target protein and a polynucleotide encoding the MRS variant;

2) introducing the expression vector of step 1) simultaneously into a host cell to prepare a transformant; And

3) culturing the transformant of step 2) to express pM-labeled target protein,

Thereby providing a method for introducing pM into the target protein.

Since the methionyl tRNA synthetase (MRS) mutant of the present invention is a mutant capable of biosynthesizing a target protein into which photomethionine (pM) has been introduced more effectively than the wild-type MRS, pM-labeled proteins can be used as an important tool for analyzing other polymers that specifically bind to specific proteins in the future.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of the production of a pET-28at vector for cloning a photomethionine (pM) -labeled protein. 6xH represents a histidine tag, and tc represents a thrombin cleavage site.
FIG. 2 is a schematic diagram showing the preparation of pET-GFP / FIH plasmid for expressing GFP / FIH junction protein.
FIG. 3 is a diagram illustrating the production of a pBAD-MRSwt plasmid in which a methionyl tRNA synthase (MRS) (MRS 1-548) having a carboxy terminal partially cleaved is cloned into a pBAD / Myc-HisA vector.
FIG. 4 is a diagram illustrating the preparation of MRS variants through wild-type MRS. The black inverted triangle represents the variation of the 13, 260 and 301 sites known as the positions of the existing methionine residues and the gray inverted triangle represents the variation of the 12 and 297 regions of the present invention. The black triangle represents the stop codon. The wild-type amino acids corresponding to each position are shown below.
Figure 5 shows the increase in the expression of the pM-labeled 6xH-EGFP by the MRS variant (MRS5pm). Ara represents 0.02% L-arabinose, IPTG represents 1 mM IPTG, and M9a represents M9BV + CMS-MET. X represents the case where Escherichia coli was cultured in a medium containing no methionine nutrient, M represents Escherichia coli cultured in a medium containing methionine, and pM represents Escherichia coli cultured in a medium containing pM as a methionine nutrient . The expressed 6xH-EGFP protein is indicated by the red arrow.
Figure 6 is a diagram that predicts the binding structure of wild-type MRS or MRS5pm variant and methionine (Met) or pM. The red star of MRSwt-pM shows that there is a molecular collision when the pM approaches the binding site of the wild-type MRS in order to combine the wild-type MRS and pM, indicating that the binding is not easy.
Figure 7 shows the increase in the expression of the pM-labeled 6xH-FIH by the MRS mutant (MRS5pm). Ara represents 0.02% L-arabinose, IPTG represents 1 mM IPTG, and M9a represents M9BV + CMS-MET. M represents the case where Escherichia coli was cultured in a medium containing methionine, and pM represents the case where Escherichia coli was cultured in a medium containing pM as a methionine nutrient. The expressed 6xH-EGFP protein is indicated by the red arrow.
Figure 8 shows the purification of the pM-labeled 6xH-EGFP. 1 represents the supernatant obtained by ultrasonication and centrifugation of the cultured Escherichia coli, 2 represents a non-purified eluate which has not been bound to the chromatography by flow trough, 3 is washed with 30 mM histidine , And 4 and 5 represent purified 6xH-EGFP obtained by elution with 150 mM imidazole.
9 is a diagram showing the structure of the FIH dimer. The black circles represent moieties involved in dimer formation at the carboxy terminus, the green and red stars represent the three methionine residues present at the dimerization site, and the green circle represents the two closely related methionine residues Lt; / RTI >
Fig. 10 is a diagram showing the dimerization of FIH with or without UV irradiation at 365 nm. A represents SDS-PAGE stained with coomassie, and B represents a western blot for anti-6xH antibody.

Hereinafter, the present invention will be described in detail.

The present invention provides a mutant of methionyl tRNA synthase (MRS) for introducing photomethionine (pM) into a target protein.

The mutant MRS is a mutant in which alanine (Ala) at the 12th position from the N-terminal of the amino acid sequence of wild-type MRS is glycine, leucine at the thirteenth position is serine, tyrosine at the 260th position ) Is phenylalanine, isoleucine at position 297 is valine, and histidine at position 301 is substituted by leucine. However, the present invention is not limited thereto.

The wild-type E. coli MRS preferably comprises the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

The wild-type MRS is preferably prepared from the C-terminal deleted sequence, but is not limited thereto.

As the target protein, all the proteins as well as the enhanced green fluorescence protein (EGFP) and factor inhibiting hypoxia inducible factor (FIH) used in the present embodiment can be used.

In a specific example of the present invention, we prepared a vector for target protein cloning and used it as a target protein with 6x histidine (6xH) tagged with enhanced green fluorescence protein (EGFP), FIH factor inhibiting hypoxia inducible factor) and a gene for expressing EGFP-FIH (Fig. 1 and Fig. 2). In addition, MRS variants through point mutations were prepared by cloning the wild-type MRS gene from which the C-terminus was removed (see FIGS. 3 and 4).

Further, the present inventors transformed the above-described target protein expression vector into E. coli B834, and expressed the target protein of EGFP, FIH or GFP-FIH in a medium containing methionine or pM as a methionine nutrient, During the treatment, the target protein was expressed, but it was confirmed that the target protein was hardly expressed at the time of pM treatment.

In addition, the present inventors transfected E. coli B834 with the target protein expression vector and the MRS mutant expression vector prepared above, and expressed the target protein in a medium containing methionine or pM as a methionine nutrient source. As a result, wild-type MRS In the expression condition, target proteins of EGFP, FIH or GFP-FIH were expressed at the time of methionine treatment, but it was confirmed that the target protein was hardly expressed at the time of pM treatment (see FIGS. 5 and 7) MRS1pm and MRS3pm mutants that induced mutations at positions 260 and 301 did not show an increase in protein expression by pM as did wild-type MRS. However, when the MRS5pm mutant was expressed, it was confirmed that 6xH-EGFP protein was expressed even when pM was treated as in methionine treatment (see FIGS. 5 and 7). Thus, the present inventors have found that although binding of wild-type MRS to methionine is easy, due to the structure of pM having a diazyl group protruding compared to methionine, MRS having Ala12 and Ile297, like wild-type MRS, It is difficult to bind pM due to the lack of space available. On the other hand, the mutant MRS5pm mutant has substitution of glycine and valine for Ala12 and Ile297 residues to remove the methyl group, and it is confirmed that pM is easily accessible to the ligand binding site of MRS (See FIG. 6).

Further, in order to optimize the binding of pM, the effect of introducing pM into the target protein of EGFP, FIH or GFP-FIH is enhanced by substituting serine, phenylalanine and leucine for Leu13, Tyr260 and His301, respectively, (See Fig. 6).

In addition, the present inventors isolated the pM-labeled target protein by Ni-NTA-agarose chromatography to partially digest pM-labeled 6xH-target protein (see Fig. 8), and purified SDS-polyacrylamide gel and Western Blurring revealed that dimer of GFP-FIH target protein was formed through photoactive covalent bonding by irradiating UV at 365 nm (see FIG. 10).

Therefore, it was confirmed that the MRS mutant of the present invention can significantly biosynthesize the target protein with pM.

The present invention also provides a method for producing an MRS variant for introducing pM into a target protein produced through point mutation of wild-type MRS.

The MRS mutant has the amino acid sequence of alanine at the 12th position from the N-terminus and glycine at the 12th position, leucine at the 13th position to serine, tyrosine at the 260th position to phenylalanine, isoleucine at the 297th position to valine, Position histidine is substituted with leucine, but it is not limited thereto.

The MRS variants of the present invention can be produced by introducing a mutation into the wild-type MRS gene. Methods for introducing a desired mutation into a nucleotide sequence of a nucleic acid include point mutation, PCR using an oligonucleotide that causes degeneracy, a cell mutagenesis agent containing a nucleic acid, or any of those known to those skilled in the art Method.

The present invention also provides a reagent composition for introducing pM to a target protein comprising an MRS variant.

Since the reagent composition comprising the MRS variant of the present invention can efficiently biosynthesize the target protein into which pM has been introduced, the target protein having pM can be significantly biosynthesized using the above reagent composition.

Also, the present invention provides a method for producing an expression vector comprising the steps of: 1) preparing an expression vector comprising an expression vector comprising a polynucleotide encoding a target protein and a polynucleotide encoding the MRS variant;

2) introducing the expression vector of step 1) simultaneously into a host cell to prepare a transformant; And

3) culturing the transformant of step 2) to express pM-labeled target protein, and then introducing pM into the target protein.

The expression vector is preferably a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector, and more preferably, a plasmid vector is used, but the present invention is not limited thereto.

The host cell is preferably E. coli , prokaryote, yeast or eukaryotic cell, more preferably E. coli , but is not limited thereto.

The culture

1) transforming E. coli simultaneously with a MRS mutant expression plasmid and a target protein expression plasmid;

2) inducing the expression of MRS variant by adding L-arabinose to the transformant of step 1);

3) removing methionine from the culture medium of the transformant of step 2); And

4) expressing the pM-labeled protein in the minimal medium containing pM, but the present invention is not limited thereto.

The polynucleotide encoding the MRS variant of the present invention can be used in a manner that does not change the amino acid sequence of the antibody expressed from the coding region, taking into account the codon degeneracy of the codon or the codon preference in the organism to which the antibody is to be expressed It will be appreciated by those skilled in the art that various modifications may be made to the coding region and that various modifications or modifications may be made within the scope of not affecting the expression of the gene, You will understand. That is, as long as the polynucleotide of the present invention encodes a protein having equivalent activity, one or more nucleotide bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included in the scope of the present invention. The sequence of such polynucleotides may be short or double-stranded, and may be a DNA molecule or RNA (mRNA) molecule.

In preparing the expression vector, an expression control sequence such as a promoter, a terminator, an inhancer and the like, a sequence for membrane targeting or secretion, and the like may be used depending on the kind of the host cell for which the MRS variant is to be produced And can be variously combined according to the purpose.

The expression vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector. Suitable expression vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoter, operator, initiation codon, termination codon, polyadenylation signal and enhancer, and can be prepared variously according to the purpose. The promoter of the expression vector may be constitutive or inducible. The signal sequence includes a PhoA signal sequence, an OmpA signal sequence and the like when the host is Escherichia sp., A? -Amylase signal sequence when the host is Bacillus sp., A subtilisin signal, An interferon signal sequence, an antibody molecule signal sequence, and the like can be used in the case where the host is an animal cell. However, in the case where the host is an animal cell, an MFI signal sequence, an alpha-interferon signal sequence, But is not limited thereto. The expression vector may also comprise a selection marker for selecting a host cell containing the vector and, if replicable expression vector, a replication origin.

The MRS variant according to the present invention can be mass produced by transforming the expression vector according to the present invention into an appropriate host cell such as Escherichia coli or yeast cells and then culturing the transformed host cell. The host cell may be a prokaryote such as E. coli or Bacillus subtilis . It may also be eukaryotic cells derived from yeast, insect cells, plant cells, animal cells such as Saccharomyces cerevisiae . More preferably, the animal cells may be autologous or allogeneic animal cells. Appropriate culture methods and medium conditions depending on the type of host cell can be easily selected by those skilled in the art from known techniques known to those skilled in the art. Any method known to those skilled in the art may be used for introducing the expression vector into the host cell.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Example  1> target protein Cloning  Manufacture of vectors

Three methionine residues were removed between NdeI and BamHI of the pET-28a (+) vector (Novagen, USA) to produce a vector for target protein cloning.

Specifically, a forward primer (TET-Xba-F) (SEQ ID NO: 3: 5'-TTCCCCTCTAGAAATAATTTTGTTTAAC-3 ') introducing a 6x histidine tag (6xH) at the N-terminus using pET- And reverse primer (ET-Bam-R) (SEQ ID NO: 4: 5'-GCGCGGATCCGCGCGGCACCAGGCCGC-3 '). The amplified DNA was then cut with XbaI and BamHI to obtain XbaI / Inserted into the BamHI site, and named pET-28at.

As a result, as shown in FIG. 1, the pET-28at vector produced a vector capable of cleaving the 6xH region while retaining the thrombin cleavage site although the three methionine residues between NdeI and BamHI were removed. , Enhanced green fluorescence protein (EGFP) and EGFP-FIH (factor inhibiting hypoxia inducible factor) in the present invention (Fig. 1).

< Example  2> pET -28 at Lt; RTI ID = 0.0 &gt; Cloning

In order to express the target protein, the protein expression gene was cloned into the pET-28at vector prepared in Example 1 above.

Specifically, a forward primer (GFP-Bam-F) (SEQ ID NO: 5'-CGGGATCCATGGTGAGCAAGGGCGAG-3 ') and a reverse primer (SEQ ID NO: 5) were constructed using pEGFP-N3 (BD Biosciences Clontech, USA) Was amplified by PCR using primer (GFP-Xho-R) (SEQ ID NO: 6: 5'-CCGCTCGAGTTACTTGTACAGCTCGTC-3 ') and inserted into the BamHI / XhoI site of pET-28at vector and named pET-GFP.

FH reverse primer (SEQ ID NO: 7: 5'-CGCCGCTGTCCCGCCGAGAGTGATCCCG (SEQ ID NO: 7)) was prepared using pEGFP-N3 (BD Biosciences Clontech, USA) as a template and GFP-Bam-F forward primer and GFP / FIH- 3 ') was used to amplify the EGFP gene to be ligated with FIH. A GFP / FIH-F forward primer (SEQ ID NO: 3) was constructed by using pET-FIH (Lee et al., Biol Chem, 278, 7558-63, FIH gene to be ligated with EGFP was amplified using primers 8: 5'-ACTCTCGGCGGGACAGCGGCGGAGGCTG-3 'and FIH-Xho-R reverse primer (SEQ ID NO: 9: 5'-TTCCCTCGAGACCCCTGGCAGGCTAG-3'). The GFP-FIH junction gene was amplified by PCR using GFP-Bam-F and FIH-Xho-R primers by mixing two kinds of amplified DNAs and inserted into the BamHI and XhoI sites of the pET-28at vector. -GFP / FIH (Fig. 2).

< Example  3> Carboxy  Truncated MRS Cloning

To produce MRS (methionyl tRNA synthase) for the synthesis of pM (photometionine) labeled protein, a mutant of the wild-life MRS gene was cloned to prepare the mutant.

Specifically, the MRS-Nco-F forward primer (SEQ ID NO: 10: 5'-AATTCCATGGCTCAAGTCGCGAA-3 ') and the MRS-Kpn-R reverse primer (SEQ ID NO: 11) were prepared using E. coli BL21 genomic DNA extract as a template, : 5'-CAGCGGTACCTTATTCTTTAGAGGCTTCCACC-3 '), the E. coli MRS gene consisting of the nucleotide sequence of SEQ ID NO: 2 was amplified by PCR and inserted into the NcoI / KpnI site of the pBAD / Myc-HisA vector (Invitrogen, USA) This was named pBAD-MRSwt (Fig. 3).

In order to prepare mutant MRS from wild-type MRS, various point mutants were prepared by the point mutagenesis method using the pBAD-MRSwt vector as a template (Fig. 4).

Specifically, the primers used for the preparation of MRS variants are summarized in Table 1. The PCR product was purified using the primer pair containing the mutation production as shown in FIG. 4, and the PCR product was PCR amplified using the BAD-F and MRS-Kpn-R primers as templates. Thereafter, it was digested with NcoI / KpnI and inserted into NcoI / KpnI of pBAD / Myc-HisA vector to prepare pBAD-MRS1m. Other mutants were also prepared by using the same primer pairs as described above to prepare pBAD-MRS2m, pBAD-MRS3m, pBAD-MRS4m and pBAD-MRS5m (FIG.

The base sequence of the primers used for MRS and mutant cloning name Base sequence MRS-Nco-F (SEQ ID NO: 10) 5'-AATTCCATGGCTCAAGTCGCGAA-3 ' MRS-Kpn-R (SEQ ID NO: 11) 5'-CAGCGGTACCTTATTCTTTAGAGGCTTCCACC-3 ' BAD-F (SEQ ID NO: 12) 5'-ATGCCATAGCATTTTTATCCA-3 ' MRS-A12G-F (SEQ ID NO: 13) 5'-GACGTGCGGCCTGCCGTACGCTAACGGCTCAATCC-3 ' MRS-A12G-R (SEQ ID NO: 14) 5'-ACGGCAGGCCGCACGTCACCAGAATTTTCTT-3 ' MRS-L13G-F (SEQ ID NO: 15) 5'-GGGCCGTACGCTAACGGCTCAATC-3 ' MRS-L13G-R (SEQ ID NO: 16) 5'-GATTGAGCCGTTAGCGTACGGCCCTGCGCACGTCACCAG-3 ' MRS-AL / GS-F (SEQ ID NO: 17) 5'-GACGTGCGGCTCGCCGTACGCTAACGGCTCAATC-3 ' MRS-AL / GS-R (SEQ ID NO: 18) 5'-ACGGCGAGCCGCACGTCACCAGAATTTTCTTCGC-3 ' MRS-Y260F-F (SEQ ID NO: 19) 5'-ACCGATTGGCTTCATGGGTTCTTTCAAGAATCTGTGCGA-3 ' MRS-Y260F-R (SEQ ID NO: 20) 5'-AAGAACCCATGAAGCCAATCGGTGCGTCCAGC-3 ' MRS-I297V-F (SEQ ID NO: 21) 5'-GGTAAAGATGTTGTTTACTTCCTGAGCCTGTTCTGGCC-3 ' MRS-I297V-R (SEQ ID NO: 22) 5'-GGCTCAGGAAGTAAACAACATCTTTACCGATGAAGTGGTACAG-3 ' MRS-H301L-F (SEQ ID NO: 23) 5'-GATATTGTTTACTTCCTGAGCCTGTTCTGGCCTGC-3 ' MRS-H301L-R (SEQ ID NO: 24) 5'-CAGAACAGGCTCAGGAAGTAAACAATATCTTTACC-3 '

MRS-Nco-F and MRS-Kpn-R are primer sets for amplifying 1 to 548 sequences of MRS and introducing them into the pBAD / Myc-HisA vector.

MRS-A12G-F and MRS-A12G-R are primer sets for replacing 12th alanine with glycine.

● MRS-L13G-F and MRS-L13G-R are primer sets for replacing thirteenth leucine with glycine.

MRS-AL / GS-F and MRS-AL / GS-R are primer sets for glycine-serine substitution of the 12th and 13th alanine-glycine.

MRS-Y260F-F and MRS-Y260F-R are primer sets for replacing 260th tyrosine with phenylalanine.

MRS-I297V-F and MRS-I297V-R are primer sets for substituting 297th isoleucine with valine.

MRS-H301L-F and MRS-H301L-R are primer sets for replacing the 301st histidine with leucine.

< Example  4> Methionine dependent nutrition E. coli ( E. coli  B834) pM  Identification of marker protein expression

In order to confirm the expression of pM-labeled protein when no external MRS was expressed, the expression of pM-labeled EGFP, FIH or GFP / FIH protein was confirmed in E. coli B834, a methionine-dependent tympanic E. coli strain.

Specifically, pET-GFP, pET-FIH or pET-GFP / FIH vector prepared in Example 2 was transformed into E. coli B834 in order to express a target protein having a 6xH tag , Kanamycin-resistant colonies were selected and cultured in LB medium containing 30 / / ml of kanamycin for one day. Then, the cultured Escherichia coli was obtained by centrifugation at 2000 xg for 15 to 20 minutes, and _{twice with} M9B (48 mM Na ₂ HPO ₄ , 22 mM KH ₂ PO ₄ , 9 mM NaCl, and 19 mM NH ₄ Cl) after the cleaning was completed, M9BV + 1/2 CMS- MET solution (M9B + 0.4% _{glucose, 2 mM MgSO 4, 0.1 mM} CaCl 2, 0.05 mM MnCl 2, 0.1 M FeCl 3, 1 ㎎ / ㎖ thiamin (thiamine), 0.2 0.2 mg / ml folic acid, 0.2 mg / ml choline chloride and 0.02 mg / ml riboflavine + 375 占 퐂 / ml CSM-MET (methionine, 19 amino acid mixture excepting)) was diluted to 1/5 and dispersed. Dissolved E. coli was further cultured at 37 ° C for 2 to 3 hours, 375 μg / ml of CSM-MET and 50 μg / ml of methionine or pM were added, and the cells were treated with 1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) RTI ID = 0.0 &gt; C &lt; / RTI &gt; for 12-16 hours. The cells were then centrifuged, washed with PBS, and the water removed as much as possible with a micropipette, and the wet cells were diluted in distilled water to a concentration of 100 mg / ml. Then, 5 占 퐇 was mixed with an equal volume of 2xSDS buffer solution and heated at 100 占 폚 for 2 minutes to analyze protein expression by SDS-polyacrylamide gel electrophoresis.

As a result, it was confirmed that the target protein was expressed during methionine treatment, but the target protein was hardly expressed during pM treatment.

< Example  5> E. coli  From B834 MRS On by pM  Cover 6 xH -E GFP  Confirmation of protein expression.

6xF-EGFP protein having a molecular weight of about 28 KDa and containing 7 methionines in the molecule and the MRS mutant prepared in Example 3 were used to express pM-labeled 6xH-EGFP protein in E. coli B834 Respectively.

Specifically, pBAD-MRSwt, pBAD-MRS1m to pBAD-MRS5m and pET-GFP prepared in Example 3 were transformed into E. coli B834, colonies having resistance to both ampicillin and kanamycin were selected Ampicillin and kanamycin for one day. The cultured Escherichia coli was obtained by centrifugation at 2000 x g for 15 to 20 minutes, diluted 1/20 in LB medium containing ampicillin and kanamycin and dispersed. The dispersed Escherichia coli was further cultured for 1 hour, and 0.02% L-arabinose was treated at 37 캜 for 2 hours to express wild-type MRS or MRS mutant. Then, the E. coli was washed twice with M9B, diluted with 1/2 to 1/3 of the M9BV + 1/2 CMS-MET solution to express the target protein EGFP, Expression of the target protein was induced in the same manner. After the incubation, the wet cells obtained by centrifugation were diluted with water to a concentration of 100 mg / ml, and then 5 μl was analyzed for protein expression by SDS-polyacrylamide gel electrophoresis.

As a result, as shown in FIG. 5, the expression of the protein during methionine treatment was high in the condition that the wild-type MRS was expressed, but the expression of EGFP protein was hardly expressed in the pM treatment Respectively. In addition, it was confirmed that the MRS1pm and MRS3pm mutants that induced the mutations at the previously known positions 13, 260 and 301 did not show an increase in protein expression by pM as in the wild-type MRS. However, when the MRS5pm mutant was expressed, it was confirmed that the 6xH-EGFP protein was expressed even when pM was treated as in methionine treatment (FIG. 5). In addition, when 6xH-EGFP was not treated with methionine or pM, the protein was not expressed by IPTG, and pM was contained in 6xH-EGFP expressed during pM treatment (Fig. 5).

As shown in FIG. 6, although the binding between wild type MRS and methionine is easy, due to the structure of pM having a diazyl group protruding compared to methionine, MRS having Ala12 and Ile297, such as wild-type MRS or MRS1pm and MRS3pm mutants, It is difficult to bind pM because there is not enough space for pM to access the ligand binding site. On the other hand, the MRS5pm mutant was found to have an effect of facilitating pM access to the ligand binding site of MRS by replacing Ala12 and Ile297 residues with glycine and valine, respectively, in order to remove the methyl group (Fig. 6). In addition, in order to optimize the binding of pM, it was confirmed that the effect of introducing pM into target proteins such as 6xH-EGFP was improved by substituting serine, phenylalanine and leucine for serine, Leu13, Tyr260 and His301, (Fig. 6).

< Example  6> E. coli  From B834 MRS On by pM  Cover 6 xH - FIH  Protein expression.

The 6xH-FIH protein having pM labeled in E. coli B834 was expressed using 6xFIH having a molecular weight of about 45 KDa and containing 10 methionines in the molecule and the MRS variant prepared in Example 3 above.

Specifically, pBAD-MRSwt or pBAD-MRS1m to pBAD-MRS5m and pET-FIH were transformed into E. coli B834, and MRS and FIH proteins were expressed in the same manner as in Example 5 above. After the incubation, the wet cells obtained by centrifugation were diluted with water to a concentration of 100 mg / ml, and then 5 μl was analyzed for protein expression by SDS-polyacrylamide gel electrophoresis.

As a result, as shown in Fig. 7, when the MRS5pm mutant was expressed, 6xH-FIH protein was significantly expressed when pM was treated than when methionine was treated. In addition, when the MRS4pm mutant was expressed, the pM-labeled protein was expressed in 6xH-FIH, but the pM-labeled protein was less expressed in the xH-FIH compared to the wild type and MRS5pm mutant (FIG. 7). Since the MRS4pm mutant is a mutant in which only four amino acid residues are substituted except that Leu13 is replaced with serine in the 5 amino acid residues substituted in the MRS5pm mutant, the above result indicates that the Leu13 residue is substituted with serine in the expression of the pM labeled protein by the MRS mutant It is important that

< Example  7> pM  Cover 6 xH -E GFP  And 6 xH - FIH  Protein fractionation purification

To purify the expressed pM-labeled protein, the pM-labeled 6xH-target protein was partially purified by Ni-NTA-agarose chromatography.

Specifically, E. coli expressing the pM-labeled protein including the MRS 5pm mutant cultured in Example 5 or 6 was centrifuged to isolate pM-labeled 6xH-target protein, and cells were obtained . Subsequently, 1 ml of a cell destruction buffer (20 mM Tris-HCl, 300 mM NaCl, 2 mM 2-mercaptoethanol, 1 mM PMSF and 5% glycerol) 2 ml, followed by ultrasonic disruption. The disrupted solution was centrifuged at 15,000 rpm for 20 minutes to obtain a supernatant, which was partially purified by Ni-NTA-agarose chromatography.

As a result, it was confirmed that the 6xHis-tagged target protein was partially purified at a concentration of 150 mM imidazole in the step gradient Ni-NTA-agarose chromatography as shown in FIG. 8 (FIG. 8).

< Example  8> pM  Cover 6 xH -E GFP  And 6 xH - FIH  Photoactive covalent binding analysis of proteins

To confirm that the expressed target protein has the photoactivity of the labeled pM, the photoactive covalent bonding of the expressed target protein was analyzed.

Specifically, the pM labeled protein purified in Example 7 was irradiated with UV at a wavelength of 365 nm to confirm dimer formation between proteins.

As a result, it was confirmed that no dimer was formed between the EGFP proteins known to be present as a monomer. Further, as shown in FIG. 9, it is known that 6xH-FIH forms a dimer at the C-terminus, and there are three methionine residues at the dimer formation site, two residues of FIH methionine residue corresponding to each other And is known to show a very close structure (Fig. 9). However, unlike the IPTG induction conditions (Lee et al., Biol Chem, 278, 7558-63, 2003) in the LB medium, the FIH protein was found to be present as a precipitate upon UV irradiation under the induction conditions induced by the culture of the present invention and IPTG Respectively.

< Example  9> pM  Cover 6 xH - GFP / FIH  Partial separation purification of proteins and photoactive covalent binding analysis

In order to overcome the absence of photoactive covalent bonds in the protein of Example 9, GFP / FIH protein expressed by binding EGFP to the amino terminus of FIH was expressed in E. coli.

Specifically, pBAD-MRS5m and pET-GFP / FIH prepared in Example 2 were transformed into E. coli B834, and the MRS mutant and the GFP / FIH protein were reacted in the same manner as in Example 5, Lt; / RTI &gt; Subsequently, the 6xH-GFP / FIH protein labeled with pM was partially purified by Ni-NTA-agarose chromatography in the same manner as in Example 7, Dimer formation was confirmed on a SDS-polyacrylamide gel followed by Coomassie brilliant blue R-250 staining and westeren blotting analysis.

For Western blotting, the proteins were transferred to a nitrocellulose (NC) membrane after SDS-PAGE, and the protein bound to 6xH specific mouse monoclonal antibody (Aviva Systems biology, USA) and horse-radish peroxidase A mouse IgG goat antibody (Millipore, USA) was sequenced. Then, Amersham ECL Western Blotting Detection Reagent (GE Healthcare, USA) was processed and confirmed.

As a result, as shown in FIG. 10, a small amount of 6xH-GFP / FIH protein was isolated and purified (FIG. 10A). Since 6xH was present only in the artificially inserted protein GFP-FIH, the protein was not completely purified It is known that there are only a few bands reacting to the 6xH specific antibody at the KDa position, and that the 6xH-GFP / FIH protein labeled with pM upon UV irradiation at 386 nm forms a dimer, Blotting (Fig. 10B).

<110> Korea Research Institute of Bioscience and Biotechnology <120> Methionyl tRNA synthase mutants for photoactive methionine          mimetics labeled protein biosynthesis <130> 2013P-04-051 <160> 24 <170> Kopatentin 2.0 <210> 1 <211> 548 <212> PRT <213> Escherichia coli <400> 1 Thr Gln Val Ala Lys Lys Ile Leu Val Thr Cys Ala Leu Pro Tyr Ala   1 5 10 15 Asn Gly Ser Ile His Leu Gly His Met Leu Glu His Ile Gln Ala Asp              20 25 30 Val Trp Val Arg Tyr Gln Arg Met Met Arg Gly His Glu Val Asn Phe Ile          35 40 45 Cys Ala Asp Asp Ala His Gly Thr Pro Ile Met Leu Lys Ala Gln Gln      50 55 60 Leu Gly Ile Thr Pro Glu Gln Met Ile Gly Glu Met Ser Gln Glu His  65 70 75 80 Gln Thr Asp Phe Ala Gly Phe Asn Ile Ser Tyr Asp Asn Tyr His Ser                  85 90 95 Thr His Ser Glu Glu Asn Arg Gln Leu Ser Glu Leu Ile Tyr Ser Arg             100 105 110 Leu Lys Glu Asn Gly Phe Ile Lys Asn Arg Thr Ile Ser Gln Leu Tyr         115 120 125 Asp Pro Glu Lys Gly Met Phe Leu Pro Asp Arg Phe Val Lys Gly Thr     130 135 140 Cys Pro Lys Cys Lys Ser Pro Asp Gln Tyr Gly Asp Asn Cys Glu Val 145 150 155 160 Cys Gly Ala Thr Tyr Ser Pro Thr Glu Leu Ile Glu Pro Lys Ser Val                 165 170 175 Val Ser Gly Ala Thr Pro Val Met Arg Asp Ser Glu His Phe Phe Phe             180 185 190 Asp Leu Pro Ser Phe Ser Glu Met Leu Gln Ala Trp Thr Arg Ser Gly         195 200 205 Ala Leu Gln Glu Gln Val Ala Asn Lys Met Gln Glu Trp Phe Glu Ser     210 215 220 Gly Leu Gln Gln Trp Asp Ile Ser Arg Asp Ala Pro Tyr Phe Gly Phe 225 230 235 240 Glu Ile Pro Asn Ala Pro Gly Lys Tyr Phe Tyr Val Trp Leu Asp Ala                 245 250 255 Pro Ile Gly Tyr Met Gly Ser Phe Lys Asn Leu Cys Asp Lys Arg Gly             260 265 270 Asp Ser Val Ser Phe Asp Glu Tyr Trp Lys Lys Asp Ser Thr Ala Glu         275 280 285 Leu Tyr His Phe Ile Gly Lys Asp Ile Val Tyr Phe His Ser Leu Phe     290 295 300 Trp Pro Ala Met Leu Glu Gly Ser Asn Phe Arg Lys Pro Ser Asn Leu 305 310 315 320 Phe Val His Gly Tyr Val Thr Val Asn Gly Ala Lys Met Ser Ser Ser Ser                 325 330 335 Arg Gly Thr Phe Ile Lys Ala Ser Thr Trp Leu Asn His Phe Asp Ala             340 345 350 Asp Ser Leu Arg Tyr Tyr Tyr Thr Ala Lys Leu Ser Ser Arg Ile Asp         355 360 365 Asp Ile Asp Leu Asn Leu Glu Asp Phe Val Gln Arg Val Asn Ala Asp     370 375 380 Ile Val Asn Lys Val Val Asn Leu Ala Ser Arg Asn Ala Gly Phe Ile 385 390 395 400 Asn Lys Arg Phe Asp Gly Val Leu Ala Ser Glu Leu Ala Asp Pro Gln                 405 410 415 Leu Tyr Lys Thr Phe Thr Asp Ala Ala Glu Val Ile Gly Glu Ala Trp             420 425 430 Glu Ser Arg Glu Phe Gly Lys Ala Val Arg Glu Ile Met Ala Leu Ala         435 440 445 Asp Leu Ala Asn Arg Tyr Val Asp Glu Gln Ala Pro Trp Val Val Ala     450 455 460 Lys Gln Glu Gly Arg Asp Ala Asp Leu Gln Ala Ile Cys Ser Met Gly 465 470 475 480 Ile Asn Leu Phe Arg Val Leu Met Thr Tyr Leu Lys Pro Val Leu Pro                 485 490 495 Lys Leu Thr Glu Ala Gla Ala Phe Leu Asn Thr Glu Leu Thr Trp             500 505 510 Asp Gly Ile Gln Gln Pro Leu Leu Gly His Lys Val Asn Pro Phe Lys         515 520 525 Ala Leu Tyr Asn Arg Ile Asp Met Arg Gln Val Glu Ala Leu Val Glu     530 535 540 Ala Ser Lys Glu 545 <210> 2 <211> 1644 <212> DNA <213> Escherichia coli <400> 2 actcaagtcg cgaagaaaat tctggtgacg tgcgcactgc cgtacgctaa cggctcaatc 60 cacctcggcc atatgctgga gcacatccag gctgatgtct gggtccgtta ccagcgaatg 120 cgcggccacg aggtcaactt catctgcgcc gacgatgccc acggtacacc gatcatgctg 180 aaagctcagc agcttggtat caccccggag cagatgattg gcgaaatgag tcaggagcat 240 cagactgatt tcgcaggctt taacatcagc tatgacaact atcactcgac gcacagcgaa 300 gagaaccgcc agttgtcaga acttatctac tctcgcctga aagaaaacgg ttttattaaa 360 aaccgcacca tctctcagct gtacgatccg gaaaaaggca tgttcctgcc ggaccgtttt 420 gtgaaaggca cctgcccgaa atgtaaatcc ccggatcaat acggcgataa ctgcgaagtc 480 tgcggcgcga cctacagccc gactgaactg atcgagccga aatcggtggt ttctggcgct 540 acgccggtaa tgcgtgattc tgaacacttc ttctttgatc tgccctcttt cagcgaaatg 600 ttgcaggcat ggacccgcag cggtgcgttg caggagcagg tggcaaataa aatgcaggag 660 tggtttgaat ctggcctgca acagtgggat atctcccgcg acgcccctta cttcggtttt 720 gaaattccga acgcgccggg caaatatttc tacgtctggc tggacgcacc gattggctac 780 atgggttctt tcaagaatct gtgcgacaag cgcggcgaca gcgtaagctt cgatgaatac 840 tggaagaaag actccaccgc cgagctgtac cacttcatcg gtaaagatat tgtttacttc 900 cacagcctgt tctggcctgc catgctggaa ggcagcaact tccgcaagcc gtccaacctg 960 tttgttcatg gctatgtgac ggtgaacggc gcaaagatgt ccaagtctcg cggcaccttt 1020 attaaagcca gcacctggct gaatcatttt gacgcagaca gcctgcgtta ctactacact 1080 gcgaaactct cttcgcgcat tgatgatatc gatctcaacc tggaagattt cgttcagcgt 1140 gtgaatgccg atatcgttaa caaagtggtt aacctggcct cccgtaatgc gggctttatc 1200 aacaagcgtt ttgacggcgt gctggcaagc gaactggctg acccgcagtt gtacaaaacc 1260 ttcactgatg ccgctgaagt gattggtgaa gcgtgggaaa gccgtgaatt tggtaaagcc 1320 gtgcgcgaaa tcatggcgct ggctgatctg gctaaccgct atgtcgatga acaggctccg 1380 tgggtggtgg cgaaacagga aggccgcgat gccgacctgc aggcaatttg ctcaatgggc 1440 atcaacctgt tccgcgtgct gatgacttac ctgaagccgg tactgccgaa actgaccgag 1500 cgtgcagaag cattcctcaa tacggaactg acctgggatg gtatccagca accgctgctg 1560 ggccacaaag tgaatccgtt caaggcgctg tataaccgca tcgatatgag gcaggttgaa 1620 gcactggtgg aagcctctaa agaa 1644 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> TET-Xba-F <400> 3 ttcccctcta gaaataattt tgtttaac 28 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> ET-Bam-R <400> 4 gcgcggatcc gcgcggcacc aggccgc 27 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> GFP-Bam-F <400> 5 cgggatccat ggtgagcaag ggcgag 26 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GFP-Xho-R <400> 6 ccgctcgagt tacttgtaca gctcgtc 27 <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> GFP / FIH-R <400> 7 cgccgctgtc ccgccgagag tgatcccg 28 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> GFP / FIH-F <400> 8 actctcggcg ggacagcggc ggaggctg 28 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> FIH-Xho-R <400> 9 ttccctcgag acccctggca ggctag 26 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MRS-Nco-F <400> 10 aattccatgg ctcaagtcgc gaa 23 <210> 11 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> MRS-Kpn-R <400> 11 cagcggtacc ttattcttta gaggcttcca cc 32 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> BAD-F <400> 12 atgccatagc atttttatcc a 21 <210> 13 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> MRS-A12G-F <400> 13 gacgtgcggc ctgccgtacg ctaacggctc aatcc 35 <210> 14 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> MRS-A12G-R <400> 14 acggcaggcc gcacgtcacc agaattttct t 31 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> MRS-L13G-F <400> 15 gggccgtacg ctaacggctc aatc 24 <210> 16 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> MRS-L13G-R <400> 16 gattgagccg ttagcgtacg gccctgcgca cgtcaccag 39 <210> 17 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> MRS-AL / GS-F <400> 17 gacgtgcggc tcgccgtacg ctaacggctc aatc 34 <210> 18 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> MRS-AL / GS-R <400> 18 acggcgagcc gcacgtcacc agaattttct tcgc 34 <210> 19 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> MRS-Y260F-F <400> 19 accgattggc ttcatgggtt ctttcaagaa tctgtgcga 39 <210> 20 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> MRS-Y260F-R <400> 20 aagaacccat gaagccaatc ggtgcgtcca gc 32 <210> 21 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> MRS-I297V-F <400> 21 ggtaaagatg ttgtttactt cctgagcctg ttctggcc 38 <210> 22 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> MRS-I297V-R <400> 22 ggctcaggaa gtaaacaaca tctttaccga tgaagtggta cag 43 <210> 23 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> MRS-H301L-F <400> 23 gatattgttt acttcctgag cctgttctgg cctgc 35 <210> 24 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> MRS-H301L-R <400> 24 cagaacaggc tcaggaagta aacaatatct ttacc 35

Claims (9)

The alanine (Ala) at the 12th position from the N-terminal of the amino acid sequence of the methionyl tRNA synthase (MRS) consisting of the amino acid sequence of SEQ ID NO: 1 is replaced by glycine and the leucine at the thirteenth position Tyrosine at the 260th position is substituted with phenylalanine, isoleucine at the 297th position is valine, and histidine at the 301th position is substituted by leucine. MRS variants for the introduction of photomethionine (pM) into the target protein, consisting of the amino acid sequence of the gene.
delete delete The MRS consisting of the amino acid sequence of SEQ ID NO: 1 is converted to phenylalanine at tyrosine at the 260th position, alanine at the 12th position to glycine at the 13th position, serine at the 13th position, Preparing an amino acid sequence in which the isoleucine valine at position 297 and the histidine at position 301 are substituted with leucine.
delete A reagent composition for introducing pM to a target protein comprising the MRS variant of claim 1.
1) preparing an expression vector comprising a polynucleotide encoding a target protein and an expression vector comprising a polynucleotide encoding the MRS variant of claim 1;
2) introducing the expression vector of step 1) simultaneously into a host cell to prepare a transformant; And
3) A method for introducing pM into a target protein, comprising the step of culturing the transformant of step 2) to express a target protein having pM.
8. The method according to claim 7, wherein the vector of step 1) is a plasmid vector.
The method according to claim 7, wherein the host cell of step 2) is E. coli , prokaryote, yeast or eukaryotic cell.

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