Patent Application
20090155844
The present invention relates to a method of synthesizing a tRNA and a DNA construct therefor, particularly to a method of synthesizing a suppressor tRNA corresponding to a non-natural amino acid and a DNA construct therefor, as well as a method of producing a non-natural amino acid-incorporated protein using the above.
A non-natural amino acid-incorporated protein (hereinafter also referred to as “alloprotein”) in which an amino acid residue at a desired position in a protein is replaced with an amino acid other than 20 different amino acids involved in normal protein synthesis (a non-natural amino acid) could offer an effective means of analyzing the function and structure of a protein. Meanwhile, lysine derivatives include amino acids, such as acetyl-lysine, methyl-lysine etc., which are synthesized by post-translational modification. Such amino acids are well-known particularly as those involved in regulation of gene expression by histone and also known as those involved in regulation of transcriptional activation, regulation of protein-protein interaction, and suppression/promotion of ubiquitination for many types of proteins. It is expected that many findings concerning acetylation, methylation etc. of lysine would be available if those lysine derivatives could be introduced site-specifically into a eukaryote.
A pyrrolysyl tRNA synthetase (Py1RS) is a novel aminoacyl tRNA synthetase (aaRS) found from a methanogenic archaebacterium (Methanosarcina). A corresponding tRNA (pyrrolysine tRNA) is a suppressor tRNA, which has a unique secondary structure such as an unusually small D loop etc. Recently, it was found that in Escherichia coli, Py1RS and pyrrolysine tRNA do not interact with endogenous aaRS and tRNA (orthogonality), and pyrrolysine could be introduced amber codon-specifically into a protein (Non-Patent Document 1). Further, it has been reported that a wild-type Py1RS can bind a non-natural amino acid such as Nε-Boc-L-lysine to pyrrolysine tRNA in Escherichia coli (Non-Patent Document 1).
On the other hand, in a mammalian cell, an enzyme that phosphorylates a tyrosine residue in a protein (tyrosine kinase) plays an important role in transducing a signal such as growth stimulating factor from an extracellular region into a nucleus. The tyrosine kinases include one capable of phosphorylating a tyrosine derivative and one incapable of phosphorylating a tyrosine derivative. For example, it was shown that a Src kinase phosphorylates an iodotyrosine residue but an EGF receptor cannot do. Thus, it is useful in examining interaction of a desired protein with various tyrosine kinases in a cell if an alloprotein, the desired protein into which a tyrosine derivative is incorporated, could be synthesized in a mammalian cell. For example, it is important in analysis of signal transduction mechanism to examine which tyrosine kinase phosphorylates the desired protein. Further, these non-natural amino acid-incorporated proteins could be useful in themselves as material for analysis of the function and structure of a protein, and be a substance having a novel bioactivity
As an expression method of an alloprotein like the above in an animal cell, there has been developed a method of expressing in an animal cell (A) a mutant tyrosyl tRNA synthetase (hereinafter referred to as “mutant TyrRS”), which is a variant of a tyrosyl tRNA synthetase derived from Escherichia coli and has an increased specificity to a non-natural tyrosine derivative as compared with the specificity to a tyrosine, (B) a suppressor tRNA originating from eubacterium such as bacillus, mycoplasma, and staphylococcus capable of binding to the above tyrosine derivative in the presence of the above mutant tyrosyl tRNA synthetase, and (C) a desired protein gene subjected to nonsense mutation or frame shift mutation at a desired position; and incorporating the above tyrosine derivative into a position of nonsense mutation or frame shift mutation of the above protein (Patent Document 1 and Non-Patent Document 2).
Hereupon, it is required that the above suppressor tRNA originating from the non-eukaryote is transcribed by an RNA polymerase in a eukaryotic cell. In contrast to one kind of RNA polymerase in prokaryotic cells, it is known that in eukaryotic cells, three different kind of RNA polymerases I, II, and III (poll, polII, and polIII) act sharing the functions. Poll synthesizes ribosomal RNA, PolII synthesizes mRNA, and PolIII synthesizes 5SrRNA, tRNA, U6 small nuclear RNA (snRNA) etc. Therefore, tRNA in a eukaryotic cell is synthesized by transcription by RNA polymerase III. Genes transcribed by the RNA polymerase III are classified broadly into three groups according to characters of their promoter structures, the groups including, as their representative genes, a 5SrRNA gene (Type I promoter), a tRNA gene (Type II promoter), and a U6 small nuclear RNA gene (Type III promoter), respectively. The type II promoter, which transcribes a tRNA, is an internal promoter made up of two regions in a tRNA coding sequence, the consensus sequences of which are known as box A and box B. The consensus sequence of the box A consists of the positions 8-19: TRGCNNAGYNGG (SED ID NO:1), and the consensus sequence of the box B consists of the positions 52-62: GGTTCGANTCC (SED ID NO:2). Accordingly, for example, the suppressor tyrosine tRNA of Bacillus stearothermophilus, although it originates from a prokaryote, can be expressed in an animal cell without any alterations, because of the presence of the box A and box B in its suppressor tyrosine tRNA coding sequence (refer to Non-patent Document 3, for example).
Here, incorporation (taking-in) of an amino acid into the position of the nonsense mutation in the above protein is referred to as suppression. Because there are only three different types of stop codons, it is three types of non-natural amino acids at the maximum that can be incorporated into one type of protein. In vitro experiments have developed artificial base pairs in addition to naturally occurring base pairs (refer to Non-patent Documents 4 and 5), and an RNA containing artificial base pairs as mentioned above can be transcribed in vitro by using an RNA polymerase of T7 bacteriophage. It is expected that the following could be achieved: increase in the number of codon types, which are now 43, by using artificial base pairs in codons encoding amino acids, and introduction of a plurality of non-natural amino acids into one type of protein by getting the codons that do not encode natural amino acids to encode non-natural amino acids.
In the case of the above-mentioned non-eukaryote-originating suppressor tRNA being expressed in a eukaryotic cell, however, the problem is raised that, if the tRNA has sequences significantly different from the consensus sequences of the eukaryote in place of box A and box B, the sequences do not function as internal promoter, achieving only an extremely small amount of synthesis by transcription, or hardly causing transcription itself, in the eukaryotic cell. For example, the D loop of a pyrrolysine tRNA originating from methanogenic archaebacterium, which lacks several bases and is unusually small, does not function as internal promoter in a eukaryotic cell. Further, a suppressor tyrosine tRNA of Escherichia coli has the box B consensus sequence in its sequence but does not contain the box A consensus sequence. Introduction of the boxes A and B into those tRNAs results in loss of their functions as tRNA so that alloproteins with incorporated lysine derivative or tyrosine derivative cannot be synthesized even if a pyrrolysine tRNA or an E. coli's suppressor tyrosine tRNA with the boxes A and B being incorporated was used.
On the other hand, it is unknown whether or not those suppressor tRNAs having no internal promoter would function as tRNA in cases where the suppressor tRNAs are expressed using an external promoter in a eukaryotic cell. That is, although it is required that base modification and formation of 3-dimensional structure etc. after transcription are normally conducted in order that tRNA functions, it remains unknown what intracellular localization a tRNA transcribed by external promoter other than type II promoter would present, and whether it would undergo post-transcriptional modification or not, and further whether it would present biological functions or not.
After investigations and considerations, the inventors have found out that a pyrrolysine tRNA originating from methanogenic archaebacterium or a suppressor tyrosine tRNA of Escherichia coli can be efficiently expressed in an animal cell by binding to its 5′ end a promoter sequence of a eukaryotic tRNA nucleotide sequence or U1 and U6 snRNA gene(s). Further, it has been found out that the tRNA can be efficiently expressed by binding a bacteriophage-originating promoter sequence to the 5′ end of the tRNA gene and introducing the promoter together with a RNA polymerase capable of transcription into an animal cell. The present invention has been accomplished based on those findings.
In accordance with a first aspect of the present invention, there is provided a DNA construct comprising a non-eukaryote-originating suppressor tRNA gene containing no internal promoter sequence that functions in a eukaryotic cell, and a eukaryote-originating promoter linked to the 5′ end of the tRNA gene. It is preferred that the tRNA gene is a pyrrolysine tRNA gene originating from archaebacteria and/or a suppressor tyrosine tRNA gene originating from Escherichia coli, and the DNA construct further comprises a transcription terminator sequence linked to the 3′ end of said tRNA gene. In a further preferable exemplary embodiment, the eukaryote-originating promoter is a nucleotide sequence that induces transcription by RNA polymerase II or III. The nucleotide sequence that induces the transcription by the RNA polymerase II is preferably a promoter of a U1 snRNA gene, for example. Also, it is particularly preferred that the nucleotide sequence that induces the transcription by the RNA polymerase III is a promoter of a eukaryotic tRNA gene such as a human valine tRNA nucleotide sequence, or a promoter of a U6 snRNA gene, for example.
In accordance with a second aspect of the present invention, there is provided a method of synthesizing a suppressor tRNA comprising: causing the DNA construct to undergo transcription in a eukaryotic cell; and there is provided a recombinant eukaryotic cell that is transformed or transfected by the DNA construct; and a method of synthesizing an aminoacyl-tRNA comprising expressing a tRNA transcribed by the DNA construct, and an aminoacyl-tRNA synthetase corresponding to said tRNA.
In accordance with a third aspect of the present invention, there is provided a process for producing a non-natural amino acid incorporated-protein comprising: expressing, in the presence of the non-natural amino acid in a eukaryotic cell, (a) an aminoacy-tRNA synthetase for the non-natural amino acid, (b) a tRNA which is capable of binding to the non-natural amino acid in the presence of the aminoacyl-tRNA synthetase, and transcribed by the DNA construct, and (c) a desired protein that has a nonsense mutation or frame shift mutation at a desired position.
In accordance with a forth aspect of the present invention, there is provided a DNA construct comprising: a non-eukaryote-originating suppressor tRNA gene containing no internal promoter sequence that functions in a eukaryotic cell, and a promoter originating from bacteriophage linked to the 5′ end of the tRNA gene. It is preferred that the tRNA gene is a pyrrolysine tRNA gene originating from archaebacteria and/or a suppressor tyrosine tRNA gene originating from Escherichia coli, and the DNA construct further comprises a transcription terminator sequence linked to the 3′ end of the tRNA gene. In addition, preferably, the bacteriophage-originating promoter is, but not restricted to, a T7 promoter, T3 promoter, or SP6 promoter.
In accordance with a fifth aspect of the present invention, there are provided a method of synthesizing a suppressor tRNA comprising: causing a DNA construct to undergo transcription in a eukaryotic cell, the DNA construct comprising a non-eukaryote-originating suppressor tRNA gene containing no internal promoter sequence that functions in a eukaryotic cell, and a promoter originating from bacteriophage linked to the 5′ end of said tRNA gene; and a recombinant eukaryotic cell being transformed or transfected by the DNA construct and a gene expressing an RNA polymerase corresponding to the bacteriophage-originating promoter.
In accordance with a sixth aspect of the present invention, there is provided a process for producing a non-natural amino acid-incorporated protein comprising: preparing a DNA construct comprising a non-eukaryote-originating suppressor tRNA gene containing no internal promoter sequence that functions in a eukaryotic cell, and a bacteriophage-originating promoter operably linked to the 5′ terminal region of the tRNA gene, wherein the suppressor tRNA is capable of binding to the non-natural amino acid in the presence of an aminoacyl-tRNA synthetase for the non-natural amino acid; and expressing, in the presence of the non-natural amino acid in a eukaryotic cell, (a) a tRNA transcribed from the DNA construct, (b) an aminoacyl-tRNA synthetase for the non-natural amino acid, and (c) a desired protein that has a nonsense mutation or frame shift mutation at a desired position. Preferably, the tRNA gene is, but not restricted to, a pyrrolysine tRNA gene originating from archaebacteria and/or a suppressor tyrosine tRNA gene originating from Escherichia coli. Further, it is preferable that the non-natural amino acid is, but not restricted to, a lysine derivative or a tyrosine derivative.
In accordance with a seventh aspect of the present invention, there is provided a process for producing a non-natural amino acid-incorporated protein comprising: preparing a DNA construct comprising a non-eukaryote-originating suppressor tRNA gene containing no internal promoter sequence that functions in a eukaryotic cell, and a bacteriophage-originating promoter operably linked to the 5′ terminal region of the tRNA gene, wherein the suppressor tRNA is capable of binding to the non-natural amino acid in the presence of an aminoacyl-tRNA synthetase for the non-natural amino acid; and expressing, in the presence of the DNA construct and the non-natural amino acid in a eukaryotic cell, (a) an RNA polymerase corresponding to the bacteriophage-originating promoter, (b) an aminoacyl-tRNA synthetase for the non-natural amino acid, and (c) a desired protein that has a nonsense mutation or frame shift mutation at a desired position. Preferably, the tRNA gene is, but not restricted to, a pyrrolysine tRNA gene originating from archaebacteria and/or a suppressor tyrosine tRNA gene originating from Escherichia coli. Further, it is preferable that the non-natural amino acid is, but not restricted to, a lysine derivative or a tyrosine derivative.
Using a process in the present invention, it allows a non-eukaryote-originating tRNA, an aminoacyl-tRNA, to be efficiently expressed in a eukaryotic cell, and a non-eukaryote-originating suppressor tRNA containing no internal promoter sequences (box A, box B) that function in a eukaryotic cell to be expressed in a eukaryotic cell. It is expected that using process of the present invention, it allows expression of tRNA containing an artificial non-natural base in a eukaryotic cell, and synthesis of an alloprotein containing 4 or more different types of non-natural amino acids.
Further, using a process in the present invention, it allows synthesis of an alloprotein into which there is incorporated a lysine derivative such as particularly in eukaryotes presented NE-acetyl-lysine, Nε-trimethyl-lysine, Nε-t-butoxycarbonyl-lysine, fluorescent group-containing Nε-2-methylamino-benzoyl-lysine etc., by using a wild-type aminoacyl-tRNA synthetase originating from archaebacteria.
Non-natural (Non-naturally occurring) amino acid as may be used herein includes, for example, lysine derivative or tyrosine derivative. Lysine derivative, a non-natural amino acid, is preferably ones, the hydrogen atom bonded to the nitrogen atom at ε position of which is replaced with another atom or atomic group. Lysine derivative includes pyrrolysine, Nε-t-butoxycarbonyl-lysine (Nε-Boc-lysine), Nε-acetyl-lysine, Nε-trimethyl-lysine, and Nε-2-methylamino-benzoyl-lysine (Nma-lysine), for example. Site-specific incorporation of methyllysine or acetyllysine, which is a modified lysine presented in a eukaryote, into a protein could produce many findings regarding methylation or acetylation of lysine. Such alloprotein with the lysine derivative incorporated is useful as material for analysis of function and structure of the protein, and could offer a target for drug development. Tyrosine derivative includes 3- or 4-substituted tyrosine made up of a tyrosine having a substituent at 3- or 4-position of a phenyl group thereof. 3-Substituted tyrosine includes 3-halogenated tyrosine such as 3-iodotyrosine and 3-bromotyrosine. 4-Substituted tyrosine includes 4-acetyl-L-phenylalanine, 4-benzoyl-L-phenylalanine, 4-azido-L-phenylalanine, O-methyl-L-tyrosine, 4-iodo-L-phenylalanine etc. Those amino acids can be prepared by known methods and are commercially available.
Aminoacyl-tRNA synthetase as used herein is tRNA synthetase capable of recognizing a non-natural amino acid and specifically recognizing a suppressor tRNA to produce a suppressor tRNA connected to such non-natural amino acid.
In a preferred exemplary embodiment, Py1RS originating from methanogenic archaebacterium is provided that is able to recognize a lysine derivative as amino acid and specifically recognize a pyrrolysine tRNA (SEQ ID NO:4) used as tRNA in combination to produce a suppressor tRNA connected by such lysine derivative. Methanogenic archaebacterium is preferably Methanosarcina mazei (M. mazei). Py1RS is expressed in a eukaryotic cell, preferably in an animal cell, particularly preferably in a mammalian cell. In order to express Py1RS in a cell, for example, a plasmid may be introduced into the mammalian cell which plasmid is constructed such that a DNA sequence made up of a Methanosarcina mazei-originating wild-type gene, added with FLAG tag etc. at its N terminal region, is amplified using PCR, followed by incorporation of the resultant DNA sequence into NheI-BamHI site of commercially available pcDNA3.1 (Invitrogen), pAGE107 (Cytotechnology, 3, 133 (1990)), pAGE103 [J. Biochem. 101, 1307 (1987)] etc.
In other exemplary embodiments, there can be used various variants of Escherichia coli-originating TyrRS capable of specifically recognizing a tyrosine derivative to produce a suppressor tRNA (SEQ ID NO:5) connected with the tyrosine derivative. For example, the Escherichia coli-originating TyrRS variant (V37C195) specifically recognizes 3-iodotyrosine. Alternatively, it has been reported that a TyrRS variant made up of Escherichia coli-originating TyrRS with introduced mutation of 5 amino acids at positions 37, 126, 182, 185 and 186 recognized non-natural amino acid such as 4-azido-L-phenylalanine and 4-benzoyl-L-phenylalanine etc. (Chin, J. W. Et al., Science, 301, 964-967, 2003). Escherichia coli-originating TyrRS (wild-type) does not react with tRNATyr of eukaryotes, and prokaryote-originating tRNATyr does not react with TyrRS of eukaryotes.
(tRNA)
It is required for tRNA used in combination with the above aminoacyl-tRNA synthetase to satisfy the requirement that it is assigned to a nonsense codon other than codons assigned to usual 20 different amino acids, and recognized only by the above non-natural amino acid-specific aminoacyl-tRNA synthetase but not recognized by an aminoacyl-tRNA synthetase normally present in a host cell (orthogonal tRNA); and to be expressed in a eukaryotic cell. In a case where the aminoacyl-tRNA synthetase is Py1RS, the corresponding pyrrolysine tRNA is a non-eukaryotic cell-originating tRNA that has an anti-codon complementary to a nonsense codon and a 3-dimensional structure for functioning as suppressor tRNA, and is expressed in a eukaryotic cell. That is, in this case, the tRNA is a suppressor tRNA that satisfies the requirement that it is assigned to a nonsense codon other than codons assigned to usual 20 different amino acids, and recognized only by the above lysine derivative-specific Py1RS but not recognized by an aaRS normally present in a host cell (orthogonality); and is expressed in an animal cell.
Here, nonsense codon includes UAG (amber), UAA (ochre), UGA (opal) etc., but UAG (amber) is preferably used. Instead of nonsense codon, codon made up of 4 or more bases (preferably 4 or 5 bases) (hereinafter referred to as “frame shift codon”) may be used.
As mentioned above, expression of tRNA in a eukaryotic cell requires two internal promoters in a tRNA coding sequence, the consensus sequence of which is known as box A and box B.
(Synthesis of tRNA, Amino Acyl-tRNA)
In a method of synthesizing aminoacyl-tRNA according to the present invention, a non-eukaryote-originating tRNA containing no internal promoter sequence that functions in a eukaryotic cell, a eukaryote-originating promoter being linked to the 5′ end of the tRNA is caused to undergo transcription in a eukaryotic cell, preferably in an animal cell, particularly preferably in a mammalian cell, each of which contains an aminoacyl-tRNA synthetase. In this case, it is preferable that a transcription terminator sequence is linked to the 3′ end of the tRNA. To be more specific, an aminoacyl-tRNA of the present invention is obtained in the following manner: the sequence of a Methanosarcina mazei-originating wild-type pyrrolysine tRNA was synthesized from DNA primers, the 5′ end of which a eukaryote-originating promoter is linked to and the 3′ end of which a transcription terminator sequence is linked to, to be incorporated into, for example, pcDNA3.1 or pCR4Blunt-TOPO (both available from Invitrogen), and the resulting plasmid is introduced into an animal cell to express the tRNA, followed by transcription and processing in the animal cell.
As the above eukaryote-derived promoter, there can be used a nucleotide sequence that induces transcription by an RNA polymerase II or III. The nucleotide sequence that induces transcription by an RNA polymerase II is preferably a U1snRNA gene promoter. However, it has been reported that a U6snRNA gene promoter with mutated TATA box region acts as promoter of the U1snRNA gene promoter-type and thus such promoter may be used. The nucleotide sequence that induces transcription by an RNA polymerase III promoter is preferably a eukaryote-originating tRNA gene or U6snRNA gene promoter. In this case, it is preferred that the eukaryote-originating tRNA gene is linked via a linker to the 5′ end of a wild-type pyrrolysine tRNA gene. The linker includes, but not restricted to, a linker cleaved by BglII, XbaI, XhoI etc. The tRNA gene linked to the 5′ end is one originating from a eukaryote, which includes, but not restricted to, an animal, a plant, an insect etc. Thereamong, human-originating tRNA gene is preferable. An amino acid to which the tRNA should be linked may be any one of usual 20 different naturally-occurring amino acids, preferably valine among them.
Human U6 small nuclear RNA (snRNA) is an RNA species which is abundantly present in a spliceosome that is formed at the stage of splicing by pre-mRNA and reaches 4-5×105 copies per cell. Its U6 promoter is believed to drive transcription of a small heterologous RNA, the activity of the transcription being higher than the activity of transcription using a tRNA promoter. Although the U6 promoter and the tRNA promoter are both transcribed by PolII, both are different from each other in location: i.e., the U6 promoter is located at 5′ upstream of the structural gene whereas the tRNA promoter is located in the interior of its own structural gene. Human U6snRNA promoter has distinctive promoter elements known as enhancer region (or distal promoter region) and core region (or proximal promoter region), and is preferably a nucleotide sequence, which is formed of the nucleotide sequence set forth in SEQ ID NO:3 or a nucleotide sequence being at least 30%, 50%, 70%, 80%, 90% or 95% homologous to the nucleotide sequence set forth in SEQ ID NO:3, and which has the activity of transcription by an RNA polymerase III in a mammalian cell. The degree of homology between nucleotide sequences can be represented by percentage of identity of two appropriately aligned nucleotide sequences, which means incidence of accurately identical amino acids between the sequences. Appropriate alignment between sequences for identity comparison may be determined using, for example, BLAST algorithm (Altschul SF J Mol Biol 1990 Oct. 5; 215(3):403-10).
In the present invention, further, the above tRNA can be efficiently transcribed in a eukaryotic cell also by using a bacteriophage-originating promoter. To be specific, possible promoter includes, but not restricted to, Escherichia coli-originating T7 promoter, T3 promoter and SP6 promoter. These promoters may be inserted into any positions within the 5′ terminal region of the above tRNA gene, but the insertion position is preferably 10-50 bp upstream from the transcription initiation site of the gene. In the case of bacteriophage-originating promoter being used, it is required to use a eukaryotic cell in which a bacteriophage-originating RNA polymerase corresponding to such promoter is expressed. To be specific, T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase may be used as the above promoter, which is not restricted thereto. It has been reported that when expressed in a mammalian cell, T7 RNA polymerase caused transcription of RNA from DNA containing a T7 promoter sequence, and the amount of the transcribed RNA reached a maximum of 20% of the total RNAs in the cell. It has been reported that in order to prepare a large amount of RNAs as in the case of a T7 RNA polymerase, a T3 RNA polymerase and a SP6 RNA polymerase were used, the promoters thereof being as short as 20 bp or less like the T7 promoter, and the RNA polymerases each having substantially the same ability of RNA transcription (as the T7 RNA polymerase) in a mammalian cell. As the promoter of bacteriophage-originating RNA polymerase, there is preferred the T7 promoter of the nucleotide sequence set forth in SEQ ID NO:13, or a sequence formed of a nucleotide sequence being at least 70%, 80%, 90% or 95% homologous to the nucleotide sequence set forth in SEQ ID NO:13 and being capable of inducing transcription by a T7 RNA polymerase in a mammalian cell.
(Protein into which a Non-Natural Amino Acid is Incorporated)
In the present invention, proteins into which a non-natural amino acid is incorporated are not restricted to particular types. Such proteins may be any proteins capable of expression and further be heterologous recombinant proteins. Types of the proteins include, for example, so-called signaling related proteins, receptors, growth factors, cell cycle related factors, transcription factors, translation factors, transport related proteins, secretory proteins, cytoskeletal proteins, enzymes, chaperones, or disease related proteins, where the diseases include cancers, diabetes or genetic disease etc.
In the present invention, it is required to introduce a nonsense codon (an amber codon in the case of a suppressor tRNA being an amber suppressor) or a frame shift codon into a site into which a non-natural amino acid, in particular a lysine derivative or a tyrosine derivative, is to be incorporated, whereby a non-natural amino acid, in particular a lysine derivative, can be specifically incorporated into the nonsense codon (amber codon) site or the frame shift codon site. As used herein, it is referred to as “frame shift mutation” that frame shift in an amino acid sequence to be translated is caused by deletion or insertion of 1, 2, or 4 bases, and aberrant codon formed at the mutated site is referred to as “frame shift codon”. Preferably, frame shift codon is codon formed of 4 or 5 bases. It has been tried to extend genetic code by using 4-base codon in various host cells. In the case of Escherichia coli, for example, 4-base codon of AGGA is used as alternative codon that is usable without causing much disturbance of cell function (Anderson, J. C. et al., Proc. Natl. Acad. Sci., USA 101, 7566-7571).
Methods for performing site-specific mutagenesis of a protein may be any well-known methods, and are not restricted to a particular one. For example, such mutagenesis may be conducted as required according to a method described in Gene 152, 271-275 (1995); Methods Enzymol. 100, 468-500 (1983); Nucleic Acids Res. 12, 9441-9456 (1984); Proc. Natl. Acad. Sci. USA 82, 488-492 (1985) or “Saiboukougaku bessatsu ‘Sinsaiboukougakujikken protocol’, Shyujyunshya, 241-248 (1993)”, or a method using “QuickChange Site-Directed Mutagenesis Kit”
In the present invention, expression can be performed in an animal cell, and thus a non-natural amino acid can be incorporated into such protein that, in Escherichia coli or a cell-free protein system, is not or less expressed, or cannot undergo post-translational modification necessary for changing to an active form. Various types of such proteins are known to a person of ordinary skill in the art. For example, there may be synthesized an alloprotein of, but not restricted to, a tyrosine kinase type receptor such as human EGFR etc. (Cell, 110, 775-787 (2002)), human Groucho/TLE1 protein (Structure 10, 751-761(2002)), rat muscle-specific kinase (Structure 10, 1187-1196 (2002)).
In the method of the present invention, an alloprotein is expressed in an animal cell so that a non-natural amino acid, in particular a lysine derivative, can be incorporated into a carbohydrate chain-linked glycoprotein. Particularly, in the case of a type of glycoprotein whose pattern of addition of carbohydrate chain in a cell-free protein system is different from its original (natural) pattern, a system in an animal cell of the present invention is thought to be an effective measure to obtain an alloprotein to which is added a glycoprotein of a pattern of interest (an original pattern).
A protein for incorporation of a non-natural amino acid, in particular a lysine derivative, may be expressed, for example, as follows: a gene having a sequence constructed such that its codon corresponding to the position of a desired amino acid of a desired protein is replaced with a nonsense codon or a frame shift codon and a desired tag is added to the C terminus thereof is integrated into BamHI-XhoI site of pcDNA4/TO etc. to produce a plasmid, which is introduced into an animal cell, resulting in expression thereof.
An animal cell as host (cell) used in the present invention is preferably a mammalian cell in which gene recombination system is established. Examples of useful mammalian host cell system include a Chinese hamster ovary (CHO) cell and a COS cell. More unique examples include SV40-transformed simian kidney CV1 system (COS-7, ATCC CRL 1651); human embryo kidney system (293 cell or subcloned 293 cell for growth in suspension culture, J. Gen Virol., 36:59 (1977)); Chinese hamster ovary cell/-DHFR (CHO, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse Sertoli's cell (TM4, Biol. Reprod., 23:243-251(1980)); human lung cell (W138, ATCC CCL 75); human liver cell (Hep G2, HB 8065); and mouse breast cancer (MMT 060562, ATCC CCL51). The expression system for each of those host cells is established, and it is within the technical skill of a person of ordinary skill in the art to select an appropriate host cell (from among them).
Method for introducing a vector into the above host cell includes, for example, electroporation (Nucleic, Acids Res. 15, 1311-1326 (1987)), calcium phosphate method (Mol. Cell. Biol. 7, 2745-2752 (1987)), lipofection method (Cell 7, 1025-1037 (1994); Lamb, Nature Genetics 5, 22-30 (1993)), etc. These methods may be conducted, for example, in accordance with a method described in Molecular Cloning 3rd edition, Cold Spring Harbor Laboratory Press (2001) etc. In accordance with one exemplary embodiment of the present invention, there is provided a recombinant eukaryotic cell, preferably a recombinant mammalian cell, transformed or transfected with an expression vector of the above non-eukaryote-originating suppressor tRNA.
(Method for Producing a Protein with Incorporated Non-Natural Amino Acid)
As an example, expression of an alloprotein with incorporated lysine derivative is explained below. An animal cell is incubated under appropriate conditions in a medium (culture) suitable for the growth of the animal cell (for example, Opti-MEMI (Gibco BRL) etc. in the case of a CHO cell), the animal cell containing (A) an expression vector expressing an aminoacyl-tRNA synthetase, in particular a Py1RS in the animal cell; (B) an expression vector expressing in the animal cell a Methanosarcina mazei-originating pyrrolysine tRNA capable of binding to a non-natural amino acid, in particular a lysine derivative, in the presence of the above aminoacyl-tRNA synthetase, in particular Py1RS; (C) an expression vector expressing a desired protein subjected to nonsense mutation or frame shift mutation at a desired position; and a non-natural amino acid, in particular a lysine derivative. In the case of a CHO cell, for example, the cell is incubated at ca. 37 degrees Celsius for ca. 24 hours.
Alternatively, in the case of the above pyrrolysine tRNA expressed by using a bacteriophage-originating promoter, it is preferable to introduce, in addition to the above (A) to (C), (D) a vector expressing in an animal cell an RNA polymerase gene capable of transcription of the above bacteriophage-derived promoter. For example, as RNA polymerases for transcription of the above T7 promoter, T3 promoter, and SP6 promoter, there are known a T7 RNA polymerase, a T3 RNA polymerase, and SP6 RNA polymerase, respectively.
Some examples of the present invention are detailed below but it should not be understood that the present invention is restricted to the examples as mentioned below.
In these examples, there were conducted experiments for incorporation of a lysine derivative or a tyrosine derivative into 111 position of human Grb2 and 91 position of β-galactosidase. In this regard, the Grb2 is a protein involved in canceration by interaction with an epidermal growth factor receptor in a cell.
Py1RS expression plasmid was constructed as follow: a DNA sequence (SEQ ID NO:8) made up of a Methanosarcina mazei-originating wild type Py1RS gene, the N terminus region of which a FLAG tag was linked to, was amplified by PCR, followed by incorporation of the DNA sequence into NheI-BamHI site of pcDNA3.1 to generate the plasmid.
On the other hand, there has been reported the expression plasmid pEYSM1 of 3-iodo-L-tyrosine specific mutant (TyrRS (V37C195)) of Escherichia coli tyrosyl tRNA synthetase (supra., Non-Patent Document 2). Transfection into a mammalian culture cell, of this plasmid together with an expression plasmid of a suppressor tRNA followed by addition of 3-iodo-L-tyrosine to a cell culture solution allows incorporation of the 3-iodo-L-tylosine into the amber codon site of a protein gene with amber mutation. Method of preparing the above expression plasmid is described in the above Patent Document 1 and Non-Patent Document 2, the contents of both documents being incorporated herein by reference. In addition, there has been reported mutant(s) specific to 4-azido-L-phenylalanine and 4-benzoyl-L-phenylalanine (Chin et al., supra). Those mutants (variants) TyrRS were cloned into multiple cloning site of pcDNA4/TO.
(Construction of Suppressor tRNA Expression Plasmid)
A sequence (SEQ ID NO: 11) was synthesized from a DNA primer. This sequence was made up of Methanosarcina mazei-derived wild type pyrrolysine tRNA gene, the 5′ end of which a human valine tRNA gene was linked to via a linker (SEQ ID NO:10), further the 5′ and 3′ ends of which a leader sequence and a transcription termination sequence, respectively, were linked to. This sequence was introduced into pCR4Blunt-TOPO, resulting in construction of a tRNAVAL-tRNAPy1 tandem expression plasmid. For Escherichia coli-originating suppressor tRNATyr, a tRNAVAL-tRNATyr tandem expression plasmid was constructed in the similar manner.
A tRNA expression plasmid with a U6 promoter was constructed in the following method. With a pcDNA3.1 vector being as template, PCR was performed using a primer made up of a CMV enhancer region the 5′ side of which EcoRI site was added to and the 3′ side of which a portion of the 5′ side sequence of a U6 promoter was added to. And with an siSTRIKE being as template, PCR was performed using a primer made up of a U6 promoter region at the 5′ side of which there was contained a portion of the 3′ side sequence of a CMV enhancer and to the 3′ side of which XbaI site was added. The two different PCR amplification fragments were joined to each other by overlap PCR to produce a DNA fragment formed of EcoRI site/CMV enhancer/U6 promoter/XbaI site, followed by treating the produced fragment treated with EcoRI and XbaI and then cloned into pUC119.
The above plasmid prepared and treated with XbaI and HindIII was joined to a fragment containing a tRNAPy1-terminator isolated from the previously prepared tRNAVAL-tRNAPy1 tandem expression plasmid by XbaI and HindIII digestion, as a result of which there was obtained an expression plasmid having a DNA fragment of a nucleotide sequence set forth in SEQ ID NO:6. As control, there was constructed a plasmid made up of pcDNA3.1+Zeo to the multiple cloning site of which three tandem tRNAPy1 were linked.
Likewise, using Escherichia coli suppressor tyrosine tRNA instead of Methanosarcina mazei-originating wild type pyrrolysine tRNAPy1, there was constructed a DNA fragment (SEQ ID NO:7) that CMV enhancer and promoter of human U6 snRNA were linked to, to produce an expression plasmid in the similar manner mentioned above. Here, it has been reported that CMV enhancer activates RNA transcription from U6 promoter.
Using Quick Change site-directed mutagenesis kit (Stratagene), the leucine codon at position 111 of human grb2 was converted to an amber codon (grb2 (111 amber)). Subsequently, a gene (SEQ ID NO:12) constructed such that FLAG tag (DYKDDDDK) was added to the C terminus thereof was incorporated into the BamHI-XhoI site of pcDNA4/TO to produce a plasmid for detection of suppression.
Likewise, a tyrosine codon at position 91 of Escherichia coli's β-galactosidase (lacZ) was converted to an amber codon and cloned (lacZ (91 amber)) into a multiple cloning site of pcDNA3.1+ (Zeo resistant).
(Introduction of Gene into Cell and Suppression Reaction)
Chinese hamster ovary cells cultivated in a 2.0 ml culture scale 6-well plate (CHO cells; as subculture medium, DMEM/F-12 (Gibco), 10% FBS (ICN), 1/100 penicillin-streptomycin (Gibco) were used) were provided with 0.5 μg/well of three (different) types of expression plasmids: Py1RS, tRNAPy1 linked to human valine tRNA gene promoter, and grb2 (111 amber) in various combinations thereof (see RESULT), and transfection was conducted under 90% confluent state. The transfection was performed according to a method using Lipofectamine 2000 (Invitrogen) and the manual (or instruction) for the method (from Invitrogen). On the transfection, Opti-MEM (Gibco) was used as culture medium. The transfected cell culture medium (solution) was replaced with DMEM/F-12 (Gibco) in the presence or absence of 1 mM Nε-Boc-lysine (Bachem), induced expression was caused by addition of 1 μg/mL tetracycline, and incubation was conducted at 37 degrees Celsius for ca. 20 hours in a CO2 incubator.
The above cultured cells from which the cell culture medium (solution) was removed were washed with buffer solution, followed by lysis of the cells to recover proteins. SDS-polyacrylamide gel electrophoresis was performed to separate the proteins from each other according to molecular weights thereof, followed by electroblotting (100 V, 1 hour) to membrane. Regarding antibodies, anti-FLAG M2 (Sigma) was used as primary antibody, and sheep-originating whole antibody conjugated with horseradish peroxidase for anti-mouse IgG (Amersham) was used as secondary antibody. As a detection reagent, ECL western blotting detection reagent (Amersham) was used. Measurement was conducted using a cooled CCD camera LAS 1000 plus (Fuji Film).
Subsequently, human Grb2 gene, wild type TyrRS, and Py1RS, which were prepared using a similar method in the above Example, were expressed and were subjected to suppression by Methanosarcina mazei-originating wild type pyrrolysine tRNAPy1 having U6 promoter or Escherichia coli suppressor tRNATyr.
Chinese hamster ovary cells (CHO-TRex cells) were given to 24-well plate by 1.2×105 cells/well and incubated in DMEM/F-12 culture media (Gibco) containing 10% fetal bovine serum (ICN) and 1/100 penicillin-streptomycin (Gibco). Next day, when the media was 95% confluent, transfection was conducted by using 0.4 μg lacZ (91 amber), 0.2 μg Py1RS expression plasmids, and three different suppressor tRNAPy1 expression plasmids (each containing human valine tRNA, U6 promoter, and CMV enhancer) prepared in the above Example. The transfection was conducted using 2 μl Lipofectamine 2000 (Invitrogen) according to the manual (instruction) thereof. On the transfection, Opti-MEM (Gibco) was used as culture medium.
The transducted cell culture medium was replaced with DMEM/F-12 (Gibco) in the presence or absence of 1 mM Boc-lysine (Bachem), induced expression was caused by addition of 1 μg/mL tetracycline, and incubation was conducted at 37 degrees Celsius for ca. 20 hours in a CO2 incubator. Next day, proteins were recovered from the cells, and lacZ enzyme activities thereof were examined using a reporter assay kit β-Gal (TOYOBO). The result is shown in
By a method similar to that of Example 3, U6 promoter-linked Escherichia coli suppressor tRNATyr gene and three different mutant TyrRS expression plasmids were expressed, and lacZ amber suppression was performed by addition of three types of tyrosine derivatives of iodotyrosine (IY), azidophenylalanine (AzPhe), and parabenzoylphenylalanine (pBpa). The result is shown in
T7 RNA polymerase gene was amplified by PCR and cloned into between EcoRI and XhoI of pcDNA4/TO to produce T7 RNA polymerase expression plasmid. In order to produce T7-tRNATyr gene, first, there was conducted PCR by using U6-tRNATyr (SEQ ID NO:7) as template, thereby adding T7 promoter to tRNATyr sequence, to be cloned into pCR4blunt-TOPO. Then, T7-tRNATyr gene was cut out by treatment of EcoRI, to be cloned into EcoRI site of pBR322. The so prepared T7-tRNATyr gene and a portion of (so prepared) plasmid sequence (SEQ ID NO:15) were amplified by PCR, and DNA obtained was purified and used to transform cells. In order to construct tRNAPy1 expression plasmid, the pBR322 into which T7-tRNATyr gene was cloned as mentioned above was treated with XbaI and HindIII, followed by isolation of a fragment containing tRNAPy1-terminator from tRNA expression plasmid with U6 promoter by means of XbaI and HindIII digestions, to couple them with each other. The so prepared T7-tRNAPy1 gene and a portion of (so prepared) plasmid sequence (SEQ ID NO:14) were amplified by PCR, and DNA obtained was purified and used to transform cells.
Using a method similar to that of Example 3, transfection was performed by using 0.2 μg T7-tRNAPy1 expression plasmid, 0.1 μg Py1RS expression plasmid, 0.4 μg lacZ (91 amber) expression plasmid, and 0.3 μg T7 RNA polymerase expression plasmid to conduct lacZ amber suppression, except that incubation was conducted in DMEM/F-12 culture medium (Gibco) without 1/100 penicillin-streptomycin (Gibco). The result is shown in
Using a method similar to that of Example 3, transfection was performed by using 0.18 μg T7-tRNATyr gene DNA, 0.1 μg TyrRS expression plasmid, 0.4 μg lacZ (91 amber) expression plasmid, and 0.3 μg T7 RNA polymerase expression plasmid to conduct lacZ amber suppression. However, incubation was conducted in DMEM/F-12 culture medium (Gibco) without 1/100 penicillin-streptomycin (Gibco). The result is shown in
Construction of tRNA expression plasmid by U1snRNA type-promoter was conducted according to the following method. Using the previously prepared U6-tRNA Try [sic] (SEQ ID NO:7) as template, the region from 198 bases upstream of U6 promoter transcription initiation site to upstream of TATA box was amplified by using the following primers:
This region includes transcription element PSE, wherein the amplified DNA fragment has EcoRV site added at its 5′ end and XhoI site added at its 3′ end. The PCR product was once integrated into EcoRV-XhoI site of plasmid pcDNA3.1+. Vector-originating EcoO109I and NotI sites are present downstream of XhoI site. A sequence downstream of TATA box of U6 promoter and a terminator for stopping transcription by polymerase III were inserted into between these XhoI and EcoO109I. After insertion of tRNATyr sequence into EcoO109I site, 3′ box, which is a terminator of polymerase II, was further inserted into NotI site. The whole region from EcoRV through 3′ box constitutes PSE-tRNATyr gene (SEQ ID NO:18). This gene was amplified by PCR and cloned into pCR4blunt-TOPO to produce a plasmid, which corresponds to PSE-tRNATyr expression plasmid. The so prepared PSE-tRNATyr gene has U6 promoter from which TATA sequence is removed so that transcription by RNA polymerase II is caused (Das et al., Nature 1995, Vol. 374, pp. 657-660). In addition, PCR amplification was performed using the following two primers having CMV enhancers similar to those of U6 promoter:
and 5′ phosphorylation was further conducted, followed by insertion into EcoRI site of PSE-tRNATyr gene to produce CMV-DSE-PSE-tRNATyr.
Using a method similar to that of Example 3, transfection was conducted by using 0.2 μg of PSE-tRNATyr expression plasmid or CMV-PSE-tRNATyr expression plasmid, 0.2 μg of TyrRS expression plasmid, and 0.4 μg of lacZ (91 amber) expression plasmid. On the day following the transfection, cells were stained using β-Galactosidase Staining Kit (Mirus) to examine whether amber suppression of lacZ was caused. A cell having caused suppression is expected to be stained blue.
The present invention is able to effectively produce alloprotein(s) into which there is incorporated non-natural amino acid such as lysine derivative, tyrosine derivative etc.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2006-045788 | Feb 2006 | JP | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP2007/053304 | Feb 2007 | US |
| Child | 12196129 | US |
