JP2012120506A - Amv reverse transcriptase gene, amv reverse transcriptase encoded by the gene, and method for producing amv transcriptase using the gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new avian myeloblastosis virus (AMV) reverse transcriptase gene expressible in an industrial scale, and a method for producing the enzyme using the same.SOLUTION: There are provided a DNA which is a gene encoding the beta form of the AMV reverse transcriptase and comprises a specific base sequence, and the AMV reverse transcriptase beta form encoded with the DNA.

Description

  The present invention relates to a novel AMV reverse transcriptase gene and a method for producing AMV reverse transcriptase using the same.

  Reverse transcriptase is an RNA-dependent DNA polymerase and was discovered as an essential factor for retroviral growth. This enzyme has an activity to synthesize DNA (reverse transcription) using single-stranded RNA as a template, and is used as a genetic engineering reagent or an enzyme for gene diagnosis essential for cDNA synthesis and the like.

  Reverse transcriptase derived from avian myeloblastoma virus (AMV) is a heterodimer consisting of an alpha chain having a molecular weight of about 63 kDa and a beta chain having a molecular weight of about 95 kDa, and is known as the ASLV family. This alpha chain is formed by proteolytic processing of the beta chain (Non-patent Document 1). This reverse transcriptase is reported to be expressed in Escherichia coli or eukaryotic insect cells (Patent Document 1), and it is reported that this reverse transcriptase is expressed in Escherichia coli by co-expression with a chaperone. However, a larger amount of expression is required from the viewpoint of industrial use.

Special Table 2002/534125 JP 2002-315584 A Le Grice S. F. J. et al. , 1993 reverse Transscriptase, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 163

  Accordingly, an object of the present invention is to provide a novel AMV reverse transcriptase gene capable of being expressed on an industrial scale (producing an AMV enzyme on an industrial scale) and a method for producing an AMV reverse transcriptase using the same. It is to provide.

  The present inventors reviewed the gene sequence of AMV reverse transcriptase to make it possible to produce (manufacture) on an industrial scale using Escherichia coli, and converted the DNA sequence to correspond to a codon that E. coli can easily translate. As a result, the inventors have found that the object can be achieved, and have completed the present invention. That is, the present invention is a gene encoding an AMV reverse transcriptase beta, which hybridizes under stringent conditions with DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 3, substantially homologous thereto, or these DNAs. DNA, which is a gene encoding AMV reverse transcriptase alpha body, comprising the nucleotide sequence of SEQ ID NO: 4, substantially homologous thereto, or DNA that hybridizes under stringent conditions with these DNAs A gene encoding a protein having the methionine N-terminal amino acid of AMV reverse transcriptase beta, comprising the nucleotide sequence set forth in SEQ ID NO: 5, DNA substantially homologous thereto, or stringent with these DNAs DNA that hybridizes under various conditions, and AMV reverse transcriptase A gene encoding a protein having the N-terminal amino acid of methionine as a methionine, which hybridizes under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 6, substantially homologous thereto, or these DNAs DNA.

  The present invention also relates to an AMV reverse transcriptase beta encoded by a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 3 or a DNA substantially homologous thereto, or a protein substantially homologous thereto, and a base represented by SEQ ID NO: 4. An AMV reverse transcriptase alpha-form encoded by DNA comprising a sequence or DNA substantially homologous thereto or a protein substantially homologous thereto, and comprising DNA comprising the nucleotide sequence of SEQ ID NO: 5 or substantially homologous thereto A protein encoded by DNA and having a methionine N-terminal amino acid of the AMV reverse transcriptase beta or a protein substantially homologous thereto, a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 6 or a DNA substantially homologous thereto Tan with N-terminal amino acid of encoded AMV reverse transcriptase alpha form as methionine A click protein or proteins substantially homologous. Hereinafter, the present invention will be described in detail.

  The “AMV reverse transcriptase gene” of the present invention is a gene encoding AMV reverse transcriptase, which can produce AMV reverse transcriptase by its expression, that is, transcription into mRNA and translation of the transcribed mRNA. is there. The natural AMV reverse transcriptase gene is a DNA having a full length of 2.6 k base and having the base sequence described in SEQ ID NO: 1. The gene having the base sequence described in SEQ ID NO: 1 is a gene encoding AMV reverse transcriptase beta, but AMV reverse transcriptase has a lower alpha body than beta, and the alpha body gene is This is a DNA (SEQ ID NO: 2) consisting of about 1.7k base on the 5 ′ end side of the gene having the base sequence shown in SEQ ID NO: 1. The functional structures of the proteins encoded by these genes have been recorded in a public database (National Center for Biotechnology Information).

  Among the AMV reverse transcriptase genes of the present invention, the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 3 is a gene encoding AMV reverse transcriptase beta, and the nucleotide sequence (codon encoding amino acid residue) is DNA converted from a natural (virus) type codon to an E. coli type codon. The DNA consisting of the base sequence described in SEQ ID NO: 4 is a gene encoding AMV reverse transcriptase alpha body, and the base sequence (codon encoding amino acid residue) is changed from a natural (virus) type codon to an E. coli type codon. It is converted DNA. The DNA consisting of the base sequence described in SEQ ID NO: 5 is a gene encoding a protein in which the N-terminal amino acid of AMV reverse transcriptase beta is methionine, and the base sequence (codon encoding the amino acid residue) is natural ( Virus) DNA converted from type codon to E. coli type codon. The DNA consisting of the base sequence shown in SEQ ID NO: 6 is a gene encoding a protein in which the N-terminal amino acid of AMV reverse transcriptase alpha is methionine, and the base sequence (codon encoding the amino acid residue) is natural. DNA converted from (virus) type codon to E. coli type codon.

  Here, “converted from a natural codon to an E. coli codon” means that in any gene, an E. coli rare codon (Acodon) found in mRNA transcribed from the AMV reverse transcriptase gene that AMV naturally has by transcription. rare codon), that is, a codon having a low frequency of use in E. coli generates an mRNA that is replaced with a codon of high frequency of use in E. coli without changing the amino acid encoded by the codon. The level of frequency of use of codons in E. coli can be estimated by analyzing genomic genes with reference to a public database (http://www.kazusa.or.jp/codon/), for example. In addition, as codons frequently used in E. coli, arginine (Arg) codons AGA, AGG, CGG and CGA, isoleucine (Ile) codon AUA, leucine (Leu) codon CUA, glycine (Gly) codon GGA, proline (Pro) Codon CCC can be exemplified. In this way, the gene of the present invention can be expressed on an industrial scale by using E. coli by changing the codon in the base sequence so as to transcribe the mRNA that has been replaced with the frequently used codon in E. coli. It is.

  Codon conversion can be performed by changing a portion (codon) of a DNA base sequence corresponding to the codon (transcribed to the codon). For example, Site-directed mutationage method can be used for DNA base sequence conversion. It can carry out using conventionally well-known mutagenesis methods, such as. Among them, the DNAWorks method (see Nucleic acid Res., 30, 43, 2002) in which a synthetic oligonucleotide and PCR are combined can be exemplified as a particularly preferable method for codon conversion. This method is a method of synthesizing a full-length gene by synthesizing an oligonucleotide group consisting of several tens of bases from an amino acid sequence and assembling the synthetic oligonucleotide by PCR.

  The AMV reverse transcriptase gene of the present invention, in addition to the four types of DNA described above, is partly a protein that is substantially identical to AMV reverse transcriptase by its expression, and forms an AMV reverse transcriptase. Is a DNA encoding an AMV reverse transcriptase-like protein in which an amino acid other than the amino acid forming AMV reverse transcriptase is added, or a predetermined amino acid forming AMV reverse transcriptase is substituted with another amino acid Furthermore, DNA that hybridizes under stringent conditions to the four types of DNA or DNA encoding the AMV reverse transcriptase-like protein (hereinafter these DNAs are collectively referred to as “substantially homologous to the four types of DNA”. Sometimes referred to as “DNA”). The “protein substantially the same as AMV reverse transcriptase” specifically means one of the three main enzyme activities of AMV reverse transcriptase called reverse transcriptase activity, DNA polymerase activity or RNase H activity. The above refers to a protein that is equivalent to or better than AMV. For example, when analysis is performed using a known sequence comparison algorithm between each of the above-mentioned genes, or when measurement is performed by manual alignment and visual test in which the method itself is known, and alignment is performed with maximum correspondence. A gene whose identity is found in 75% or more, preferably 85%, more preferably 95% or more of each base sequence can be exemplified as a gene encoding such a protein. Examples of the known analysis method include an analysis local homology algorithm (Smith et al., Adv. Appl. Math., 2, 482, 1981) and a homology alignment algorithm (Needlman et al., J. Mol. Biol., 48,). 443, 1970), similarity search method (Person et al., Proc. Natl. Acd. Sci. USA, 85, 2444, 1988). Among them, the known Pileup method and BLAST algorithm (Altschul et al., J.MOL.BIOL., 215,403, 1990) can be exemplified as preferable methods for defining the substantially homologous DNA of the present invention. The “stringent condition” refers to a condition in which DNA does not easily form a double strand compared to a normal state. For example, 50% (v / v) formamide, 0.1% bovine at 42 ° C. Examples include conditions in which serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer (pH 6.5), 150 mM sodium chloride and 75 mM sodium citrate coexist.

  The AMV reverse transcriptase of the present invention is a protein (including an AMV reverse transcriptase-like protein) obtained by expressing the aforementioned AMV reverse transcriptase gene. For the expression of the AMV reverse transcriptase gene, a conventionally known method can be used. For example, the DNA of the present invention is transformed into a suitable cell or a plasmid. Insert it into a vector and use it. Insertion into a plasmid vector may be performed by genetic engineering at an appropriate position of the plasmid vector. The appropriate position herein can be freely determined within a range that does not destroy the replication function of the plasmid vector, the desired antibiotic marker, or the region related to transmissibility. When inserting into a plasmid vector, for example, a DNA sequence having a promoter function may be added. Examples of promoters that function in E. coli include lac promoter, trc promoter, T7 promoter, and the like. Among these, the lac promoter that can easily regulate transcription activity is a suitable promoter.

  The plasmid vector is not particularly limited as long as it is stably present in the cell to be transformed and can be replicated. However, since the base sequence of the gene of the present invention is converted to a codon frequently used in E. coli, it is known as a plasmid vector that can be stably present and replicated in E. coli, pUC system, pBR system, pET. It is preferable to use a system, a broad host range (Broad-Host-Range) plasmid vector or the like.

  As described above, K12 E. coli is used as the host. E. coli typified by E. coli JM109 strain is preferred, but a mutant E. coli mutant strain can also be used. For the mutation treatment, a method using a conventionally known mutation means such as nitrosoguanidine, ethyl methanesulfonate, ultraviolet light or radiation can be used.

  AMV reverse transcriptase can be produced by introducing the plasmid vector described above into E. coli or the like according to a known technique and culturing the E. coli according to a known technique. As a method for introducing a plasmid vector or the like into Escherichia coli or the like, for example, “Vectors for cloning genes”, Method in Enzymology, 216, p. 469-631, 1992, Academic Press, “Other Bacterial systems”, Method in Enzymology, 204, p. 305-636, 1991, Academic Press).

  Examples of the method for culturing E. coli include a method of culturing in an LB (Luria Broth) medium supplemented with necessary nutrient sources. More specifically, for example, in order to select and culture only Escherichia coli containing a plasmid vector, it is possible to exemplify using a medium containing a selection agent based on the configuration of the plasmid vector as a medium. For example, when a plasmid vector having an ampicillin resistance gene is used, it can be exemplified that ampicillin is added to the medium. In addition to carbon, nitrogen, and inorganic salt sources, an appropriate nutrient source may be added to the medium. Further, one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, and dithiothreitol may be added. The culture temperature may be, for example, about 20 ° C. to 40 ° C., preferably 25 ° C. to 35 ° C., more preferably about 30 ° C. It can be exemplified that the pH is about 6.8 to 7.4, preferably 7.0.

  When an inducible promoter such as the above-mentioned lac promoter is used to express an AMV reverse transcriptase gene by induction, the culture is performed to such an extent that good AMV reverse transcriptase productivity can be obtained. It is preferable to perform the operation. Examples of the standard include measuring the turbidity (absorbance at 660 nm) of a culture solution, performing an induction operation during a growth period of about 0.5 to 1.0, and then culturing. Examples of the lac promoter inducer include IPTG, but may be appropriately selected and used according to the promoter to be used. When the lac promoter is used, the concentration of IPTG added may be about 0.1 to 1.0 mM in the final concentration, but is preferably 0.15 mM.

  When extracting AMV reverse transcriptase from the culture solution, an extraction method may be appropriately selected depending on the form of expression. For example, when AMV reverse transcriptase accumulates in the host as in E. coli, the cells can be collected by centrifugation or the like, and then disrupted and extracted by an enzyme treatment agent or ultrasonic disruption. At this stage, various proteins are mixed, but only AMV reverse transcriptase can be obtained by applying ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, etc. alone or in combination. Can be extracted.

  By converting the natural (viral) codon of the AMV reverse transcriptase gene into an E. coli type codon, expression on an industrial scale (production of AMV reverse transcriptase on an industrial scale) is possible using E. coli as a host. It becomes possible.

FIG. 1 to FIG. 6 are diagrams comparing the gene sequences of the natural (virus) codon and E. coli codon of AMV reverse transcriptase beta. In the figure, AMVbetaEcoli is a sequence of a DNA sequence converted to an E. coli type codon, and AMVbeta is a sequence of a natural (virus) type AMV beta. The AMV alpha body has base numbers 1 to 1716. FIG. 1 to FIG. 6 are diagrams comparing the gene sequences of the natural (virus) codon and E. coli codon of AMV reverse transcriptase beta. FIG. 1 to FIG. 6 are diagrams comparing the gene sequences of the natural (virus) codon and E. coli codon of AMV reverse transcriptase beta. FIG. 1 to FIG. 6 are diagrams comparing the gene sequences of the natural (virus) codon and E. coli codon of AMV reverse transcriptase beta. FIG. 1 to FIG. 6 are diagrams comparing the gene sequences of the natural (virus) codon and E. coli codon of AMV reverse transcriptase beta. FIG. 1 to FIG. 6 are diagrams comparing the gene sequences of the natural (virus) codon and E. coli codon of AMV reverse transcriptase beta. FIG. 7 shows the structure of the plasmid vector pTrcAMVB. FIG. 8 shows the structure of the plasmid vector pTrcAMVA. FIG. 9 shows the structure of the plasmid vector pTrcAMVBwild. FIG. 10 shows the structure of the plasmid vector pTrcAMVAwild. FIG. 11 is a diagram showing a result of an electrophoretic image showing a difference in expression level.

  Hereinafter, embodiments of the present invention will be described in detail with reference to examples.

  The following examples are provided to illustrate one embodiment of the present invention and are not intended to limit the present invention.

Example 1 Design of DNA sequence encoding AMV reverse transcriptase Based on the amino acid sequence of AMV reverse transcriptase described in SEQ ID NO: 1, the codon was converted to E. coli by the DNAWorks method. Results of alignment of the codon-converted DNA sequence and the natural (virus) codon are shown in FIGS. As shown in FIGS. 1 to 6, DNA sequence conversion was performed with the amino acid sequence as it was, and the similarity between the DNA sequences was 74%.

Example 2 Preparation of DNA sequence encoding AMV reverse transcriptase 120 oligonucleotides were synthesized in order to prepare a DNA sequence converted from a natural (virus) type codon to an E. coli type codon. The synthesized oligonucleotides are shown in SEQ ID NOs: 7 to 126.

  A two-step PCR reaction was then performed to generate DNA encoding the full-length AMV reverse transcriptase from the oligonucleotide. The reaction solution of the first stage PCR reaction is as shown in Table 1. After heat treatment at 94 ° C. for 5 minutes, the first step at 94 ° C. for 30 seconds, the second step at 62 ° C. for 30 seconds, 72 ° C., 1 A third step of 25 minutes was performed, followed by a fourth step of 7 minutes at 72 ° C. The DNA mix in Table 1 is a solution obtained by sampling and mixing 120 kinds of synthesized 50 pmol / μL oligonucleotides, respectively.

第二段階目のＰＣＲ反応は、第一段階目のＰＣＲ反応の反応液を用いて、表２の反応液組成で行った。プライマーの配列は、配列番号７（５’―ＡＴＧＡＣＧＧＴＧＧＣＧＴＴＡＣＡＣＣＴＧ―３’）と配列番号１２６（５’―ＴＴＡＧＧＣＡＡＡＣＡＧＣＧＧＡＧＡＧＧＣＴＴＣＧＴＣＣＴＴＣＴＴＣＧＴＣＡＣＴＴＣＧＴＣＴ―３’）のオリゴヌクレオチドを用いた。９４℃、５分の熱処理後、９４℃、３０秒間の第一ステップ、６５℃、３０秒間の第二ステップ、７２℃、１分間の第三ステップを２５サイクル行い、最後に、７２℃、７分の第四ステップを行った。反応終了後、０．９％のアガロース電気泳動で確認したところ、設計通りのサイズのＤＮＡバンドを確認することができた。

The PCR reaction in the second stage was performed with the reaction liquid composition shown in Table 2 using the reaction liquid of the PCR reaction in the first stage. As the primer sequence, the oligonucleotides of SEQ ID NO: 7 (5′-ATGACGGTGGCGTATACACCTG-3 ′) and SEQ ID NO: 126 (5′-TTAGCAAACAGCGGAGAGGCTTCGTCCTTTCTCTCTCACTTCGTCT-3 ′) were used. After heat treatment at 94 ° C. for 5 minutes, 25 cycles of 94 ° C., 30 second first step, 65 ° C., 30 second second step, 72 ° C., 1 minute third step, and finally 72 ° C., 7 The fourth step of minutes was done. When the reaction was confirmed by 0.9% agarose electrophoresis, a DNA band of the designed size could be confirmed.

次いで、目的バンドから市販の試薬（Ｑｉａｑｕｉｃｋ Ｇｅｌｅｘｔｒａｃｔｉｏｎ ｋｉｔ：キアゲン社）を用いてＤＮＡを抽出後、抽出したＤＮＡの５’末端を市販の試薬（ＴＡＫＡＲＡ ＢＫＬ Ｋｉｔ：タカラバイオ社）を用いてリン酸化し、制限酵素ＳｍａＩで消化したｐＴｒｃ９９ａプラスミドベクターに挿入しＥ．ｃｏｌｉ ＪＭ１０９株（タカラバイオ社）を形質転換した。調製したプラスミドベクターは制限解析及び配列決定により調査し、これをｐＴｒｃＡＭＶＢとした。

Next, after DNA is extracted from the target band using a commercially available reagent (Qiaquick Gelation kit: Qiagen), the 5 ′ end of the extracted DNA is phosphorylated using a commercially available reagent (TAKARA BKL Kit: Takara Bio Inc.). And inserted into the pTrc99a plasmid vector digested with the restriction enzyme SmaI. E. coli strain JM109 (Takara Bio Inc.) was transformed. The prepared plasmid vector was investigated by restriction analysis and sequencing, and this was designated as pTrcAMVB.

  Furthermore, a gene encoding an AMV reverse transcriptase alpha body was produced using the PCR method using pTrcAMVB as a template. The sequences of the PCR primers used are SEQ ID NO: 7 (5'-ATGACGGTGGCGTTACACCTG-3 ') and SEQ ID NO: 127 (5'-TTAATACGCTTGAAACGGTGCCCTGCTCATCCCGCC-3'). After heat treatment at 94 ° C. for 5 minutes, 25 cycles of 94 ° C., 30 second first step, 65 ° C., 30 second second step, 72 ° C., 1 minute third step, and finally 72 ° C., 7 The fourth step of minutes was done. When the reaction was confirmed by 0.9% agarose electrophoresis, a DNA band of the designed size could be confirmed.

  Next, after DNA is extracted from the target band using a commercially available reagent (Qiaquick Gelation kit: Qiagen), the 5 ′ end of the extracted DNA is phosphorylated using a commercially available reagent (TAKARA BKL Kit: Takara Bio Inc.). And inserted into the pTrc99a plasmid vector digested with the restriction enzyme SmaI. E. coli strain JM109 (Takara Bio Inc.) was transformed. The prepared plasmid vector was investigated by restriction analysis and sequencing, and this was designated as pTrcAMVA. FIG. 7 shows the structure of pTrcAMVB, and FIG. 8 shows the structure of pTrcAMVA.

  A gene encoding AMV reverse transcriptase beta was extracted from an E. coli clone (ATCC 31990) carrying a gene encoding AMV reverse transcriptase beta on a plasmid using the PCR method. The sequences of the PCR primers used are SEQ ID NO: 128 (5'-ATGACTGTTGCGCTACATCTGGCTATTCCGCTC-3 ') and SEQ ID NO: 129 (5'-TTATGCAAAAAGGGGCTCGCTCCATC-3'). After heat treatment at 94 ° C. for 5 minutes, 25 cycles of 94 ° C., 30 second first step, 65 ° C., 30 second second step, 72 ° C., 1 minute third step, and finally 72 ° C., 7 The fourth step of minutes was done. When the reaction was confirmed by 0.9% agarose electrophoresis, a DNA band of the designed size could be confirmed. Next, after DNA is extracted from the target band using a commercially available reagent (Qiaquick Gelation kit: Qiagen), the 5 ′ end of the extracted DNA is phosphorylated using a commercially available reagent (TAKARA BKL Kit: Takara Bio Inc.). And inserted into the pTrc99a plasmid vector digested with the restriction enzyme SmaI. E. coli strain JM109 (Takara Bio Inc.) was transformed. After replicating the plasmid vector, the replicated plasmid vector was examined by restriction analysis and sequencing, and this was designated as pTrcAMVBwild.

  Using the PCR method, a gene encoding AMV reverse transcriptase alpha was extracted from an E. coli clone (ATCC 31990) carrying a gene encoding AMV reverse transcriptase beta on the plasmid. The sequences of the PCR primers used are SEQ ID NO: 128 (5'-ATGACTGTTGCGCTACATCTGGCTATTCCGCTC-3 ') and SEQ ID NO: 130 (5'-TTAATACGCTTGAAAGGTGCTCTGCTCTCTGCC-3'). After heat treatment at 94 ° C. for 5 minutes, 25 cycles of 94 ° C., 30 second first step, 65 ° C., 30 second second step, 72 ° C., 1 minute third step, and finally 72 ° C., 7 The fourth step of minutes was done. When the reaction was confirmed by 0.9% agarose electrophoresis, a DNA band of the designed size could be confirmed.

Next, after DNA is extracted from the target band using a commercially available reagent (Qiaquick Gelation kit: Qiagen), the 5 ′ end of the extracted DNA is phosphorylated using a commercially available reagent (TAKARA BKL Kit: Takara Bio Inc.). And inserted into the pTrc99a plasmid vector digested with the restriction enzyme SmaI. E. coli strain JM109 (Takara Bio Inc.) was transformed. The transformed Escherichia coli was cultured and the plasmid vector was replicated. Then, the replicated plasmid vector was examined by restriction analysis and sequencing, and this was designated as pTrcAMVAwild. FIG. 9 shows the structure of pTrcAMVBwild, and FIG. 10 shows the structure of pTrcAMVAwild.
Example 3 Confirmation of Sequence The sequence of DNA inserted into the plasmid vectors pTrcAMVB and pTrcAMVA prepared in Example 2 was converted into a commercially available reagent kit based on the chain terminator method (Big Dye Terminator Cycle Sequencing Reaction Kit, PE Applied Biosystems). ) Was used for a cycle sequencing reaction and analyzed with a fully automatic DNA sequencer ABI Prism 3700 DNA Analyzer (PE Applied Biosystems). In addition, 120 kinds of synthesized oligonucleotides were appropriately used as sequencing primers. As a result, it was confirmed that the base sequences of the DNA inserted into pTrcAMVB and pTrcAMVA were as designed.

Example 4 Induction of AMV Reverse Transcriptase Expression Using the plasmid vector pTrcAMVB, E. coli was cultured in LB agar medium supplemented with 50 μg / mL antibiotic ampicillin. E. coli JM109 (Takara Bio Inc.) was transformed. After culturing at 37 ° C. for 18 hours, an arbitrary colony that appeared was picked up and inoculated into LB liquid medium supplemented with 50 μg / mL antibiotic ampicillin. After culturing at 37 ° C., when the value of OD660 nm reached about 0.5, 0.15 mM IPTG (manufactured by Takara Bio Inc.) was added. A strain transformed with the plasmid vector pTrcAMVB was designated as JM109 / pTrcAMVB. The plasmid vector pTrcAMVA was similarly operated, and a transformant was designated as JM109 / pTrcAMVA.

  In the same manner, plasmid vector pTrcAMVBwild and plasmid vector pTrcAMVAwild were also transformed into JM109 / pTrcAMVBwild and JM109 / pTrcAMVAwild, respectively.

  The culture solution was sampled at an appropriate interval from the addition of IPTG, and a protein solution was prepared from each transformant. Preparation was performed with a commercially available kit (Bugbuster protein extraction Kit, Novagen), and the prepared protein solution was subjected to SDS-PAGE. The result of the electrophoresis image is shown in FIG. As shown in FIG. 11, although the amino acid sequences of the proteins to be expressed are the same, the expression level was significantly increased in codons matched to the host E. coli as compared to natural (virus) type codons.

Claims (8)

A gene encoding an AMV reverse transcriptase beta, which is a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 3, a DNA substantially homologous thereto, or a DNA that hybridizes under stringent conditions with these DNAs. A gene encoding an AMV reverse transcriptase alpha body, which is a DNA comprising the nucleotide sequence of SEQ ID NO: 4, a DNA substantially homologous thereto, or a DNA that hybridizes under stringent conditions with these DNAs. A gene encoding a protein having a methionine N-terminal amino acid of AMV reverse transcriptase beta, comprising a DNA comprising the nucleotide sequence of SEQ ID NO: 5, a DNA substantially homologous thereto, or a stringent condition with these DNAs DNA that hybridizes with A gene encoding a protein having an N-terminal amino acid of the AMV reverse transcriptase alpha methionine as a methionine, a DNA comprising the nucleotide sequence of SEQ ID NO: 6, a DNA substantially homologous thereto, or a stringent condition with these DNAs DNA that hybridizes with An AMV reverse transcriptase beta or a protein substantially homologous thereto, encoded by DNA consisting of the base sequence set forth in SEQ ID NO: 3 or DNA substantially homologous thereto. An AMV reverse transcriptase alpha body encoded by a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 4 or a DNA substantially homologous thereto or a protein substantially homologous thereto. A protein having a methionine as the N-terminal amino acid of an AMV reverse transcriptase beta encoded by a DNA comprising the nucleotide sequence of SEQ ID NO: 5 or a DNA substantially homologous thereto, or a protein substantially homologous thereto. A protein having the methionine N-terminal amino acid of the AMV reverse transcriptase alpha encoded by a DNA comprising the nucleotide sequence of SEQ ID NO: 6 or a DNA substantially homologous thereto, or a protein substantially homologous thereto.

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