JP7753659B2 - Avian myeloblastoma virus reverse transcriptase expression vector and method for producing said enzyme using said vector - Google Patents

Description

The present invention relates to a vector for expressing avian myeloblastoma virus reverse transcriptase in a host, and a method for producing the enzyme using the vector. In particular, the present invention relates to a vector capable of expressing the enzyme in large quantities in a host, and a method for producing the enzyme using the vector.

Among reverse transcriptases, avian myeloblastoma virus (AMV) reverse transcriptase is used as a genetic engineering reagent for cDNA synthesis and as an enzyme for genetic diagnosis.

A long-standing method for producing AMV reverse transcriptase involves growing AMV in chickens and extracting and purifying the enzyme from the resulting AMV (Patent Document 1). However, this method poses productivity challenges.

A known method for producing AMV reverse transcriptase without using AMV is a production method using genetically modified Escherichia coli, which is capable of stable mass production of proteins. Examples include a production method using an expression vector in which a gene encoding AMV reverse transcriptase is inserted into a pKK177-3-based plasmid vector (David C. LaPorte et al., J. Biol. Chem., 260, 15291-15297, 1985) having a ColE1-type replication origin (Patent Document 2), and a production method using an expression vector in which a gene encoding the enzyme is inserted into a pTrc99A plasmid vector (GenBank No. M22744), which also has a ColE1-type replication origin (Patent Document 3). However, further improvement in expression level is required for industrial mass production of AMV reverse transcriptase.

Japanese Patent Application Publication No. 60-091981 Japanese Patent Application Laid-Open No. 2002-315584 JP 2013-146235 A

The object of the present invention is to provide a plasmid vector capable of expressing large amounts of avian myeloblastoma virus reverse transcriptase, which can be used to produce the enzyme using recombinant Escherichia coli.

As a result of extensive research to solve the above-mentioned problems, the inventors discovered that optimizing the replication origin in a plasmid vector makes it possible to express large amounts of avian myeloblastoma virus (AMV) reverse transcriptase, leading to the completion of the present invention.

That is, the present invention encompasses the following aspects <1> to <4>.

<1> A plasmid vector for expressing an AMV reverse transcriptase in a host, the vector comprising a replication origin and a gene encoding the enzyme, wherein the host is Escherichia coli, and the replication origin is a polynucleotide shown in (1) or (2) below:
(1) A polynucleotide comprising at least the sequence set forth in SEQ ID NO: 7;
(2) A polynucleotide that contains at least the sequence set forth in SEQ ID NO: 7, but further contains one or more of the following substitutions, deletions, insertions, and additions of one or more nucleotides at one or more positions other than the 148th thymine, and that functions as a replication origin.

<2> The vector described in <1>, which does not contain a gene encoding a Rom/Rop protein, or which contains the gene but whose expression is inhibited.

<3> A transformant obtained by transforming Escherichia coli with the vector described in <1> or <2>.

<4> A method for producing AMV reverse transcriptase, comprising the steps of expressing AMV reverse transcriptase by culturing the transformant described in <3>, and recovering the expressed enzyme from the resulting culture.

The present invention is described in detail below.

In the present invention, the AMV reverse transcriptase refers to a reverse transcriptase contained in AMV (Avian Myeloblastosis Virus, also known as avian myeloblastosis virus). Examples of AMV reverse transcriptase include:
(I) A polypeptide containing at least the amino acid residues from threonine at position 8 to alanine at position 865 of the amino acid sequence set forth in SEQ ID NO: 1 (GenBank No. AAB31929), or
(II) A polypeptide comprising at least the amino acid residues from threonine at position 8 to alanine at position 865 of the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that one or more of the following substitutions, deletions, insertions, and additions of one or more amino acid residues occur at one or more positions within the amino acid sequence, and which has reverse transcriptase activity, DNA polymerase activity, and RNase H activity;
(III) A polypeptide comprising at least the amino acid residues from threonine at position 8 to alanine at position 865 of the amino acid sequence set forth in SEQ ID NO: 1, having 70% or more homology to the amino acid residues, and having reverse transcriptase activity, DNA polymerase activity, and RNase H activity.

An example of (II) is a polypeptide comprising at least the amino acid sequence set forth in SEQ ID NO: 2. The polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 is a naturally occurring variant of AMV reverse transcriptase, and specifically, it is a polypeptide consisting of amino acid residues from threonine at position 8 to alanine at position 865 of the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the following three amino acid residue substitutions have occurred within these amino acid residues:
Substitution of arginine at position 280 of SEQ ID NO: 1 (position 273 of SEQ ID NO: 2) with methionine;
Substitution of arginine at position 311 of SEQ ID NO: 1 (position 304 of SEQ ID NO: 2) with glutamine;
Substitution of aspartic acid at position 402 of SEQ ID NO: 1 (position 395 of SEQ ID NO: 2) with glutamic acid.

In (III) above, homology refers to similarity or identity, and can be determined using an alignment program such as BLAST (Basic Local Alignment Search Tool). For example, "amino acid residue identity" may refer to identity between amino acid sequences calculated using blastp, specifically, identity between amino acid sequences calculated using blastp with default parameters. Homology may be 70% or more, and may also be 80% or more, 90% or more, or 95% or more.

The AMV reverse transcriptase mentioned above is a polypeptide called a β chain. On the other hand, AMV reverse transcriptase also has an α chain, which is a polypeptide with a lower molecular weight than the β chain. An example of the AMV reverse transcriptase α chain is:
(i) a polypeptide consisting of amino acid residues from threonine at position 8 to tyrosine at position 579 in the amino acid sequence set forth in SEQ ID NO: 1;
(ii) A polypeptide comprising at least the amino acid residues from threonine at position 8 to tyrosine at position 579 of the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that one or more of the following substitutions, deletions, insertions, and additions of one or more amino acid residues occur at one or more positions within the amino acid sequence, and which has reverse transcriptase activity, DNA polymerase activity, and RNase H activity;
(iii) A polypeptide comprising at least the amino acid residues from threonine at position 8 to tyrosine at position 579 in the amino acid sequence set forth in SEQ ID NO: 1, with a homology of 70% or more to said amino acid residues, and having reverse transcriptase activity, DNA polymerase activity, and RNase H activity.
An example of (ii) above is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 3, which is a polypeptide consisting of the N-terminal 572 residues of the amino acid sequence set forth in SEQ ID NO: 2.

AMV reverse transcriptase is known to form the above-mentioned β chain monomer or homodimer, the above-mentioned α chain monomer or homodimer, and a heterodimer of the α chain and β chain (hereinafter also referred to as "αβ body"). The AMV reverse transcriptase expressed in a host using the vector of the present invention may have any of these forms.

The AMV reverse transcriptase of the present invention may also have added to its N-terminus and/or C-terminus a tag peptide to facilitate analysis and purification, a signal peptide to promote secretory expression in E. coli, or a peptide derived from the start codon or multi-cloning site of a vector. One example is a polypeptide consisting of a sequence in which a peptide derived from the multi-cloning site (methionine-glutamic acid-phenylalanine) and a methionine derived from the start codon are added to the N-terminus of the aforementioned AMV reverse transcriptase.

In the present invention, a gene encoding AMV reverse transcriptase (hereinafter also referred to as an AMV reverse transcriptase gene) may be a gene containing a sequence substantially homologous to the nucleotide sequence of the gene to the extent that a protein substantially identical to AMV reverse transcriptase can be expressed, or a gene containing a sequence capable of hybridizing to the nucleotide sequence of the gene under stringent conditions. Specifically, "a protein substantially identical to AMV reverse transcriptase" refers to a protein that has at least one of the three enzymatic activities of AMV reverse transcriptase (reverse transcriptase activity, DNA polymerase activity, and RNase H activity) equal to or greater than that of native AMV reverse transcriptase. For example, the protein may be translated from a gene containing a nucleotide sequence that shares at least 70% homology (preferably 80% or more, 90% or more, or 95% or more) with the nucleotide sequence of the AMV reverse transcriptase gene inherent in AMV. Additionally, "stringent conditions" herein refer to conditions under which DNA is less likely to form double strands than under normal conditions, and specific examples include conditions at 42°C in the presence of 50% (v/v) formamide, 0.1% (w/v) bovine serum albumin (BSA), 0.1% (w/v) Ficoll (trade name), 0.1% (w/v) polyvinylpyrrolidone, 50 mmol/L sodium phosphate buffer (pH 6.5), 150 mmol/L sodium chloride, or 75 mmol/L sodium citrate.

Examples of the AMV reverse transcriptase gene of the present invention include:
A polynucleotide (SEQ ID NO: 4) encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, which is AMV reverse transcriptase (β chain), and which is optimized for codon usage in the host Escherichia coli;
An example is a polynucleotide (SEQ ID NO: 5) that encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 3, which is the AMV reverse transcriptase α chain, and is optimized for codon usage in the host, E. coli.

The position of the AMV reverse transcriptase gene in the vector of the present invention may be appropriately determined as long as it does not disrupt the replication function of the plasmid vector, the desired antibiotic marker, or regions involved in transmissibility.

The vector of the present invention is a polynucleotide that functions as a replication origin, and is either (1) a polynucleotide that contains at least the sequence set forth in SEQ ID NO: 7, or (2) a polynucleotide that contains at least the sequence set forth in SEQ ID NO: 7, but further contains one or more of substitution, deletion, insertion, and addition of one or more nucleotides at one or more positions other than the 148th thymine, and that functions as a replication origin.
The polynucleotide consisting of the sequence set forth in SEQ ID NO:7 (hereinafter also referred to as "modified ColE1 replication origin") is a polynucleotide in which the cytosine base at position 148, located immediately upstream of the RNAI transcription initiation site (-1 site) of the ColE1 replication origin consisting of the nucleotide sequence set forth in SEQ ID NO:6, has been substituted with a thymine base (Figure 1), and has the same sequence as the replication origin of a pUC-based plasmid vector, which is a multicopy plasmid vector. Note that the modified ColE1 replication origin may further undergo one or more of the following substitutions, deletions, insertions, and additions of one or several nucleotides at one or several positions, as long as it has the above substitution and has substantially the same function as the ColE1 replication origin. In this specification, "one or several" means, for example, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.

The vector of the present invention is, for example,
It may be prepared by inserting the AMV reverse transcriptase gene into a plasmid vector having a modified ColE1 replication origin, such as a pUC-based plasmid vector, by a known method.
The vector may be prepared by inserting the AMV reverse transcriptase gene into a plasmid vector having a ColE1 replication origin, such as a pBR or pET plasmid vector, by a known method, and then introducing the aforementioned nucleotide substitution (substitution of the cytosine base at position 148 of SEQ ID NO: 6 with a thymine base).
The vector may also be prepared by inserting the AMV reverse transcriptase gene into a plasmid vector having a modified ColE1 replication origin or a replication origin other than the ColE1 replication origin, such as a pRSF-based, pACYC-based, or pSC101-based plasmid vector, by a known method, and then replacing the replication origin with the modified ColE1 replication origin.

It is preferable to create the vector of the present invention by inserting the AMV reverse transcriptase gene into the pTrc99A plasmid vector and then introducing the aforementioned nucleotide substitution (substitution of the cytosine base at position 148 of SEQ ID NO: 6 with a thymine base), as this allows the vector to exist stably in transformed E. coli.

It is also preferable that the vector of the present invention is a vector that does not express Rom/Rop proteins. Specifically, it is an embodiment that does not contain a gene encoding Rom/Rop proteins, or an embodiment that contains the gene but whose expression is inhibited. Rom/Rop proteins form homodimers and form complexes with RNAI and RNAII of the ColE1-type plasmid replication machinery, negatively regulating replication. In other words, the use of a vector that does not express Rom/Rop proteins is expected to have the effect of increasing the expression level of AMV reverse transcriptase in the host (E. coli).

To prepare a vector of the present invention that does not express a Rom/Rop protein, the vector of the present invention may be prepared from a plasmid vector that does not contain a gene encoding a Rom/Rop protein, such as a pTrc99A plasmid vector. Furthermore, when preparing a vector of the present invention from a plasmid vector that contains a gene encoding a Rom/Rop protein, such as a pBR322 plasmid vector or a pET-based plasmid vector, the vector may be prepared by, for example, deleting a specific site in the gene encoding the Rom/Rop protein, substituting a specific site in the gene with another nucleotide, disrupting the gene by inserting one or several bases into a specific site in the gene, or inserting a polynucleotide that inhibits the transcription or translation of the Rom/Rop protein.

The vector of the present invention may further include a promoter that controls the expression of AMV reverse transcriptase. Examples of such promoters include the trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, and lpp promoter. Among these, the use of the tac promoter or T7 promoter as the promoter is preferred, as it increases the expression level of AMV reverse transcriptase in the host.

The vector of the present invention is a vector for expressing AMV reverse transcriptase in E. coli. Preferred forms of the E. coli include E. coli MV1184 strain, E. coli GM31 strain, E. coli HB101 strain, E. coli JM101 strain, E. coli BL21 (DE3) strain, and E. coli W3110 strain. Of these, E. coli W3110 strain is particularly preferred. It is also possible to use mutant E. coli strains that have been mutated using conventional methods such as chemicals such as nitrosoguanidine or ethyl methanesulfonate, ultraviolet light, or radiation.

Escherichia coli can be transformed using the vector of the present invention using known methods (e.g., the methods described in Method in Enzymology, 216, 469-631, 1992, Academic Press and Method in Enzymology, 204, 305-636, 1991, Academic Press).

The transformant of the present invention obtained by the above-mentioned method can be cultured using known methods, such as methods using LB (Luria-Bertani) medium or 2YT medium. If the vector of the present invention contains a drug resistance gene, adding a drug corresponding to the gene to the medium as a selection agent is preferable, as it allows the transformant of the present invention to be selected and cultured. For example, if the vector of the present invention contains an ampicillin resistance gene, ampicillin or carbenicillin can be added to the medium. The medium may also contain carbon, nitrogen, inorganic salts, and appropriate nutrient sources. The culture temperature may be, for example, between 10°C and 40°C, preferably between 25°C and 37°C, and more preferably around 37°C. The pH may be, for example, between 5.9 and 8.1, and preferably around 6.0. The culture time may be, for example, 3 hours or more, preferably 16 hours or more, more preferably 50 hours or more, and even more preferably 72 hours or more.

When the vector of the present invention has an inducible promoter such as the lac promoter or tac promoter, an inducer such as IPTG (isopropyl-β-thiogalactopyranoside) may be added to the medium to induce expression of AMV reverse transcriptase. In this case, the inducer may be added at the start of culture to induce expression of AMV reverse transcriptase, or the inducer may be added after the cells have grown to a level that achieves good AMV reverse transcriptase productivity, to induce expression of AMV reverse transcriptase. An example of a method is to perform the induction procedure by adding the inducer during the growth period when the turbidity (absorbance at 660 nm) of the culture medium is approximately 0.1 to 20.0, more preferably during the growth period when the turbidity is 0.5 to 10.0, followed by continued culture. The concentration of IPTG added may be selected appropriately from a range of approximately 0.1 mmol/L to 10.0 mmol/L inclusive, but is preferably in the range of 1.0 mmol/L to 10.0 mmol/L inclusive, and more preferably around 5.0 mmol/L. It is preferable to lower the culture temperature after the induction procedure (after addition of the inducer) to a temperature lower than the optimal culture temperature for E. coli, specifically in the range of 10°C to 30°C inclusive, more preferably in the range of 15°C to 25°C inclusive, and even more preferably around 25°C. Specifically, the method described in JP 2014-113106 A is preferred because it allows for efficient production of AMV reverse transcriptase.

When expressing the αβ form of AMV reverse transcriptase in E. coli using the vector of the present invention, the gene for the AMV reverse transcriptase α chain and the gene for the AMV reverse transcriptase β chain may be co-expressed in the transformant of the present invention. In this case, expression may be achieved using a transformant of the present invention obtained by transformation with the vector of the present invention containing the α chain gene and the β chain gene, or using a transformant containing the vector of the present invention containing the α chain gene and the vector of the present invention containing the β chain gene, using selection pressure such as a selection agent. In the latter case, inserting one of the AMV reverse transcriptase genes into a plasmid vector having a modified ColE1 replication origin and the other into a plasmid vector having a replication origin compatible with the modified ColE1 replication origin is preferred, as it allows stable maintenance of both plasmid vectors and allows adjustment of the balance in the expression levels of the genes encoding AMV reverse transcriptase. Another method for producing the αβ form of AMV reverse transcriptase is to use recombinant E. coli (transformant) transformed with a plasmid vector having a modified ColE1 replication origin and an AMV reverse transcriptase β chain gene inserted therein, to express the AMV reverse transcriptase β chain, and then produce the α chain using endogenous E. coli protease, thereby producing the αβ form. This method is preferred because it allows for easy construction of the vector of the present invention.

To extract AMV reverse transcriptase from the culture medium of the transformant obtained by the above-mentioned method, an appropriate extraction method can be selected depending on the form of expression by the transformant. For example, if the expressed AMV reverse transcriptase accumulates in E. coli, the cells can be collected by centrifugation or other procedures, and then disrupted using an enzyme treatment agent or ultrasonic disruption, followed by extraction. Although various contaminants, such as nucleic acids, are present during the extraction step, AMV reverse transcriptase can be efficiently precipitated and recovered by adding a nucleic acid removal agent such as streptomycin hydrochloride or polymin P (polyethyleneimine) and co-precipitating the nucleic acids. Furthermore, AMV reverse transcriptase can be highly purified by using ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, or other chromatographic methods, either alone or in combination. As an example of purifying AMV reverse transcriptase using chromatography, AMV reverse transcriptase is purified using hydrophobic interaction chromatography incorporating polypropylene glycol groups, and then further purified using ion exchange chromatography using a phosphocellulose carrier, allowing the AMV reverse transcriptase α chain, AMV reverse transcriptase β chain, and αβ form of AMV reverse transcriptase to be separated and purified.

The vector of the present invention for expressing the avian myeloblastoma virus reverse transcriptase in E. coli is characterized by using a modified ColE1 replication origin of a pUC-based plasmid vector as the replication origin. Transformants obtained by transformation with a plasmid vector that uses a modified ColE1 replication origin as the replication origin exhibited approximately three times the bacterial growth rate compared to transformants obtained by transformation with a conventional vector. Furthermore, transformants (recombinant E. coli) obtained by transformation with the vector of the present invention exhibited approximately six times the expression level of the enzyme compared to transformants obtained by transformation with a conventional vector. Therefore, the present invention can be said to be useful for the industrial production of the enzyme.

FIG. 1 is a diagram comparing the base sequences of the ColE1 replication origin and the modified ColE1 replication origin used in the vector of the present invention. FIG. 1 shows the structure of the plasmid vector pTrcK-AMVEb. FIG. 1 shows the results of Western blotting analysis of the expression level of AMV reverse transcriptase in recombinant Escherichia coli obtained by transformation with each plasmid vector.

The present invention will be explained in more detail below using examples and comparative examples, but the present invention is not limited to these examples.

Example 1 Construction of an Avian Myeloblastoma Virus (AMV) Reverse Transcriptase Expression Plasmid Vector Having a Modified ColE1 Origin of Replication (1) A polynucleotide was synthesized in which an initiation codon (ATG) was added to the 5'-end of the AMV reverse transcriptase gene consisting of the nucleotide sequence set forth in SEQ ID NO:4, and a termination codon (TAA) was added to the 3'-end.

(2) The polynucleotide synthesized in (1) was inserted into the pTrc99A plasmid vector (with the antibiotic resistance gene replaced from ampicillin resistance to kanamycin resistance) that had been previously digested with the restriction enzymes EcoRI and KpnI, and the vector was then used to transform Escherichia coli JM109 strain (Takara Bio).

(3) From the transformants obtained in (2), recombinant plasmids were extracted using a QIAprep Spin Miniprep Kit (Qiagen), and the plasmid vector pTrcK-AMVEb containing a ColE1-type replication origin (SEQ ID NO: 6) was prepared (Figure 2).

(4) Using the Q5 mutagenesis kit (New England BioLabs), the cytosine base located at position -1 (position 148 of SEQ ID NO: 6) from the RNAI transcription start site of the plasmid vector pTrcK-AMVEb prepared in (3) was replaced with a thymine base to construct the plasmid vector pTrcK-pUC-AMVEb containing a modified ColE1-type replication origin (SEQ ID NO: 7) (Figure 1).

(5) The plasmid vectors obtained in (3) and (4) were used to transform Escherichia coli W3110, respectively, to generate AMV reverse transcriptase-producing Escherichia coli.

Comparative Example 1 Construction of AMV reverse transcriptase expression plasmid vectors having various replication origins AMV reverse transcriptase-producing Escherichia coli was prepared in the same manner as in Example 1(4), except that the replication origin in Example 1 was replaced with any of the replication origins shown in Table 1: RSF1030 type (SEQ ID NO: 8), CloDF13 type (SEQ ID NO: 9), and p15A type (SEQ ID NO: 10).

Example 2 Expression of AMV reverse transcriptase (1) The AMV reverse transcriptase-producing Escherichia coli strains prepared in Example 1 and Comparative Example 1 were inoculated into 3 mL of 2YT medium (tryptone 16 g/L, yeast extract 10 g/L, sodium chloride 5 g/L) containing an appropriate amount of kanamycin, and pre-cultured overnight at 30°C and 200 rpm.

(2) 0.2 mL of the preculture solution from (1) was inoculated into 20 mL of 2YT-PNa medium (tryptone 16 g/L, yeast extract 10 g/L, sodium dihydrogen phosphate dihydrate 14.5 g/L, disodium hydrogen phosphate dodecahydrate 2.5 g/L) containing an appropriate amount of kanamycin, and cultured at 37°C and 150 rpm for 3 hours.

(3) A portion of the culture medium was collected and the OD at 600 nm was measured using a spectrophotometer. IPTG (isopropyl-β-thiogalactopyranoside) was then added to the remaining culture medium to a final concentration of 5 mmol/L, and the medium was further cultured at 25°C and 150 rpm for 3 days.

(4) A portion of the culture medium was collected and the OD at 600 nm was measured using a spectrophotometer. The culture medium was then centrifuged to obtain E. coli (transformants) expressing AMV reverse transcriptase.

Of the transformants obtained, a comparison of bacterial cell growth rates based on OD600nm was made between transformants obtained by transformation with pTrcK-AMVEb (replication origin: ColE1 type (SEQ ID NO: 6)) and transformants obtained by transformation with pTrcK-pUC-AMVEb (replication origin: modified ColE1 type (SEQ ID NO: 7)). The results are shown in Table 2. The transformant obtained by transformation with pTrcK-pUC-AMVEb (microbial cell growth rate: 18.8) showed improved bacterial cell growth rate upon IPTG induction compared to the transformant obtained by transformation with pTrcK-AMVEb (microbial cell growth rate: 6.5).

Example 3 Evaluation of AMV Reverse Transcriptase Expression Level (1) Each E. coli strain recovered in Example 2(4) was suspended in 2 mL of disruption buffer (glycerol 10% (w/v), potassium phosphate 20 mmol/L, dithiothreitol 4 mmol/L, pH 7.2) and disrupted using an ultrasonic disrupter UD-100 (Tomy Seiko).

(2) The cell lysate obtained in (1) was added to an SDS-PAGE sample buffer containing dithiothreitol, heat-treated at 98°C for 5 minutes, and then electrophoresed using a 5% (w/v) to 20% (w/v) concentration gradient SDS-polyacrylamide gel.

(3) After electrophoresis, the proteins in the gel were transferred to a PVDF membrane using the Trans-Blot Turbo Transfer System (Bio-Rad).

(4) The membrane from (3) was stained using anti-AMV reverse transcriptase rabbit polyclonal antibody as the primary antibody, anti-rabbit IgG antibody-HRP (Bethyl) as the secondary antibody, and Super Signal West Pico PLUS (Thermo Fisher) as the color reagent.

(5) The membrane stained in (4) was photographed using an Amersham Imager 680 (Cytiva), and the brightness of each band was quantitatively analyzed using the analysis software provided with the instrument.

Figure 3 shows the results of Western blotting analysis of the expression level of AMV reverse transcriptase in transformants (recombinant E. coli) obtained by transformation with plasmid vectors containing various replication origins, expressed relative to the expression level (corresponding to band brightness) in transformants obtained by transformation with pTrcK-AMVEb (replication origin: ColE1 type (SEQ ID NO: 6)), set at 1. Transformants obtained by transformation with pTrcK-pUC-AMVEb (replication origin: modified ColE1 type (SEQ ID NO: 7)) showed approximately six-fold higher expression of AMV reverse transcriptase. On the other hand, transformants obtained by transformation with pTrcK-RSF-AMVEb (origin of replication: RSF1030 type (SEQ ID NO: 8)), pTrcK-Clo-AMVEb (origin of replication: CloDF13 type (SEQ ID NO: 9)), or pTrcK-p15A-AMVEb (origin of replication: p15A type (SEQ ID NO: 10)) showed reduced or no expression of AMV reverse transcriptase. These results demonstrate that the expression level of the enzyme is improved by using a modified ColE1-type replication origin (SEQ ID NO: 6) as the replication origin in a vector for expressing AMV reverse transcriptase in E. coli, in which at least the 148th cytosine base of the ColE1-type replication origin (SEQ ID NO: 6) has been replaced with a thymine base.

The nucleotide sequence set forth in SEQ ID NO:7 is the same sequence as the replication origin of pUC-based plasmid vectors, and it has been reported that this replication origin has a copy number 1.5 times higher than the ColE1 replication origin (SEQ ID NO:6) (Molecular Microbiology, 6, 3385-3393, 1992). However, in this example, the effect of the modified ColE1 replication origin on improving expression levels was approximately 6 times higher than the value reported in the literature (1.5 times). Furthermore, although the literature reports that the copy number decreases to the same level as the ColE1 replication origin (SEQ ID NO:6) when the culture temperature is set to 30°C or below, in this example, the effect of improving expression levels was confirmed even at a culture temperature of 25°C.

The vector of the present invention enables large-scale expression of AMV reverse transcriptase using E. coli as a host, enabling efficient production of AMV reverse transcriptase on an industrial scale.

Claims (2)

A method for producing avian myeloblastoma virus reverse transcriptase, comprising the steps of: culturing a transformant to express the avian myeloblastoma virus reverse transcriptase; and recovering the expressed enzyme from the resulting culture,
The transformant is
a plasmid vector for expressing avian myeloblastoma virus reverse transcriptase in a host, the vector comprising a replication origin and a gene encoding the enzyme, wherein the host is Escherichia coli, and the replication origin is a polynucleotide comprising at least the sequence set forth in SEQ ID NO:7; and a transformant obtained by transforming Escherichia coli with the vector,
an inducer for inducing expression of the enzyme is added during the cultivation of the Escherichia coli, and the cultivation temperature after the addition of the inducer is 25°C;
Method for producing avian myeloblastoma virus reverse transcriptase. The production method described in claim 1, wherein the vector does not contain a gene encoding a Rom/Rop protein, or contains the gene but expression of the gene is inhibited.