JP6070180B2 - Enzyme screening method - Google Patents

Description

  The present invention relates to an enzyme screening method. In particular, the present invention relates to an enzyme having at least one of (1) RNA-dependent DNA polymerase activity, (2) DNA-dependent DNA polymerase activity, and (3) ribonuclease H activity obtained by genetic engineering techniques. The present invention relates to a method for efficiently screening for an enzyme having a desired performance from a transformant capable of expressing.

  Reverse transcriptase, discovered as an essential factor for retrovirus growth, has RNA-dependent DNA polymerase activity that synthesizes DNA (reverse transcription) using single-stranded RNA as a template, and is essential for cDNA synthesis, etc. It is used as a genetic engineering reagent or gene diagnostic enzyme.

  Avian myeloblastoma virus (AMV) reverse transcriptase, one of the reverse transcriptases, is a heterocomplex composed of an α chain with a molecular weight of about 63 kDa and a β chain with a molecular weight of about 95 kDa, known as the ASLV family. Yes. Among these, the α chain is formed from the β chain by proteolytic processing (Non-patent Document 1). It is known that the hetero complex of α chain and β chain of AMV reverse transcriptase is more stable and useful than the case of only α chain or β chain (Patent Document 1).

  Reverse transcriptase is known to have not only RNA-dependent DNA polymerase activity but also DNA-dependent DNA polymerase activity and ribonuclease H (RNase H) activity. The above-mentioned avian myeloblastoma virus (AMV) reverse transcriptase Is an example. Such an enzyme having a plurality of activities is used as an enzyme used in gene amplification methods such as the TRC method and NASBA method because it can perform a plurality of reactions with one enzyme (Patent Documents 2 and 3). ). In the reaction using reverse transcriptase, an enzyme capable of performing reverse transcription reaction at a higher temperature is desired in order to improve the efficiency and accuracy of amplification.

JP 2003-334095 A JP 2006-14632 A JP-T-4-507197 JP 2012-120506 A JP 2012-074418 A

Le Grice S. F. J. et al. , Reverse Transscriptase, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 163 (1993)

  An object of the present invention is to obtain an enzyme having one or more activities among (1) RNA-dependent DNA polymerase activity, (2) DNA-dependent DNA polymerase activity, and (3) ribonuclease H activity with improved performance. A screening method is provided.

  The inventors of the present invention have disclosed a polymorphism encoding an enzyme having one or more of (1) RNA-dependent DNA polymerase activity, (2) DNA-dependent DNA polymerase activity, and (3) ribonuclease H (RNase H) activity. In screening for an enzyme obtained from a transformant obtained by introducing a mutation into a nucleotide and transforming a host with a vector containing the polynucleotide into which the mutation has been introduced, nucleic acid synthesis using the enzyme is performed and evaluated. Thus, it was found that the enzyme having the desired performance was obtained, and the present invention was completed.

That is, the first aspect of the present invention is:
Encodes an enzyme having one or more, preferably two or more, of (A) (1) RNA-dependent DNA polymerase activity, (2) DNA-dependent DNA polymerase activity, and (3) ribonuclease H (RNase H) activity Introducing a mutation into the polynucleotide to be
(B) inserting the polynucleotide into which the mutation is introduced into a vector;
(C) transforming a host with a vector having the polynucleotide inserted therein;
(D) culturing the obtained transformant and expressing the enzyme;
(E) extracting the enzyme from the culture solution of the transformant;
(F) evaluating the performance of the enzyme;
In the method for screening an enzyme, the nucleic acid synthesis that requires one or more, preferably two or more of the activities (1) to (3) is performed for the performance evaluation of the enzyme in the step (F). It is the said screening method evaluated by this.

  The second aspect of the present invention is the screening method according to the first aspect, wherein the enzyme is an enzyme having all the activities (1) to (3).

  The third aspect of the present invention is the screening method according to the second aspect, wherein the enzyme is avian myeloblastoma virus (AMV) reverse transcriptase.

In the fourth aspect of the present invention, the performance evaluation of the enzyme in the step (F) is complementary to the first primer having a sequence homologous to a part of the specific base sequence and the part of the specific base sequence. From the following (I), using a second primer having a typical sequence (wherein either one of the first or second primers has an RNA polymerase promoter sequence added to its 5 ′ end) The screening method according to any one of the first to third aspects, wherein the evaluation is performed by synthesizing an RNA containing the specific base sequence or a sequence complementary to the specific base sequence by the step (V).
(I) a step of synthesizing cDNA complementary to a specific base sequence or a sequence complementary to the sequence with an enzyme having RNA-dependent DNA polymerase activity;
(II) a step of degrading RNA-DNA double-stranded RNA by an enzyme having RNase H activity (generation of single-stranded DNA),
(III) A double-stranded DNA having a promoter sequence capable of transcribing a specific base sequence using the single-stranded DNA as a template or RNA complementary to the sequence is generated by an enzyme having DNA-dependent DNA polymerase activity. The process of
(IV) generating an RNA transcript using the double-stranded DNA as a template from the promoter sequence by an enzyme having DNA-dependent RNA polymerase activity;
(V) A step of generating RNA transcripts in a chain by using the RNA transcript as a template for cDNA synthesis in the step (I).
Furthermore, a fifth aspect of the present invention is an enzyme obtained by the screening method according to any one of the first to fourth aspects.

The present invention includes a step of introducing a mutation into a polynucleotide encoding an enzyme having one or more of RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity, and ribonuclease H activity; A step of inserting a nucleotide into an expression vector, a step of transforming a host with the expression vector having the polynucleotide inserted therein, a step of culturing the obtained transformant and expressing the enzyme,
In the method for screening an enzyme comprising the steps of extracting the enzyme from the culture solution of the transformant and evaluating the performance of the enzyme, the performance evaluation of the enzyme is performed by measuring at least one of the three activities. It is characterized by performing nucleic acid synthesis that requires activity. According to the present invention, it is possible to obtain an enzyme having a desired performance necessary for the nucleic acid synthesis.

It is a figure which shows the mutation site | part illustrated by the amino acid sequence (sequence number 1, upper stage) of wild type AMV reverse transcriptase (beta) chain | strand, and this invention. It is a figure which shows the structure of plasmid vector pTrcAMVB. It is a figure which shows the calibration curve which estimates the number of units of reverse transcriptase from the increase time of fluorescence intensity. It is a figure which shows the result of the heat resistance evaluation of the avian myeloblastoma virus (AMV) reverse transcriptase which has a wild type and the modification or mutation of this invention.

Hereinafter, the present invention will be described in detail.
In the present invention, RNA-dependent DNA polymerase activity is an activity of synthesizing a DNA strand having a complementary base sequence using a single-stranded RNA strand as a template. C. It is an enzyme activity possessed by an enzyme classified as 2.7.7.7. In the present invention, the DNA-dependent DNA polymerase activity is an activity of synthesizing a DNA strand having a base sequence complementary to a single-stranded DNA strand as a template. C. It is an enzyme activity possessed by an enzyme classified as 2.7.7.79. In the present invention, ribonuclease H (RNase H) activity is a ribonuclease that cleaves an RNA strand forming a hybrid duplex of DNA and RNA to produce a single-stranded DNA. C. This is an enzyme activity possessed by an enzyme classified as 3.1.26.4.

  In the present invention, the enzyme having one or more enzyme activities of (1) RNA-dependent DNA polymerase activity, (2) DNA-dependent DNA polymerase activity, and (3) RNase H activity is any of (1) to (3) From the viewpoint of screening an enzyme having an activity or an enzyme having any combination of (1) to (3), and synthesizing nucleic acid in a chain manner, (1) RNA-dependent DNA polymerase activity and (2) A combination of DNA-dependent DNA polymerase activity is preferred, and from the viewpoint of obtaining an enzyme useful for RT-PCR, an enzyme having a combination of (1) RNA-dependent DNA polymerase activity and (3) RNase H activity is preferred. From the viewpoint of screening for enzymes useful as enzymes used in the TRC method and NASBA method, the activities of (1) to (3) Enzymes with all, for example, murine leukemia virus (MMLV) reverse transcriptase or avian bone marrow neuroblastoma virus (AMV) reverse transcriptase are preferred, AMV reverse transcriptase is preferable.

  The total length of the polynucleotide encoding AMV reverse transcriptase inherent to AMV is 2574 bases, and the nucleotide sequence is shown in SEQ ID NO: 3. The polynucleotide encoding the avian myeloblastoma virus (AMV) reverse transcriptase having the base sequence set forth in SEQ ID NO: 3 is a gene encoding the AMV reverse transcriptase gene reverse transcriptase β chain, but the AMV reverse The transcriptase gene reverse transcriptase also has an α chain having a lower molecular weight than the β chain, and its nucleotide sequence consists of 1716 bases on the 5 ′ end side of the base sequence described in SEQ ID NO: 3 (SEQ ID NO: 4). As an example of a polynucleotide encoding a more preferable AMV reverse transcriptase when the host is Escherichia coli, a β-chain gene (SEQ ID NO: 5) converted using an E. coli codon using the method described in Patent Document 4 is used. Examples thereof include the gene for the sequence and α chain (SEQ ID NO: 6).

In the screening method of the present invention, the method for screening an enzyme having one or more activities among the above (1) to (3) includes the following steps (A) to (F):
(A) introducing a mutation into a polynucleotide encoding an enzyme having one or more of the activities (1) to (3);
(B) inserting the polynucleotide into which the mutation is introduced into a vector;
(C) transforming a host with a vector having the polynucleotide inserted therein;
(D) culturing the obtained transformant and expressing the enzyme;
(E) extracting the enzyme from the culture solution of the transformant;
(F) a step of evaluating the performance of the enzyme, and by appropriately selecting the conditions in step (F), the desired performance, for example, any one or more of (1) to (3) improved Enzymes having activity, improved heat resistance, performance not affected by surfactants and organic solvents, etc. can be screened.

Step (A) is a step of introducing a mutation at an arbitrary position of a polynucleotide encoding a wild-type enzyme or the like. The polynucleotide encoding the wild-type enzyme can be obtained by any method that can be obtained by those skilled in the art. For example, the polynucleotide can be obtained by PCR using appropriate primers prepared from the genome information of the enzyme. As a polynucleotide encoding a wild-type enzyme or the like, in addition to a polynucleotide encoding a wild-type enzyme, it encodes an enzyme having at least one of the activities (1) to (3). Polynucleotides having arbitrary deletions, substitutions, and / or additions can be used. For example, as described in Patent Document 4, a polynucleotide modified to have an E. coli codon type can also be used. The polynucleotide into which the mutation has been introduced appropriately uses known techniques such as site-directed mutagenesis, PCR using degenerate oligonucleotides, error-prone PCR, mutagenesis of nucleic acid-containing cells, and exposure to radiation. However, error-prone PCR is preferable from the viewpoint of obtaining a polynucleotide into which a mutation is introduced with high throughput. Error-prone PCR can be performed by a known method, but in general, a polymerase having no 3′-5 ′ exonuclease activity (proofreading activity) is converted into a Mn ²⁺ concentration such that a desired degree of mutation occurs. Mutations can be introduced by selecting a dNTP concentration and temperature and performing PCR on a polynucleotide encoding a wild-type enzyme or the like.

  Step (B) is a step of inserting the polynucleotide into which a mutation has been introduced into a vector, whereby a gene library can be constructed. Insertion of a polynucleotide into a vector can be performed using a ligase by a method known in the art. For example, a primer previously provided with a restriction enzyme cleavage site is cleaved with a restriction enzyme, and the vector is ligated using the ligase. It can be inserted into the desired cloning site. The type of vector to be inserted is not particularly limited, and may be an autonomously replicating vector or a vector that is integrated into the host cell genome when introduced into the host cell. The vector used in the present invention is preferably an expression vector from the viewpoint of expressing an enzyme encoded by a polynucleotide incorporated in the vector, and the polynucleotide into which the mutation is introduced in the expression vector is necessary for transcription such as a promoter. It is operably linked to elements such as Shine-Dalgarno sequence and Kozak sequence necessary for translation in the mRNA after transcription. These elements can be appropriately selected depending on the type of host. For example, when Escherichia coli is used as a host, examples of promoters that can be used include lac, trp, and tac promoters. As an expression vector, when Escherichia coli is used as a host, expression plasmids such as pTrc99A (GenBank Accession No. U13872) and pCDF-1b (manufactured by Takara Bio Inc.) can be exemplified. You may use it selecting it suitably from. Furthermore, a vector capable of adding a sequence useful for enzyme purification to the 5 'end or 3' end of the polynucleotide into which the mutation has been introduced, or both may be selected. By selecting such a vector, for example, a signal peptide can be added to make an extracellular secretory enzyme, or a tag sequence such as a histidine tag, a FLAG tag, or a Myc tag can be added to facilitate purification of the enzyme. it can. When inserting a polynucleotide into a vector to which such sequences can be added, it is necessary that the polynucleotide be operably linked to these elements. However, the types of signal peptides and tags, and the method for binding these peptides and the present enzyme are not limited to the above methods, and any peptide available to those skilled in the art can be used.

  Step (C) is a step of transforming the host with the gene library constructed by the method described in step (B) (a set of vectors into which the polynucleotide into which the mutation has been introduced is inserted). As the host used in the step (C), any host that expresses the target enzyme can be appropriately used. Among prokaryotic cells, Escherichia coli, bacteria belonging to the genus Bacillus, bacteria belonging to the genus Rhodococcus, bacteria belonging to the genus Streptomyces, bacteria belonging to the genus Staphyrococcus, Examples of eukaryotic cells include bacteria belonging to the yeast Saccharomyces, bacteria belonging to the genus Candida, bacteria belonging to the genus Pombe, bacteria belonging to the filamentous fungus Aspergillus, and the like. Furthermore, insect cells and animal cells can be exemplified. An example of a preferred host cell is E. coli that is easy to culture and handle. As the E. coli strain, any strain used in the field of genetic engineering may be used, but from the viewpoint of having high gene conversion ability, preferably E. coli MV1184 strain, E. coli GM31 strain, E. coli HB101 strain, E. coli JM101 strain, E. coli strain W3110, more preferably E. coli strain W3110. In addition, E. coli mutant strains that have been subjected to mutation treatment by a conventionally known means such as chemical substances such as nitrosoguanidine and ethyl methanesulfonate, ultraviolet rays, radiation, etc. may be used. As a method for transforming Escherichia coli using a plasmid vector, any method used in the field of genetic engineering can be used. For example, Methods in Enzymology, 216, p. 469-631, 1992, Academic Press and Method in Enzymology, 204, p. 305-636, 1991, Academic Press.

  Step (D) is a step of culturing the transformant obtained from step (C) to express an enzyme. The culture condition of the transformant may be any known condition as long as it allows the expression of the polynucleotide encoding the enzyme inserted into the vector, and after the transformant is precultured, The enzyme may be expressed by adding a corresponding inducer. For example, as a method for expressing avian myeloblastoma virus (AMV) reverse transcriptase using E. coli, 2 × YT-Pna medium (pH 6.0) (16 g / L tryptone, 10 g / L yeast extract, 5 g / L chloride) Sodium, 14.5 g / L monobasic sodium phosphate dihydrate, 2.5 g / L dibasic sodium phosphate dodecahydrate), and cultured with shaking at 37 ° C./160 rpm for 3 hours. An example is a method in which an inducing agent is added to the culture solution and the culture is performed with shaking at 25 ° C. and 160 rpm for 3 nights. The inducer to be used can be appropriately selected depending on the type of promoter used in the vector. For example, IPTG can be used if it is a vector containing a lac promoter.

  Step (E) is a step of extracting the enzyme from the culture solution of the transformant, and the enzyme expressed by a known method suitable for each host may be extracted. For example, when E. coli is sonicated under appropriate conditions and undisrupted cells are separated by centrifugation, or when an enzyme is secreted by adding a signal sequence, E. coli is not disrupted. An example is a method of separating cells by centrifugation. An extraction method using a surfactant is a preferred example because parallel processing is easy. In the step of extracting the enzyme, the extracted enzyme may be further purified. For purification of the enzyme, any purification method may be used as long as a substance that becomes an inhibitor in the subsequent nucleic acid synthesis can be separated from the enzyme, and the purification method may be diluted as long as the inhibitor is acceptable. In order to separate the polymerase from other proteins, a preferred example is a method of efficiently precipitating and recovering by coprecipitation with nucleic acid by adding streptomycin hydrochloride or polymin P. Furthermore, by applying chromatography such as ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, and affinity chromatography alone or in combination, purification can be performed with higher purity. Furthermore, when a tag sequence is added to the enzyme, it can be purified using a purification method according to the tag sequence, for example, a pull-down method or an affinity chromatography method. A more preferable example of the chromatography is a batch method suitable for parallel processing.

Step (F) is a step of evaluating the performance of the enzyme. Using the enzyme extracted in step (E), nucleic acid synthesis that requires one or more of the activities (1) to (3) above To evaluate such an enzyme. The nucleic acid synthesis method is not particularly limited as long as it requires one or more of the activities (1) to (3), and examples thereof include an RT-PCR method. Preferably, it is a method that requires a combination of two or more of the activities (1) to (3), and more preferably a TRC method or NASBA method that requires all of (1) to (3). It is. In order to evaluate the performance of an enzyme using these methods, a target RNA containing a specific base sequence, a first primer having a sequence homologous to a part of the specific base sequence, a part of the specific base sequence, A second primer having a complementary sequence (wherein either one of the first or second primers has an RNA polymerase promoter sequence added to its 5 ′ end), and an enzyme group; What is necessary is just to evaluate by synthesize | combining RNA containing the said specific base sequence or a sequence complementary to the said sequence by the process of the following (I) to (V).
(I) a step of synthesizing a cDNA complementary to a specific base sequence or a sequence complementary to the sequence by an enzyme having RNA-dependent DNA polymerase activity;
(II) a step of degrading RNA-DNA double-stranded RNA by an enzyme having RNase H activity (generation of single-stranded DNA),
(III) A double-stranded DNA having a promoter sequence capable of transcribing a specific base sequence using the single-stranded DNA as a template or RNA complementary to the sequence is generated by an enzyme having DNA-dependent DNA polymerase activity. The process of
(IV) generating an RNA transcript using the double-stranded DNA as a template from the promoter sequence by an enzyme having DNA-dependent RNA polymerase activity;
(V) A step of generating RNA transcripts in a chain by using the RNA transcript as a template for cDNA synthesis in the step (I).

In one embodiment, an RNA polymerase promoter sequence is added to the 5 ′ end of the first primer. In that case, RNA comprising the specific base sequence is synthesized by the following steps (I ′) to (V ′). You can evaluate by doing.
(I ′) a step of synthesizing a cDNA complementary to a specific base sequence by the second primer and an enzyme having RNA-dependent DNA polymerase activity;
(II ′) a step of degrading RNA-DNA double-stranded RNA by an enzyme having RNase H activity (generation of single-stranded DNA);
(III ′) a step of generating a double-stranded DNA having a promoter sequence capable of transcribing a specific base sequence using the single-stranded DNA as a template by the first primer and an enzyme having a DNA-dependent DNA polymerase activity;
(IV ′) a step of generating an RNA transcript using the double-stranded DNA as a template by an enzyme having DNA-dependent RNA polymerase activity;
(V ′) A step of generating RNA transcripts in a chain by using the RNA transcript as a template for cDNA synthesis in the step (I).

In another embodiment, an RNA polymerase promoter sequence is added to the 5 ′ end of the second primer. In this case, the second primer is complementary to the specific base sequence by the following steps (I ″) to (IX ″). What is necessary is just to evaluate by synthesize | combining RNA containing a sequence | arrangement.
(I ″) a step of synthesizing cDNA complementary to a specific base sequence by the second primer and an enzyme having RNA-dependent DNA polymerase activity;
(II ″) a step of degrading RNA-DNA double-stranded RNA by an enzyme having RNase H activity (generation of single-stranded DNA),
(III ″) a double-stranded DNA having a promoter sequence capable of transferring a complementary sequence to a specific base sequence using the single-stranded DNA as a template by the first primer and an enzyme having DNA-dependent DNA polymerase activity; Generating step,
(IV ″) generating an RNA transcript using the double-stranded DNA as a template by an enzyme having DNA-dependent RNA polymerase activity;
(V ″) a step of synthesizing a cDNA having a specific base sequence by the RNA transcript with an enzyme having a first primer and an RNA-dependent DNA polymerase activity;
(VI ″) a step of degrading RNA-DNA double-stranded RNA by an enzyme having RNase H activity (generation of single-stranded DNA),
(VII ″) a double-stranded DNA having a promoter sequence capable of transferring a sequence complementary to a specific base sequence using the single-stranded DNA as a template by the second primer and an enzyme having DNA-dependent DNA polymerase activity Generating a process,
(VIII ″) generating an RNA transcript using the double-stranded DNA as a template by an enzyme having DNA-dependent RNA polymerase activity;
(IX ″) The RNA transcript of the above (VIII ″) is used as a template for cDNA synthesis in the step (V ″), so that the RNA transcript having a sequence complementary to the specific base sequence in a chained manner. Generating.

  The above enzyme group includes one or more enzymes having activities necessary for the nucleic acid synthesis reaction, that is, (1) RNA-dependent DNA polymerase activity, (2) DNA-dependent DNA polymerase activity, and (3) ribonuclease H activity. (4) A combination with an enzyme having DNA-dependent RNA polymerase activity. When evaluating the performance of one or more activities among (1) to (3), it is necessary to add an enzyme having an activity other than the activity to be evaluated. For example, in the case of a screening method for evaluating (1) RNA-dependent DNA polymerase activity and (2) DNA-dependent DNA polymerase activity, (4) In addition to an enzyme having DNA-dependent RNA polymerase activity, (3) Ribonuclease H It is necessary to add an enzyme having activity.

  The step (F) includes a target RNA containing a specific base sequence, a first primer having a sequence homologous to a part of the specific base sequence, and a second primer having a sequence complementary to a part of the specific base sequence. A primer (wherein either the first or second primer has an RNA polymerase promoter sequence added to its 5 ′ end), and an enzyme group, as well as other components necessary for the nucleic acid synthesis reaction, For example, it is carried out continuously in one reaction vessel containing dNTPs, NTPs, and buffers and salts required for enzyme activity. As the temperature in this reaction, a temperature used in a known TRC method or NASBA method can be used, but the reaction conditions can be appropriately selected from the viewpoint of screening an enzyme having a desired performance.

  Further, the step (F) may be repeated after the step (F). When the second and subsequent steps (F) are performed, the steps (D) and (E) may be accompanied, or only the step (F) may be repeated. When the steps (D) and (E) are involved, an extraction or purification step that further increases the purity of the enzyme may be performed. The first step (F) and the second and subsequent steps (F) may be the same performance evaluation step, or may be different performance evaluation steps. For example, the first step (F) is a so-called primary screening, an enzyme having one or more activities among (1) to (3) is screened, and a desired performance is obtained in the second and subsequent steps (F). It is also possible to screen for enzymes having In yet another aspect, after obtaining an enzyme having a desired performance in the screening, the enzyme having higher performance is screened by further performing steps (A) to (F) on the polynucleotide of the enzyme. You can also.

  After nucleic acid synthesis, the enzyme activity is measured by measuring the amount of synthesized nucleic acid, and the performance of the enzyme is evaluated. Examples of the measurement of the amount of synthesized nucleic acid include separation and visualization of nucleic acid by agarose electrophoresis and ethidium bromide, and quantitative determination of products generated during the synthesis process, such as pyrophosphate. A more preferable example is real-time detection using a fluorescent probe, and a method for quantifying nucleic acid amplified by target nucleic acid synthesis by amplification or reduction of fluorescence is known.

  The mutant having the desired performance obtained by the screening method of the present invention can be applied to any function as long as the function is recognized as a difference in the measurement method, Examples include mutants with improved heat resistance, mutants not affected by surfactants, mutants not affected by organic solvents, and the like. For example, when it is desired to obtain a mutant with improved heat resistance, the temperature of the nucleic acid synthesis described above may be set higher than usual, or the enzyme before nucleic acid synthesis may be left at a constant temperature for a predetermined time. . For example, a time sufficiently long for the enzyme to work at a temperature higher than 37 ° C., preferably higher than 45 ° C., more preferably higher than 50 ° C., more preferably higher than 55 ° C., for example more than 1 minute It is held for a long time, preferably longer than 5 minutes, more preferably longer than 10 minutes, more preferably longer than 20 minutes, more preferably longer than 30 minutes.

  Hereinafter, the present invention will be described in detail using an example of a screening method for avian myeloblastoma virus (AMV) reverse transcriptase having desired performance. This example is an embodiment of the present invention. However, the present invention is not limited thereto.

（２）ＰＣＲ産物を、１％アガロース電気泳動で泳動後、エチジウムブロマイド染色を行ない、染色後のゲルから目的産物のバンドを切り出すことで精製した。
（３）精製したＡＭＶ逆転写酵素遺伝子を含むＤＮＡを制限酵素ＥｃｏＲＩおよびＨｉｎｄＩＩＩで消化後、同酵素で消化したｐＴｒｃ９９ａプラスミドにＴ４リガーゼを用いて４℃で３０分反応させ、反応後のＤＮＡ溶液をＡＭＶ逆転写酵素遺伝子変異体ライブラリーとした。
（４）さらに作製したライブラリーを定法に従い大腸菌ＨＢ１０１株に形質転換し、３７℃のＬＢ／Ｃｒｂ寒天培地（１０ｇ／Ｌ ポリペプトン、５ｇ／Ｌ 酵母エキス、１０ｇ／Ｌ ＮａＣｌ、１５ｇ／Ｌ 精製寒天、０．１ｍｇ／ｍＬ カルベニシリン（ｐＨ７．４））で一晩培養し変異体候補株を得た。 Example 1 Preparation of Avian Myeloblastoma Virus (AMV) Reverse Transcriptase Gene Mutant Library Plasmid vector pTrcAMVB having the E. coli codon-type AMV reverse transcriptase gene (SEQ ID NO: 5) by the method described in Patent Document 4. Was made. The nucleotide sequence information of the plasmid vector pTrcAMVB is shown in SEQ ID NO: 7. A mutation was introduced into the AMV reverse transcriptase gene of the prepared plasmid vector pTrcAMVB by the following procedure.
(1) An error-prone PCR reaction was performed based on the reaction solution composition shown in Table 1 using the plasmid vector pTrcAMVB as a template plasmid. For the PCR reaction, a thermal cycler (Eppendorf) was used and heated at 95 ° C. for 2 minutes, and then a temperature cycle of 95 ° C./30 seconds, 55.7 ° C./30 seconds, 72 ° C./3.5 minutes was repeated 40 times. It was.

(2) The PCR product was subjected to 1% agarose electrophoresis, purified by ethidium bromide staining, and the target product band was cut out from the stained gel.
(3) After digesting the DNA containing the purified AMV reverse transcriptase gene with restriction enzymes EcoRI and HindIII, the pTrc99a plasmid digested with the enzymes is reacted at 4 ° C. for 30 minutes using T4 ligase. An AMV reverse transcriptase gene mutant library was used.
(4) Further, the prepared library was transformed into E. coli strain HB101 according to a conventional method, and LB / Crb agar medium (10 g / L polypeptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L purified agar, By culturing overnight with 0.1 mg / mL carbenicillin (pH 7.4), mutant candidate strains were obtained.

Example 2 Cultivation of Mutant Candidate Strain 2 × YT-Pna medium (pH 6.0) (16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride) in a 96-well plate (manufactured by Greiner Japan) 0.5 g / L monobasic sodium phosphate dihydrate, 2.5 g / L dibasic sodium phosphate dodecahydrate, 0.1 mg / mL carbenicillin sodium) 200 μL / well, Example 1 The mutant candidate strain prepared in (1) was inoculated one colony at a time, and preculture was performed by shaking culture at 37 ° C. and 2000 rpm overnight. A part of the preculture was stored as a glycerol stock at -80 ° C.
2 × YT-Pna medium (pH 6.0) (16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride, 14.5 g / L primary phosphorus in a 96-well deep well plate (made by Greiner Japan) Sodium phosphate dihydrate, 2.5 g / L dibasic sodium phosphate dodecahydrate, 0.1 mg / mL carbenicillin sodium) 500 μL / well, inoculate 50 μL each of the precultured solution, The shaking culture was performed at 37 ° C. and 1500 rpm for 3 hours. Furthermore, IPTG was added to each well so that it might be set to 5 mM, and it culture | cultivated by shaking overnight at 25 degreeC * 2000rpm, and the expression of the avian myeloblastoma virus (AMV) reverse transcriptase was promoted.
The cultured cells were centrifuged at 4 ° C. and 2500 rpm for 30 minutes, the supernatant was discarded, and the cells were collected and stored at −30 ° C.

続いて各ウェルに開始溶液（１８ｍＭ ＭｇＣｌ₂、１００ｍＭ ＫＣｌ、３．８％（ｗ／ｖ） グリセロール、１０．４％（ｖ／ｖ） ＤＭＳＯ）を１０μＬ混合し、ＡＢＩ７３００ＲｅａｌＴｉｍｅ−ＰＣＲ装置（商品名）（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製）を用いて、４６℃・１５秒、４７℃・３０秒、４６℃・１５秒の温度サイクルを３０回繰り返し、サイクルごとに蛍光強度を測定した。
野生型のＡＭＶ逆転写酵素と比較し蛍光強度の増加が早いサイクルで観察される変異体を耐熱性変異株として取得した。 Example 3 Evaluation of Enzyme Activity of Avian Myeloblastoma Virus (AMV) Reverse Transcriptase Extract The extract (BugBuster (trade name) (manufactured by Merck)) solution, 50 mM Tris-HCl ( pH 7.2), 0.2 mg / mL egg white lysozyme, 25 U / mL Benzonase (trade name) (manufactured by Merck & Co., 10 mM DTT) was added at 100 μL / well, and the mixture was shaken at 25 ° C. and 1500 rpm for 1 hour, followed by 4 Centrifugation was performed at 2500 ° C. for 20 minutes, and the supernatant was collected to obtain an extract.
The enzyme activity was measured based on the nucleic acid amplification method described in Patent Document 2. 15 μL of the reaction solution (mixed reagent) having the composition described in Table 2 was dispensed into a 96-well plate for PCR (manufactured by ABI), and the extract was diluted 10-fold with 10 mM DTT aqueous solution and 5 μL of the solution was added. .

Subsequently, 10 μL of the starting solution (18 mM MgCl ₂ , 100 mM KCl, 3.8% (w / v) glycerol, 10.4% (v / v) DMSO) was mixed in each well, and the ABI7300 RealTime-PCR apparatus (trade name) (Applied Biosystems) was used, and a temperature cycle of 46 ° C. for 15 seconds, 47 ° C. for 30 seconds, and 46 ° C. for 15 seconds was repeated 30 times, and the fluorescence intensity was measured for each cycle.
A mutant in which the increase in fluorescence intensity was observed in a cycle earlier than that of the wild type AMV reverse transcriptase was obtained as a heat-resistant mutant.

Example 4 Preparation of Avian Myeloblastoma Virus (AMV) Reverse Transcriptase In order to evaluate the heat resistance of the heat resistant mutant selected in Example 3, the heat resistant mutant was cultured and purified.
(1) 2 × YT-Pna medium (pH 6.0) (16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride, 14.5 g / L monobasic sodium phosphate dihydrate in a test tube , 2.5 g / L dibasic sodium phosphate twelve hydrate, 0.1 mg / mL carbenicillin sodium) was dispensed in 2 mL, and 20 μL of the glycerol stock prepared in Example 2 was inoculated at 37 ° C./160 rpm. Preculture was performed by overnight shaking culture.

  (2) 2 × YT-Pna medium (pH 6.0) (16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride, 14.5 g / L monobasic sodium phosphate dihydrate in a 100 mL flask , 2.5 g / L dibasic sodium phosphate twelve hydrate, 0.1 mg / mL carbenicillin sodium) was dispensed 20 mL, inoculated 200 μL of the preculture, and cultured with shaking at 37 ° C./160 rpm for 3 hours. Was done. Furthermore, IPTG was added to the culture solution to 5 mM, and the culture was shaken overnight at 25 ° C. and 160 rpm to promote the expression of AMV reverse transcriptase. Thereafter, the cells were collected by centrifuging the culture solution at 4 ° C. and 8000 rpm for 30 minutes, and stored frozen at −30 ° C.

  (3) Frozen cells were suspended in 5 mL / wet cell mass of buffer A (20 mM Tris-HCl (pH 7.2), 10% (w / v) glycerol, 4 mM DTT), and sonicator (Insonator 201M, Kubota Seisakusho). Was subjected to ultrasonic crushing under conditions of 150 W and 4 ° C., and centrifuged at 4 ° C. and 8000 rpm for 30 minutes to remove unbroken cells and obtain an extract.

  (4) Streptomycin hydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in the extract of (3) so that the concentration was 0.83% (w / v), and stirred at 4 ° C. for 30 minutes. Thereafter, the AMV reverse transcriptase was recovered as a precipitate by centrifuging at 4 ° C. and 8000 rpm for 30 minutes, and resuspended in an equal amount of buffer A to the removed supernatant. The supernatant obtained by further centrifuging this resuspension at 4 ° C. and 8000 rpm for 30 minutes was recovered. 500 μL of this supernatant was mixed with 500 μL of buffer B (20 mM Tris-HCl (pH 7.2), 10% (w / v) glycerol, 4 mM DTT, 170 g / L ammonium sulfate), and then buffer C (20 mM Tris-HCl (pH 7)). .2) Mix with 500 μL of 50% slurry of Toyopearl PPG-600M (manufactured by Tosoh Corporation) equilibrated with 10% (w / v) glycerol, 4 mM DTT, 85 g / L ammonium sulfate), and invert for 10 minutes at 4 ° C. The adsorption was performed. After centrifuging at 4 ° C. and 5000 rpm for 1 minute, the unadsorbed portion was removed. Subsequently, 1.7 mL of buffer D (buffer mixed with buffer A and buffer C at 2: 8) was added to the adsorbed gel, and the mixture was stirred by inverting several times and then centrifuged at 4 ° C. and 5000 rpm for 1 minute. The gel was washed by removing the supernatant. This washing was repeated once more, 250 μL of buffer A was added to the washed gel, and the mixture was stirred by inverting at 4 ° C. for 10 minutes to elute from the gel, and then centrifuged at 4 ° C. and 5000 rpm for 1 minute to remove the gel. Kiyoshi was recovered as an AMV reverse transcriptase solution of each thermostable mutant and stored at 4 ° C.

Example 5 Evaluation of Heat Resistance of Avian Myeloblastoma Virus (AMV) Reverse Transcriptase Solution The heat resistance evaluation of the AMV reverse transcriptase solution derived from each heat resistant mutant prepared in Example 4 was performed.
(1) Add 40.3 μL of sterilized water to 3.2 μL of AMV reverse transcriptase solution, and then add 5.2 μL of DMSO (final concentration 10.4% (v / v)) and mix. Held at 0C. This enzyme reaction solution was used as a non-heated enzyme reaction solution, and a portion of this non-heated enzyme reaction solution was heated at 46 ° C. for 10 minutes using a thermal cycler (manufactured by Eppendorf), then cooled at 4 ° C. for 3 minutes and heated enzyme It was set as the reaction liquid.
(2) A mixed reagent is prepared with the composition described in Example 3, and 15 μL is dispensed into a 0.5 mL PCR tube (GeneAmp Thin-Walled Reaction Tubes, manufactured by PerkinElmer), and the non-heated enzyme reaction solution or 5 μL of the heated enzyme reaction solution was added and incubated at 46 ° C. for 2 minutes. Subsequently, 10 μL of the starting solution (18 mM MgCl ₂ , 100 mM KCl, 3.8% (w / v) glycerol, 10.4% (v / v) DMSO) was mixed in each PCR tube, and TRCRapid-160 (manufactured by Tosoh Corporation). ) Was used to measure changes in fluorescence intensity.

  (3) Fluorescence in the same manner using AMV reverse transcriptase (manufactured by Life Technology) appropriately diluted with buffer A (20 mM Tris-HCl (pH 7.2), 10% (w / v) glycerol, 4 mM DTT) A change in intensity was measured, and a calibration curve was prepared to estimate the number of reverse transcriptase units from the time when the fluorescence intensity was 1.2 times the initial fluorescence (FIG. 3). Using this calibration curve, the activity of the avian myeloblastoma virus (AMV) reverse transcriptase was estimated for each of the non-heated enzyme reaction solution and the heated enzyme reaction solution, and the activity of the heated enzyme reaction solution was determined by the activity of the non-heated enzyme reaction solution. The residual activity was calculated. As a result of comparing this residual activity with the AMV reverse transcriptase reaction solution derived from the wild type and each heat-resistant mutant, the heat resistance of the enzyme derived from each heat-resistant mutant is improved compared to that derived from the wild type. (Fig. 4).

Example 6 Determination of base sequence of avian myeloblastoma virus (AMV) reverse transcriptase gene A plasmid was extracted from a part of the cells cultured in Example 4 by a conventional method, and avian myeloblasts contained in the extracted plasmid The nucleotide sequence of the tumor virus (AMV) reverse transcriptase gene was determined by the following method.
(1) Using Big Dye Terminator v3.1 cycle Sequencing Kit (trade name) (Applied Biosystems), 2.0 μL of attached buffer, 4.0 μL of premix, 3.2 pmol of synthetic DNA primer, 500 ng of template plasmid Prepare to 20 μL with sterilized water, use a thermal cycler (Eppendorf) and heat at 96 ° C for 1 minute, then 25 cycles of 96 ° C for 10 seconds, 50 ° C for 5 seconds, 60 ° C for 4 minutes. Repeated.
(2) The sample for determining a base sequence prepared in (1) was purified by the method shown below using a Centri-Sep spin column (trade name) (manufactured by Applied Biosystems).
(2-1) 800 μL of sterilized water was added to the Centri-Sep spin column, and the gel dried by vortexing was fully hydrated.
(2-2) After confirming that there were no bubbles in the column, the column was allowed to stand at room temperature for 2 hours or more.
(2-3) The upper cap and the lower stopper were removed in order, and the sterilized water in the column was allowed to fall naturally onto the gel surface, and then centrifuged at 730 × g for 2 minutes.
(2-4) A base sequence determination sample was applied to the center of the spin column prepared in (2-1) to (2-3), and the sample was collected in a tube by centrifugation at 730 × g for 2 minutes.
(2-5) The collected sample was dried under reduced pressure and then dissolved in formamide.
(3) The base sequencing sample prepared in (2) was treated at 95 ° C. for 2 minutes, rapidly cooled on ice, and then analyzed with ABI PRISM310-DNA Analyzer (trade name) (Applied Biosystems) The sequence was determined. The synthetic DNA primers used for the base sequence determination were selected from those shown in SEQ ID NOs: 15 to 22 as necessary.
(4) The determined base sequence is GENETYX ver. Analysis was performed using 11.0.1 (trade name) (manufactured by Genetics).

As a result of the analysis, the following seven mutants were identified from the heat-resistant mutants.
(A) A mutant in which the 658th serine of the reverse transcriptase amino acid sequence described in SEQ ID NO: 1 is substituted with glycine. The avian myeloblastoma virus (AMV) reverse transcriptase having this mutation is an S658G mutant, the gene sequence of which is shown in SEQ ID NO: 23, and the amino acid sequence thereof is shown in SEQ ID NO: 24.
(B) A mutant in which the 813rd valine of the reverse transcriptase amino acid sequence described in SEQ ID NO: 1 is substituted with alanine. The avian myeloblastoma virus (AMV) reverse transcriptase having this mutation is a V813A mutant, the gene sequence of which is shown in SEQ ID NO: 25, and the amino acid sequence thereof is shown in SEQ ID NO: 26.
(C) A mutant having the mutation in which the 192nd aspartic acid is replaced with glutamic acid and the 715th threonine in the mutation of the reverse transcriptase amino acid sequence described in SEQ ID NO: 1. The avian myeloblastoma virus (AMV) reverse transcriptase having this mutation is a (D192E, T715M) mutant, the gene sequence is shown in SEQ ID NO: 27, and the amino acid sequence is shown in SEQ ID NO: 28. Further, the avian myeloblastoma virus (AMV) reverse transcriptase having a mutation in which the 192nd aspartic acid is replaced with glutamic acid is a D192E mutant, the gene sequence of which is SEQ ID NO: 29, and the amino acid sequence of SEQ ID NO: 30 The avian myeloblastoma virus (AMV) reverse transcriptase having a mutation in which the 715th threonine is replaced with methionine is used as a T715M mutant, the gene sequence is shown in SEQ ID NO: 31, and the amino acid sequence is shown in SEQ ID NO: 32. Each is shown.
(D) A mutant in which the 583rd alanine in the amino acid sequence of the reverse transcriptase described in SEQ ID NO: 1 is replaced with threonine. The avian myeloblastoma virus (AMV) reverse transcriptase having this mutation is an A583T mutant, the gene sequence of which is shown in SEQ ID NO: 33, and the amino acid sequence thereof is shown in SEQ ID NO: 34.
(E) A mutant in which the 65th serine of the reverse transcriptase amino acid sequence described in SEQ ID NO: 1 is substituted with glycine. The avian myeloblastoma virus (AMV) reverse transcriptase having this mutation is an S65G mutant, the gene sequence of which is shown in SEQ ID NO: 35, and the amino acid sequence thereof is shown in SEQ ID NO: 36.
(F) A mutant in which the 113th phenylalanine of the reverse transcriptase amino acid sequence described in SEQ ID NO: 1 is substituted with serine. The avian myeloblastoma virus (AMV) reverse transcriptase having this mutation is an F113S mutant, the gene sequence of which is shown in SEQ ID NO: 37 and the amino acid sequence of SEQ ID NO: 38.
(G) A mutant in which the 689th alanine in the amino acid sequence of the reverse transcriptase described in SEQ ID NO: 1 is substituted with valine. The avian myeloblastoma virus (AMV) reverse transcriptase having this mutation is an A689V mutant, the gene sequence is shown in SEQ ID NO: 39, and the amino acid sequence is shown in SEQ ID NO: 40.

Claims (3)

(A) (1) RNA-dependent DNA polymerase activity, a mutation in a polynucleotide encoding an enzyme having all activity of (2) DNA-dependent DNA polymerase activity, and (3) the ribonuclease H (RNase H) activity Introducing the process;
(B) inserting the polynucleotide into which the mutation is introduced into a vector;
(C) transforming a host with a vector having the polynucleotide inserted therein;
(D) culturing the obtained transformant and expressing the enzyme;
(E) extracting the enzyme from the culture solution of the transformant;
(F) evaluating the performance of the enzyme;
In the method for screening an enzyme, the performance evaluation of the enzyme in the step (F) is evaluated by performing nucleic acid synthesis that requires all of the activities (1) to (3), Screening method. The screening method according to claim 1 , wherein the enzyme is avian myeloblastoma virus (AMV) reverse transcriptase. The performance evaluation of the enzyme in the step (F) was carried out using a first primer having a sequence homologous to a part of the specific base sequence and a second primer having a sequence complementary to a part of the specific base sequence ( Here, either one of the first and second primers has an RNA polymerase promoter sequence added to the 5 ′ end thereof, and the specific base sequence is subjected to the following steps (I) to (V): Alternatively, the screening method according to claim 1 or 2 , wherein the evaluation is performed by synthesizing RNA containing a sequence complementary to the sequence.
(I) a step of synthesizing cDNA complementary to a specific base sequence or a sequence complementary to the sequence with an enzyme having RNA-dependent DNA polymerase activity;
(II) a step of degrading RNA-DNA double-stranded RNA by an enzyme having RNase H activity (generation of single-stranded DNA),
(III) A double-stranded DNA having a promoter sequence capable of transcribing a specific base sequence using the single-stranded DNA as a template or RNA complementary to the sequence is generated by an enzyme having DNA-dependent DNA polymerase activity. The process of
(IV) generating an RNA transcript using the double-stranded DNA as a template from the promoter sequence by an enzyme having DNA-dependent RNA polymerase activity;
(V) A step of generating RNA transcripts in a chain by using the RNA transcript as a template for cDNA synthesis in the step (I).

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