JPH01252291A - Vector for site-specific mutagenesis - Google Patents

Abstract

PURPOSE:To obtain a plasmid/phage fused vector collectible of mutated DNA in a high probability and capable of inserting DNA of large size, containing a gene in which an amber mutated codon in a gene region coding ampicillin resistance. CONSTITUTION:A vector pBSM 13(+) inserted with 454 nucleotide fragments from Rsa I to Dra I site of M13 DNA to restricted enzyme Nar I site of commercialized pBS vector and pBSM 13 are used as starting materials. The vector is a plasmid/phage fused vector containing an intergenic region derived from a bacteriophage M13, and an amber mutation is introduced to the vector. For instance; amber mutation is introduced, giving attention to 37 salts from Dde I site (2715) to Hpa 11 site (2752) of a gene region coding ampicillin resistance.

Description

[Detailed description of the invention]

(Field of Industrial Application) The present invention is directed to site-specific mutagenesis used in genetic engineering.
sis). (Conventional technology) In recent years, mutation methods using genetic engineering methods have become an important tool because mutant strains and mutant genes can be obtained more accurately and more frequently than mutation methods using conventional genetic methods. . Mutation by this genetic engineering method is a method of introducing a desired mutation into a certain DNA sequence, and is based on molecular biology,
This has become one of the most effective tools for research such as protein engineering research. The structure of a protein can be artificially changed by manipulating the DNA sequence of the coding region, so by changing the structure of a protein found in nature, such as an enzyme, it is possible to modify it into an enzyme with effective properties. It is becoming. The properties relate to enzyme functions such as protein action pl+, stability, heat resistance, Km value, and protease resistance. By changing these properties, it is possible to modify the enzyme to meet the design goals, and this is expected to have great potential in the future. In addition, gene expression takes place through the processes of transcription, translation, and RNA processing, but if we consider sequentially changing the base sequence at the DN^ level, the degree of expression may change. It is an important and indispensable method for improving productivity and researching the expression mechanism. In addition, site-directed mutagenesis techniques are widely used in molecular biology and genetics, such as searching for active sites in physiologically active substances such as enzymes and peptides, and elucidating secretion mechanisms of physiologically active substances. A typical procedure for site-directed mutagenesis consists of the following items: (See Figure 1) (1) Clone the DNA fragment you want to mutate into a vector that can exist as single-stranded DNA or double-stranded DNA. (2) An oligonucleotide synthesized complementary to the base portion that mismatches the region to be mutated at one location is hybridized to a circular-main-stranded combination DNA. (3) Next, using a complementary double-stranded oligonucleotide as a primer, filling-in is performed using DNA polymerase, and ligation is performed to connect the newly synthesized strand to the 5' end of the oligonucleotide. (4) H,co! double-stranded DNA that is completely complementary except for the desired mutation point. i and allow it to replicate, and the parental DNA
and a phage clone having the mutant DNA are grown. In this way, phage derived from two types of DNA are prepared, a mutant phage clone is isolated, and mutant DNA is created from this. In this case, the probability of generation of each DNA-derived phage is theoretically calculated to be 50%. However, in reality, the yield of mutant DNA is extremely low. (Problems to be Solved by the Invention) In conventional site-directed mutagenesis methods, phage DNA, that is, vectors derived from the three old phages, are used as vectors, but they have the following disadvantages:
It has been pointed out that this method cannot be used efficiently. That is, since this vector is derived from a phage, it has the disadvantage that it is difficult to insert large NA fragments, for example, DNA with a length of 5 Kb or more. Furthermore, it has been pointed out that such phage vectors have the disadvantage that mutations are likely to occur during the replication process peculiar to phages. (Means for Solving the Problems) The present inventors have developed a vector that has improved these drawbacks, namely: (1) mutant DNA can be collected with a high probability; (2) large-sized DNA can be inserted. There is, (3) compound =
The present invention has been achieved by successfully producing a vector having these advantages, such as the recombinant vector not undergoing mutation during the 3-type. That is, the present invention is a plasmid/phage fusion vector containing a gene encoding ampicillin resistance, and is a patent characterized in that an amber mutation codon is incorporated into the gene region encoding ampicillin resistance. In the present invention, a commercially available pBS vector (Stra
tagene) restriction enzyme Nar I site,
A 454 nucleotide fragment from Rsa I to Dra I site of M13 DNA was inserted into the synthesized vectors pBsM13 + and pBsM13 − (Str
Atagene) was used as a starting material. This vector contains an intergenic region derived from bacteriophage M13 and is a plasmid/phage fusion vector. This vector is characterized by a small number of base sequences (320
It has the advantage of being able to insert genes of large size (5 Kb or more), and does not suffer from the disadvantage of M13 vectors, that is, the instability that causes mutations during replication. E to hold this vector
．． When E. coli is infected with M13 helper phage, single-stranded DNA is produced in good yield. In this method, single-stranded D
Since NA can be collected, single-stranded mutations D can be generated by mutagenesis using helper phages and oligonucleotides.
Can produce N^. However, this vector has the disadvantage that it is not easy to specifically identify mutant DNA from the parent single-stranded DNA that can be produced simultaneously. The vector of the present invention is a vector that can be improved to specifically select phage clones having mutant DNA in a host and rapidly produce mutants. As mentioned above, in conventional site-directed mutagenesis using a phage vector, a vector into which an unhar mutation has been introduced, that is, an oligonucleotide incorporating the desired mutation to be created, is annealed, and the hybrid F' 11 N is filled in and ligated with an enzyme. Transform A.
When replicated, the mutant phage clones were 5upE-strain, 5
Although any upE' strain can replicate and grow, the parent phage clone uses a vector that can replicate only in the 5upE+ strain. In this vector, as mentioned above, phage clone is, large size DNA
It has the disadvantage of not being able to be inserted, and of unstable replication. The site-directed mutagenesis vector of the present invention has M
This is a plasmid/phage fusion vector with an intergenic region of 13 phages, in which an amber mutation is introduced. We focused on the gene that coats the protein and introduced mutations without altering its function. For example, Dd in the gene region encoding ampicillin resistance
e 1lpall site from I-Sai-(2715)
Focusing on the 37 bases up to (2752), an amber mutation was introduced using the method shown below. This DNA fragment (Dder-11pa I
The base sequence of I) is shown below. In order to introduce an amber mutation into this region, a DNA fragment having the following base sequence was synthesized. As shown in the base sequence of the synthetic 11NA fragment above, 985
From Dde Izyte of M13 vector, 1Ipal
Two TTG codons in the DNA fragment up to the T site were changed to CTA. This allows TT
The stop codon is changed from asparagine encoded by G to CTA. In the 5up(-) strain, this is a special tR that has the function of incorporating glutamine in response to the amber codon UAG.
Since it does not have NA, the peptide chain ends there, and the protein function is lost and it cannot be produced, making the strain unable to grow, whereas the 5up(+) strain has the characteristic of being able to grow. . This mechanism makes it possible to distinguish between mutant phage clones and parent phage clones. In other words, in the process of creating site-specific mutants, the DNA of the mutant phage clone + plasmid/phage fusion vector (pBS old 3 (+), 985M13 (
Since it is derived from the (÷) strand of -), it does not contain an amber mutation, whereas the DNA of the parent phage clone contains an amber mutation. This is because if a phage clone carrying IIJ DNA infects an Amber Sup host and cultivates it, the Amber mutation will be rescued, but the 5up (-)
It cannot grow on stocks. On the other hand, if a mutant phage clone that does not have the amber mutation is infected with the 5up (-) strain and cultured, it can grow, so only mutant phage clones can be specifically selected. The vector of the present invention includes T7 RNA bromoter, T3 RNA
The bromoter is a polylinker site (EcoRI, Sa
cI. 3ma I X BamHI,, Xba I,
SalI, 1linclI, AccI. Pst I, Sph I, tlind m)
are placed on both sides. For this reason, the gene that has undergone site-specific mutation is immediately transferred to T7RN.
A polymerase T3 RNA polymerase synthesizes and translates RNA to produce proteins, which has the advantage of being able to examine changes in function due to mutations. It has many polylinker sites, making it easy to insert foreign genes. The vector was transformed into pKMN(+) and pKMN(
−). Next, the construction procedure of pKMN(+) and pKMN(-) will be shown. (1) 985M13(+) and 985M13(
-) from 8at II (3140) -Pst
Adjustment of I (924) (989bp) First 985M
13(+), 985M13(-) to 13 to adjust the region containing the intergenic region 13, double digestion was performed with restriction enzymes AatIl and Pst■, and the digested product was subjected to agarose electrophoresis to obtain a 989bp long DNA. The fragment was isolated and purified. 985M1
The restriction enzyme maps of 3 (+) and 985M13 (-) are shown in FIGS. 2 and 3. Aat U (3140)
-Pst I (924) (989 bp) The reaction conditions for double digestion for the preparation of the (989 bp) fragment are as follows. The above mixture was reacted for 2 hours at a temperature of 37°C. (2) 8at II from pBsM13(+) (
3139) -Hpa II (2752) (388b
p), Dde I (2714) - Bgl I (
2338) (377bp), Bgl I (2337)
-Pst I (925) (1413bp) pKMN(+), pKMN(
-) In order to prepare each DNA fragment for vector construction, double digestion was performed with each restriction enzyme, the digested products were subjected to agarose electrophoresis, and separated and purified from agarose. In this way, the DN shown in Figure 4
The A fragment was trimmed 8 times. (3) Hpa II (2751) -Dde I
(2715) Synthesis of fragment pBsM13(+), l1p of pBsM13(-)
a II (2751) -Dde I (2715)
In order to impart an amber mutation to the region, the following I) NA fragment was synthesized according to a conventional method. (4) Construction of pKMN(+) and pKMN(-) of the present invention In order to construct vectors pKMN(+), pKMN, and (-) with amber mutations from the fragments prepared in the above process, a mixture of each fragment was injected into T4DNA. Ligation was performed overnight at 16°C using ligase. After ligation, the reaction solution was mixed with commercially available B, coliB
It was transformed into MH71-18MIITS (Sup(+)). Three clones were obtained from the reaction solution for producing pitMN(+), and two clones were obtained from the reaction solution for producing pKMN(-). The plasmid obtained from this clone was
The vectors were transformed into liMK30-3 (Sup(-)) and tested for clone production, but no clones were produced, confirming that these five vectors had an amber mutation. Furthermore, in order to confirm the orientation of the intergenic region, each plasmid was prepared from the transformed strain. The prepared plasmid was digested with restriction enzyme Nae I −
Double digestion was performed with Pvu I and Bam1l I, and the fragment length was examined by electrophoresis of the decomposed product to confirm the directionality. This was the expected result. The created pKMN (+) and pKMN (~) are
As shown in FIG. Next, an example will be shown in which a base sequence encoding a physiologically active substance was subjected to site-specific mutation using the site-directed mutation vector of the present invention. Example 1 By mutating two bases of the approximately 6000 bp DNA base sequence (hereinafter referred to as rII-1^) encoding rat brain membrane protein, A at position 1315 of the protein was mutated.
We tried changing rg to Gln and Arg at position 1319 to Gin. In order to determine whether such mutations were successful, one base was mutated to introduce a site that can be cleaved with the restriction enzyme Nsv I. Each step is shown below. (1) pKMN inserted with foreign gene γII-LA
Prepared γll-18 of pKMN(-) of the present invention was inserted into the polylinker site of pKMN(-) of the present invention by a conventional method to create a recombinant plasmid pKM.
N(-) (T II-IA) was inserted. This recombinant plasmid was transformed into Escherichia coli JOld01, and TIKMN(-)(711-18)/JMIOI containing the recombinant plasmid pKMN(-) (γII-LA) was prepared (21 pKMN(-) ) Preparation of 5sDNA of (Tll-18) pKMN(-) (r II-IA)
/ JMIOI as a medium (Gata! Tryptone: 16g/l
, yeast extract: 10 g/I, NaCl: 15
g/l, pH 7,6) and O, D, 1looo
= 0.3, helper phage R408 was added to moi 20 and cultured with horizontal tilting shaking (37°C for 11 h).
r) was performed. Based on this, add 50ml of medium (2xYT, ampicillin 100μB7) in a 500ml flask.
ml, 100 μg of kanamycin (7 ml, 0.01% thiamine)). After culturing, centrifuge and add 20% polyethylene glycol 6000 and 2.5% NaCl to the supernatant.
M was added to precipitate the phages. After centrifugation (8000 rpm, 15 minutes), the precipitate was centrifuged at 600 rpm.
It was added and suspended in μm Tlj buffer. Furthermore, xlOIlight Buffer 70 B 1,
Add 70 μl of DNase and RNase mixture,
Incubate at 7°C for 11 hours. After the reaction was completed, phenol treatment and chloroform treatment were repeated twice, NaCl was added to the aqueous layer, the mixture was left at -20°C, centrifuged, and the precipitate was collected. 300 μm in the precipitate
Add TE buffer and dissolve 5sDNA (15 μg).
was dissolved. Furthermore, phenol treatment and ethanol precipitation were performed, and the precipitate was added to 100 μl of distilled water to give 5 sD
A NA solution was prepared. (3) 985M13(-) with γn-IA inserted
r Preparation of II-IA 985M13(-) prepared from 985M13(-) and γII-IA by a conventional method T II-IA 15
8 g was cut with restriction enzymes Nsi 1 and old ul. After further treatment with alkaline phosphatase, acrylamide gel electrophoresis was performed. After electrophoresis, it was extracted from the gel and the linear DNA Nsi I-Mlu I fragment l-985M13(-) (γII-IA) was prepared. (4) Adjustment of mutant primers The amino acid of the gene encoding rat brain membrane protein, namely A
rg (1315)-Gln, Arg (1319)→
→ In order to change to Gln, the following primers were prepared. At the same time, a primer (lower strand) with the following DNA sequence was prepared in order to change the third codon of Phe (1320) from T to C in order to select the mutation only by restriction enzyme analysis. In this case, Phe (1320) remains unchanged and does not change to other amino acids.・The marked part is the mismatched base. When DNA is synthesized using the lower strand as a template

The part in [ ] has the following sequence, and a new N5pV site will be created in the underlined part. ↓ The above primer used for mutation (oligomer 56mer
) was labeled using the following method. Primer (12 pmol) 2 pl Kinase buffer 0.5 μm (γ-32P)-ATPO, 5 μm 3.5
μm The above mixture was reacted at 37°C for 30 minutes. 10n+M ATP (cold) 0.5μ
ml 1.0 μl The above mixture was further added and reacted at 37°C for 30 minutes. (5) Construction of gapped duplex DNA (8) Vector containing the dsDNA fragment created by hybridization of dsDNA and 5sDNA (3), namely pBsl'11 containing the Nsi I-old uI fragment
3(-) (T11-18) 0.6 μg and 5sDN81 of pKMN(-) (γ■-IA) prepared in (2)
．． 5 μg was hybridized as follows. After making the above mixture and heating it at 100°C for 3 minutes,
Incubate for an additional 5-10 minutes at °C. After this, it was frozen at -20°C. The reaction solution for sample (1) was N5ir.
-old ul fragment I-pBsM13(-) (r n
-IA) double strand is missing, and Nsi I -M
lu I fragment-pBsM13(-) (γII
-IA) and pKMN(-)(γII-LA) 5sDN
It was confirmed by agarose electrophoresis that 8 had undergone hybridization. (B) Annealing of mutant primers The hybridization reaction solutions prepared in (5) to (8) and the lahered mutant primers prepared in (4) were annealed as follows. Hybridization reaction solution 8 μm labeled mutant primer 1.51tl 1゜0
0.5 μm 10.0 μm The above mixture was prepared, heated at 65° C. for 5 minutes, and then slowly cooled to room temperature for annealing. (C) Polymerization and ligation (5)-
Polymerization and filling-in of the primer annealed DNA prepared in (B) were performed as follows. Distilled water
23 μmxlo filling buffer
4 μm Klenow fragment 1
μIT4 DNA ligase
2μ 140μm After reacting the above mixture at room temperature for 45 minutes, 0.5M
The reaction was stopped by adding 1 uI of EDTA (pH 8,0) and incubated for 65°CIO minutes. The ×10 filling buffer has the following composition. -19=625mM KCl 275mM Tris-11cI (pH7,5)1
50mM M@C1z 20mM DTT0, 5mM ATPO, 25mM of 4 dNTPs eac
After the reaction, agarose electrophoresis was performed to confirm that the mixture was filled. (D) 30 μl of DNA reaction solution filled in for transformation into E. coli 5uppressor E” strain
, xlo chloramphenicol 20μ+, water 150
p1. E. coli 5uptE" combinant cells were mixed, spread on an agar plate, incubated overnight to grow colonies, and observed the formation of a total of 38 colonies. (E) Isolation of DNA from the colonies. Twelve clones were collected from the colony, plasmids were extracted, and purified to make a 100 μm DNA solution. (F) Transformation into E. coli 5upE- strain (5
) The DNA of each clone prepared in iE) was transformed into E.coli E-strain MX30-3 by the following method. DNA
3 μ 1 Chloramphenicol (xlo)
50 μm 106 μm The above mixture was applied to an agar plate and incubated to grow colonies. The number of colonies that grew was as follows. (G) Isolation of DNA and confirmation of mutation using the restriction enzyme N5pV Pick up a colony from each plate with a toothpick and use the DNA
A was extracted (PIEG precipitation method) and a DNA solution was prepared. Adjusted IIINA? Restriction enzyme Nsν
1, and the digested solution was subjected to agarose electrophoresis to examine the cleavability with the restriction enzyme N5pV. Result 12
Among the clones, 9 clones were cleaved with N5pV, and the 3 l'J-on was not cleaved. In order to confirm that the DNA of the 9 clones had undergone mutations, DNA sequencing was performed using the Sanger method, and Arg (1315)-→Gl
It was confirmed that a mutation occurred from n, Arg (1319) to Gln. (Effects of the Invention) The site-directed mutagenesis vector of the present invention has advantages such that mutated DNA is collected with a high probability, large-sized DNA can be inserted, and the recombinant vector during replication is not mutated. have. By artificially changing the structure of a protein using such a vector for part-specific mutation, it is possible to modify the protein into one with effective properties. Furthermore, by sequentially changing the base sequence, it is possible to study the mechanism of gene expression, and its uses are wide-ranging.

[Brief explanation of the drawing]

FIG. 1 shows a typical procedure for site-directed mutagenesis. FIG. 2 shows a restriction enzyme map of 983M13 (+). FIG. 3 shows a restriction enzyme map of 983M13(-). Figure 4 does not show the three fragments of 983M13(+). FIG. 5 shows a restriction enzyme map of pKMN(+) of the present invention. FIG. 6 shows a restriction enzyme map of pKMN(-) of the present invention. Patent applicant: Toyobo Co., Ltd. -

Claims (1)

[Claims] 1. A plasmid/phage fusion vector comprising a gene encoding ampicillin resistance, the vector for site-specific mutation comprising an amber mutation codon incorporated into the gene region encoding ampicillin resistance.