JPH02447A - Human lymphotoxin synthesizing gene - Google Patents

Abstract

PURPOSE:To facilitate production of study of human lymphotoxin by providing one place of recognition site of 9 kinds of restriction enzymes to recognize 6 base pairs of palindromes, eliminating nucleotide sequence of specific part and providing specific recognition sequence. CONSTITUTION:Nucleotide sequence of human lymphotoxin is modified in the following way to give a synthetic gene. (1) A methionine codon is added to the upper stream of leucine codon, two nonsense codons are added in series to 3'-end and further ClaI to 5'-end and SalI recognition sequence to 3'-end in order to efficiently carry out translation initiation. (2) One place is provided with recognition site of restriction enzyme BssHII, EcoRI, KpnI, HinaIII, XhoI, SacI, SmaI, HpaI and BamHI to recognize 6 pairs of palindromes. (3) Chain length of 100 nucleotides is made into one block and direct repeat in the block is eliminated. (4) Palindromes sequence exceeding 6 base pairs are not made to exist in any of continuous 30 nucleotides.

Description

DETAILED DESCRIPTION OF THE INVENTION OBJECTS OF THE INVENTION The present invention relates to the human lymphotoxin synthesis gene 'IC+. Specifically, by replacing all the amino acids and peptides that make up human lymphotoxin, the overall purpose is to facilitate research on the production of improved human lymphotoxin with enhanced human lymphotoxin activity through genetic engineering. This relates to a newly constructed IJ photoxin synthesis gene.

[Conventional technology]

Human lymphotoxin and this? The encoding cDNA is known, and the amino acid and DNA sequences shown in Figure 1 have been reported (Nature, Vol. 312, 721-724).
Page, 1984).

However, what is shown here is the amino acid and cDNA sequence of naturally occurring human lymphotoxin, and the K used in research on improving human lymphotoxin using genetic engineering techniques is quite inconvenient. be. For example, when the purpose is to study the structure-activity relationship of protein proteins or to enhance the activity of biologically active proteins by exchanging amino acids or peptides, appropriate restriction enzyme recognition sites that enable complete gene conversion may be used. does not have

[Problem that the invention seeks to solve]

In view of this situation, the present invention was made to solve the above-mentioned drawbacks, and the human lymphotoxin synthesis gene of the present invention has the following characteristics.

Bs, a restriction enzyme that recognizes a 6 base pair palindrome
5Hn, EcoRI, Kpnl, Hindll
, Xhol, 5acl, Small, Hpa
It has been modified by possessing only one recognition site for 1 and BamH[.

(2) Without changing the amino acid sequence of the original human lymphotoxin, by changing codons, the direct repeats present in one block of 100 nucleotides are deleted.

(4) No matter which 30 consecutive nucleotides are taken,
Among these, halindrome sequences exceeding 6 base pairs have been deleted.

As an example of a human lymphotoxin synthesis gene that still has such characteristics, it has a c1al recognition sequence at the 5'-end and a 3'
- Care was also taken to have a 5all recognition sequence at the end.

[Means to solve all problems]

■1 Design of a synthetic gene To chemically synthesize a gene, oligonucleotides with a chain length of 20 to 40 nucleotides are synthesized, and their linkage (i.e., the linkage of oligonucleotides that are not intended for the purpose at that stage) Direct repeat f
We must erase as much as possible. In addition, the characteristics of synthetic genes are that they make it possible to study the structure-activity relationship of proteins, as well as to exchange genes with amino acids and peptides in biologically active proteins (thus, to increase activity). It is possible to introduce restriction enzyme recognition sites.

(1) Nucleotide sequence synthesis of human lymphotoxin gene If only the amino acid sequence of the protein is known, the basic nucleotide sequence of the gene can be determined by selecting appropriate codons for each amino acid. decide.

On the other hand, if the nucleotide sequence of the target protein gene or complementary DNA (cDNA) has been determined, that sequence is used as the basis for the nucleotide sequence of the synthetic gene.

In the case of human lymphotoxin, the cDNA nucleotide sequence has been reported (Figure m1) (Nature
, Vol. 312, pp. 721-724, 1984),
This was used as the basis for synthetic gene design.

(2) The N-terminal amino acid of mature human lymphotoxin modified at the 5'- and 3'-ends of the synthetic gene is leucine, which is effective for efficient translation initiation and for the target N-terminus.
A methionine codon was added upstream of the leucine codon to obtain a -terminus. On the other hand, in order to complete the termination of translation, two nonsense codons were added in series at the 3'-end.

Furthermore, in consideration of inserting the constructed synthetic gene into plasmid P, cohesive ends corresponding to both the 5'- and 3'-cohesive ends of the plasmid are added to both ends of the gene. Vector plasmid pG for human lymphotoxin gene
Since it is preferable to insert into the C1a T and Sal I recognition sites of H-L9 (Figure 8), a ClaI recognition sequence was added to the 57-end of the human lymphotoxin gene, and a Sal r recognition sequence was added to the 3'-end. (Figure 2).

(3) Insertion of restriction enzyme recognition sites One method of gene conversion is cassette conversion.
For this reason, K requires the presence of an appropriate restriction enzyme recognition site. Since it is desirable that the restriction enzyme recognition site be at one site at any site in the gene of interest, it is necessary to select an SII restriction enzyme that cleaves only one site within the gene, including the DNA sequence of the plasmid into which the gene has been integrated. It is. In the case of the human lymphotoxin gene of the present invention, 6 base pairs
All restriction enzymes that recognize Rome were selected.

Computer search within the human lymphotoxin gene (C
As a result of searching all restriction enzyme recognition sites, no suitable site was detected. Therefore, we investigated whether it is possible to introduce a restriction enzyme recognition site by nucleotide transshipment using a restriction enzyme that recognizes 6 base pairs. An example in Figure 3? As shown,
Recognition sequence of restriction enzyme B s s H [1 is GCGCGC
Possible amino acid sequences from this sequence are shown in the figure, and from these, one that matches the amino acid sequence of human lymphotoxin is selected, and 1 from the N-terminal loin.
4. The sequence 15th'/CAla-Arg matches. So, looking at the nucleotide sequence of this site, ()C
Since it is CCGT, if we change all of this to GCGCGC, what is the amino acid sequence? This part remains unchanged (・teBs5H
■This means that all of the recognition sites have been introduced.

Similarly, EeoRI, Bindll, Kpnl 1
Xhol 5Sael, Smal, Hpa I,
A recognition site for BamHl was introduced at the position shown in FIG.

(4) Elimination of direct repeats When searching for direct repeats of 5 or more nucleotides in length using a computer, a large number of d
direct repeat is discovered. An example is shown in Figure 4, but such a long chain directrepea
When t are located close to each other, the oligonucleotides containing the Gln-Hls-Pro sequence are 5er-Thr-Le when the synthesized oligonucleotides are annealed in two steps and ligated by T4 ligase to construct a gene.
Associations between oligonucleotides containing sequences of u and complementary strands and vice versa may occur. This makes it impossible to construct the desired gene, so the amino acid sequence? Direct by changing codons without changing
repeat 'i delete. about 100 nucleotides chain length f, dire present in it as one block
Eliminate most of ct repeat. However, due to nucleotide substitution, a new direct repeat
t may occur, so direct repeat
The search and deletion of t was repeated several times until it was completely deleted.

(5) Delimitation of oligonucleotides to be synthesized Since the nucleotide sequence of the gene to be synthesized has been determined by the above operations, the next step is to determine the oligonucleotides to be actually synthesized chemically.

Is the oligonucleotide chain length determined manually or with an automatic synthesizer? The length of the chain was set at around 30 nucleotides, taking into consideration the need to use an automatic synthesizer in the case of human lymphotoxin genes and the ease of purification of the oligonucleotide after synthesis (although this varies depending on the method used) (Fig. 2) U1 to U18 and L1 to L18) at ν).

The first example of an artificial gene was Khorana et al.'s tRN.
Synthesis of the A gene (Naturs Vol. 227, 2
7-3.1 (-・) (11970), at this time, adjacent DNA fragments overlapped with complementary strands to form several bases.
A gene called the yprotruding method was constructed, and most of the examples of gene synthesis to date have been studied. Human IJ of the present invention for synthesis of IJ phototoxin gene
The design of =+' nucleotides followed this method as well.

(6) Elimination of Palindromic Sequences Once the design of the oligonucleotide to be synthesized is complete, what must be investigated is the oligonucleotide region 1' (/f) within the same fragment, which is the basis of synthesis. Because the entire pin structure is created, the desired oligonucleotides cannot be assembled together.Also, if there is an IC palindromic structure at the binding site of double-stranded oligonucleotides, dilation of the same double-stranded DNA occurs, and the desired DNA Therefore, these obstacles must be eliminated by nucleotide substitution as shown in Figure 5.

With the above operations, the structure of the gene to be synthesized was designed.

(2) Synthesis of oligonucleotides and nM Oligonucleotides were synthesized by the phosphite method using an automatic synthesis image. Nucleotides include adenine-daanine-cytosine-thymine nucleoside β-
Cyanethyl phosphoramidite was used, and tetrazole was used as the activator. Since an oligonucleotide having a protecting group is obtained from an automatic synthesizer after the synthesis, the oligonucleotide is heated in concentrated ammonia water at 60° C. for 5 hours to completely remove the acyl protecting group and the phosphoric acid protecting group. Since the 57-terminus of the target oligonucleotide thus obtained has all somethoxytrityl (DMTr) groups, separation by reversed phase chromatography can be performed using this lipophilicity (Figure 7a). ). Oligonucleotides were eluted using a linear concentration gradient using CH30N as a solvent. Subsequently, the DMTr group was removed by treating the oligonucleotide with DMTr group in f80 thiacetic acid for 20 minutes at room temperature. This yielded an oligonucleotide from which the protecting group had been completely removed, which was purified by HPLC.

Purification by HPLC first involves reverse-phase HPLC to remove impurities (Figure 7b), followed by ion-exchange HPLC.
The purity is verified by analysis by C, and if necessary, fractionation is performed for purification (FIG. 7C). This operation yields oligonucleotides with a purity of approximately 100% from several OD to 10%.
Several ODs are obtained.

35 for the human) IJ nphotoxin gene of the present invention.
Species oligonucleotides (U1-U1 shown in Figure 2)
7.Ul8 and L1 to L18) were synthesized and purified.

■Construction of human lymphotoxin gene Construction of the gene takes about 10 minutes after 5'-phosphorylation of the oligonucleotide.
By annealing 0 nucleotide chain length as one block, performing the first step of ligation with T4 ligase to obtain double-stranded DNA fO blocks, and then ligating them with T4-9 gauze. carried out (8th
figure).

(1) Oligonucleotides (Ul.

Except for Ll)'i, each of the 16 oligonucleotides was phosphorylated using polynucleotide kinase in the presence of ATP. i.e. 0,100 uni
9.5μt of 50mM T oligonucleotide
ris-HCt (pH 8,0), 10 mM MgCl
2.10mM β-mercaptoethanol, 1
The 5'-end was phosphorylated by dissolving in a buffer containing mM spermine, 1 mM ATP'i gold, adding 3 units (0.5 μt) of T4 f-rinucleotide kinase, and incubating at 37°C for 90 min. oligonucleotides are obtained.

(2) Linking oligonucleotides (gene construction)
All the phosphorylated oligonucleotides were combined into a DNA block with a length of about 100 nucleotides, and 66
mM Tris-HCt (pH 7,6) -6,6m
M>AgC12-0.5mM ATP mixture (
After heating (90°C, 3 minutes), the final volume of 100 μt was slowly cooled, and 700 units of T4 DNA ligase was added.
Incubate for 2 hours at 5°C. Next, after removing all protein by phenol treatment and recovering DNA by ethanol precipitation, 10mM Tr1s-HCI
Dissolve (pi(s,o)-1 sushi in a small amount of buffer containing EDTA, add bromine phenol blue, an electrophoresis marker, and apply it to a 10% polyacrylamide gel. Approx. Electrophoresis was carried out for 2 hours. After electrophoresis, the gel was stained with ethisium bromide, and the portion of the gel corresponding to DNA K of the desired chain length was cut out. The gel was then placed in a dialysis tube and electrophoresed. DNA was eluted with DNA i and recovered by ethanol precipitation.The entire DNA block thus obtained is shown in Table 1.

Table 1 - Stage ligation and chain length Next, DN
Combine blocks A to v and add the same amount of T4 Suga-se under the same buffer conditions as in the first step of ligation.
Incubated for 2 hours at 0°C. After phenol treatment and ethanol precipitation, 5 polyacrylamide gel electrophoresis was performed, and DNA with a chain length corresponding to the target human lymphotoxin gene (522 base pairs) was recovered from the gel. Through the above operations, about 5 pmol of human lymphotoxin gene was obtained.

(2) Insertion of the human lymphotoxin gene into the vector The E. coli tryptophan promoter (Trp) was used as the collar promoter for expression of the human lymphotoxin gene. That is, plasmid pGH-L9 (Proc, Na
tl.

Acad, Sci, USA+ Volume 81 5956
~5960 Beno (1984)) was selected as a plasmid vector for insertion of the human lymphotoxin gene.

Plasmid pGH-L9 was digested with restriction enzymes C1al and Sa.
The plasmid vector in which the human growth hormone gene was completely deleted was separated by digestion with lI and subjected to 10 polyacrylamide gel electrophoresis. This vector (1 μg) and the previously obtained human lymphotoxin gene were combined at 66 mM
-Hcz (pH 7,6), 6.6 mM lact2.
05 Sushi ATP 110mM β-mercaptoetha
T4 ligase after dissolving in buffer containing nol f 350
uniti and incubation at 20° C. for 2 hours yielded cilasmid pTLym containing the human lymphotoxin gene downstream of the Trp promoter, which was recovered by ethanol precipitation (FIG. 8).

■ Expression of human lymphotoxin gene (11pTLy
Transformation of the plasmid into E. coli with Ca.
A 2+ treatment method was used.

100 t of Ca2+-treated E. coli HBIOI suspension
pTLym f was added to tt under ice-cooling, and the mixture was left for 10 minutes in water-cooling.

Subsequently, the entire culture solution was heated at 42° C. for 60 seconds, and then returned to room temperature. A 1N L-Broth culture solution was added thereto, cultured at 37°C for 40 minutes, the bacteria were collected using a refrigerated centrifuge, the supernatant was discarded, and the cells were suspended again in 0.3 times of the L-Broth culture solution. Subsequently, 100 μt of each suspension was spread on an agar medium supplemented with ampicillin, and cultured at 37° C. overnight. Through this operation, E. coli transformed by pTLym forms entire colonies on the agar medium.

10 colonies were obtained with pTLymK starting from 1 μ2 vector (pTLym-1 to pTLym-1).
TLym-10).

(2) Screening of E. coli containing the human lymphotoxin gene Since transformants of E. coli HBIOI were obtained by pTLym, it was determined whether these transformants contain the human lymphotoxin gene.
id boiling method (Molecu
larCloning; pages 365-367, C
o1d Spring Harbor Laborato
ry (published in 1982).

Part of E. coli from 10 colonies i! The cells were transplanted into J9cA liquid medium (5 mt) and cultured at 37°C overnight. Collect 0.1 ml of E. coli from the culture solution by centrifugation and lyse at 100°C in the presence of lysozyme. The lysate solution is centrifuged, the supernatant is taken, and the isopro-gnol precipitation is repeated twice to obtain the plasmid. This plasmid was dissolved in an appropriate mild S solution, and the restriction enzymes C1al and 5ai
Digestion with K and phenol treatment followed by K ])
NAk was recovered. By electrophoresis on a lyacrylamide gel, the recovered DNA was determined to be 522 base pairs of Dli.
The presence of the A fragment (corresponding to the human lymphotoxin gene) was confirmed. As a result, pTLym-1, pTLy
The presence of human lymphotoxin genes was confirmed for m-5, pTLym-8, and pTLym-10K.

(3) Expression of human lymphotoxin gene (part 1)
We investigated the production of human lymphotoxin protein in four types of E. coli transformants in which the presence of the human lymphotoxin gene has been confirmed.

100 μtf of E. coli cultured in M9CA medium
Transfer 5ml of CA medium and culture at 37°C for 1 hour. After induction by adding indole acrylic acid (IAA) to a final concentration of 40 μl/ml, the culture was continued again, and the turbidity of the culture solution (A660 u
m ) "jr: Measured (Figure 9). In Figure 9, ya 1,
Ai5 and A9 are pTLym-1 and pTLym-, respectively.
5 and pTLym-9, pGH-L9 indicates E. coli transformed with cilasmid pGH-L9, which does not contain the human lymphotoxin gene. After 8 hours and 23 hours after the induction, 1100μ of the culture solution was taken and centrifuged to collect the bacteria.Escherichia coli was then lysed at 100°C in the presence of SDS. This lysate was subjected to SDS-15% polyacrylamide gel electrophoresis, and after staining with Coomassie brilliant blue, the presence of a band corresponding to human lymphotoxin was confirmed (FIG. 10). As a result, the presence of protein was observed near the molecular weight marker protein of 21,000. As a result of gel scanner analysis, this protein was found to be 3.5% of the total E. coli protein after 8 hours of induction.
This was equivalent to 7%, and had increased to 4.79% in 23 hours. As a result of expressing the human lymphotoxin gene in E. coli as described above, it was found that human lymphotoxin was produced.

(4) Expression of human lymphotoxin gene (Part 2)
M9 medium containing 0.5% (w/v) casamino acids and 40 μl/mi ampicillin (6?/Z of Na2HP)
O4゜3t/! K) 12P○4, 2.5r, /A's N
aCA, 19/Ik NH4Cl, 0.2%
(w/v) glucose, 2mM MgSO4,0,
1mM CaCl2 and 10q/A thiamine hydrochloride)5
ml at 37°C for about 12 hours, (1) and (2) of the above V.
), a transformant of E. coli HBIOI,
E. coli HBIOI/pTLym f was precultured. Next, 2 ml of this preculture solution was added to 200 ml of the above M9 medium containing casamino acids and ampicillin,
The culture was carried out with shaking for about 24 hours.

After culturing, collect the E. coli bacteria into standard buffer C3.
0mM Tris-HCt (pi(s,o, 30mM
of NaCt), then suspended in 5 ml of standard buffer and 0.1 mM Phenylme
thyl-sulfonylfluoride, 10
mM EDTA i was added.

Next, a solution of 10 μfie H2O of lysozyme was added to the above onion suspension so that the amount of lysozyme was 11e, and the mixture was heated at 0°C.
Leave it for a minute.

Next, freezing and thawing was repeated five times using acetone cooled with dry ice, followed by centrifugation at 25,000 Y for 1 hour.

This supernatant is used as a crude extract of human lymphotoxin.

This method is the method of Nagata et al. (Nature + 2nd
84 Makiya 27, 316-320-), (1980)) made some improvements to all references.

(5) Measurement of antitumor activity of human lymphotoxin crude extract The approximate activity of the human lymphotoxin expressed this time was measured using the human lymphotoxin crude extract.

1) Determination of the amount of human lymphotoxin in the crude extract Crude extract of human lymphotoxin'e Polyacrylamide gel electrophoresis containing 0.1% (w/v) SDS C15%
(w/v) acrylamide, 0.4% (w/v) bisacrylamide, 375mM Tris-HCl (pH
8, 8)] and analyzed using Takashi MDual-wave le
When the content was measured using ngthTLC5canner C5-900, the human lymphotoxin content in the crude extract was about 5-8%.

2) Measurement of antitumor activity B, B, Aggarwal et al.'s method (The Jo
Urnal of Bio-1logical Chem
istry Vol. 260, A42345-2354 Beno, (1985)].

Crude extract of human lymphotoxin t 0.1 to 10-
It was diluted in several parts up to 6 times and dispensed into a 96-well microplate. Add 0.1 ml of L-929 cells (
3X 10 cells/m/ ) and 1 μg/ml actinomycin D in the presence of 5% CO2.
It was incubated for 8 hours. After discarding the supernatant, 96-well
After washing twice with Microplatera saline (0.9% (v/v) NaC2), 0.5% (w/v)
crystal violet (methanol:water 1:4)
t'lo.

Dispense into μt portions and leave for 20 minutes.

After discarding the crystal violet solution, the microplate was thoroughly washed three times with physiological saline, and then washed for 1 fy (w/
v) SDS aqueous solution'! Add 200 ttt and leave for 30 minutes.

After standing, the absorbance of this solution was measured at 540 nm, and the activity was calculated from this to be 2 to 4 x 10'.
3) Crude Purification of Human Lymphotoxin Crude Extract The crude extract prepared in (4) of V above was completely purified.

Q-5 epharose Fast Flow for purification
(manufactured by Falcia) ts, smt columns were used.

The buffer was 20mM Tris-Hct (pH 8,0
), 0, 1 mM Phenyl-me thylsu
l fonyl fluoride and 0.01% (w
/v) using 14aN3f, 0.2 from OM with NaC4
Eluted at 400 rnl with a gradient of 1-) end to M.

The flow rate at that time was 30 m1/h.

The crude extract was dissolved in 20 mM Tris-HCl (PF(8,
After diluting 2 times with 0), crude purification was performed using a column.

In addition, human lymphotoxin was concentrated 5 times by this operation. When the activity of this crudely purified human lymphotoxin was measured, it showed cytotoxicity of about 106 units/■.

〔effect〕

As mentioned above, the human lymphotoxin synthesis gene of the present invention is designed in such a way that the amino acids and peptides that constitute human lymphotoxin can be easily replaced by genetic engineering, and therefore cannot be improved by genetic engineering techniques. It is very useful for research on the production of photoxin.

[Brief explanation of the drawing]

FIG. 1 shows the nucleotide sequence of a known human lymphotoxin cDNA, and FIG. 2 shows an example of the nucleotide sequence of the human lymphotoxin synthesis gene of the present invention. Figure 3 shows an example of introduction of a restriction enzyme recognition site, and Figure 4 shows 1 fII of a direct repeat. Figure 5 shows an example of Malindrome structure elimination;
The figure shows the synthesis of oligonucleotides Ekt, and Figure 7 shows H
NM of oligonucleotides by PLC is shown. FIG. 8 shows an example of the construction of the human IJ nonphotoxin gene of the present invention and its insertion into a plasmid. Figure 9 shows the growth of E. coli transformants;
Figure 0 shows confirmation of the human lymphotoxin produced by SDS-15% polyacrylamide gel electrophoresis. I in the diagram
AA (E) indicates no addition of IAA, and ■ indicates addition of IAA. Also, the arrow indicates the position of the molecular weight marker of 21,000. Go to Figure 1 2. Patent Issuance Ujin Sankyo Co., Ltd.

Claims (1)

[Claims] 1. Human lymphotoxin synthesis gene having the following characteristics (1) BssHII, EcoR I, Kpn I, HindIII, which are restriction enzymes that recognize a 6 base pair palindrome.
, Xho I , Sac I , Sma I , Hpa I and BamH I have been modified to have only one recognition site. (2) Without changing the amino acid sequence of the original human lymphotoxin, by changing codons, the direct repeats present in the 100-nucleotide complex are deleted as one block. (3) Any 30 consecutive nucleotides,
Among these, palindromic sequences exceeding 6 base pairs have been deleted. 2. The human lymphotoxin synthesis gene according to claim 1, which has a Cla I recognition sequence at the 5'-end and a Sal I recognition sequence at the 3'-end. 2. Human lymphotoxin synthesis gene according to claim 1 or 2 having the following DNA sequence [There is a gene sequence] [There is a gene sequence] [There is a gene sequence]