JPH0817712B2 - Novel human TNF polypeptide variants - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel human TNF polypeptide variants and their production.
DNA related to the method and its variants. TNF (tumor necrosis factor)
Le et al. [Proc.Natl.Acad.Sci., USA,72, 3666 (1975)]
It is a more discovered bioactive substance and
Cell culture system showing strong cytotoxic activity and transfer in vivo.
Characterized as a substance that causes necrosis in transplanted tumors
[L.J.Old, Cancer Res.,41, 361 (1981)]. From 1984 to 1985, rabbit, human and mouse TNF
The isolated gene has been isolated [JP-A-60-120990, JP-A-6
0-185799, L. Fransen et al., Nucleic Acids Res.,13, 44117
(1985)], total primary structure of each TNF polypeptide.
Was clarified. Due to the isolation of the TNF gene, especially the human TNF gene
To produce human TNF in microorganisms using genetic engineering techniques.
It becomes possible to produce the human TNF
Further detailed studies have been added, and strong in vitro
Cysticidal activity and antitumor activity in vivo confirmed
[D. Pennica et al., Nature,312, 724 (1984); T. Shirai et al., Na
ture,313, 803 (1985), M. Yamada et al., J. Biotechnology,3,
141 (1985)]. No studies have been conducted on modified forms of human TNF polypeptide.
And several patent applications have been published (PCT
International patent application publication number WO 86/02381, publication number WO 86/04
606, European Patent 168214, Japanese Patent 155549, JP
62-48632). Of these, the former three are mainly 157 patent applications.
Human TNF polypeptide composed of amino acid residues
Modification or refer to specific modifications.
It is only shown. The remaining two patent applications are 155
Human TNF polypeptide composed of amino acid residues
Of the present invention.
Human TNF polypeptide variants are described in these application specifications.
Amino acid sequence differs from the specifically disclosed modified form
ing. The present inventors have found that human TNF protein consisting of 155 amino acid residues
Go from the N-terminal side of the polypeptide or its polypeptide
In a polypeptide with some amino acids deleted,
Specifically perform amino acid conversion in the amino acid sequence of
And examined the properties of the various polypeptide variants obtained.
As a result, only when the amino acid at a specific position was converted
Therefore, it was revealed that it was obtained as a soluble protein. Obedience
Therefore, an object of the present invention is to modify soluble human TNF polypeptide.
It is to provide a group of variants. Among the above-mentioned group of polypeptide variants, the present inventors have
Specific variants in vitro and in vivo in humans
Having cytotoxicity and antitumor activity comparable to TNF
I found out. Therefore, another object of the present invention is to
B) Excellent in vivo cytotoxicity and antitumor activity in vivo
To provide a human TNF polypeptide variant having the activity
It is in. In addition, in the above-mentioned polypeptide variant group, another specific
Mutants were quite surprisingly found to be
In vivo despite very low cytotoxic activity
Was found to show excellent antitumor activity. And this
The polypeptide variants of
The unfavorable effects of heat generation, etc. have been significantly reduced.
I also found that. Therefore, in addition to the present invention,
The purpose of is to show extremely low cytotoxic activity in vitro.
However, it has an excellent antitumor effect in vivo, and
Provides human TNF polypeptide variants with reduced side effects
To do. Like this mutant, in vitro
Excellent activity in vivo despite low activity
The fact that TNF has a typical biological activity of cells
Structures essential for the expression of interfering activity and antitumor activity (activity
Center) is not the same site in the TNF polypeptide molecule
It means that. Furthermore, various biological activities of TNF
It is speculated that the active centers of sex are also different.
The Other objects of the present invention will be apparent from the following description.
U. In this specification, the following abbreviations are used for simplification of description.
I will decide. A adenine C cytosine G guanine T thymine Ala alanine Arg arginine Asn asparagine Asp aspartic acid Cys cysteine Gln glutamine Glu glutamic acid Gly glycine His histidine Ile isoleucine Leu leucine trilephaneline Tyrophane serine prothrin serine proline serine prothrin proline serine prothrin proline serine proline serine proline serine prothrin Deoxyribonucleic acid cDNA Complementary DNA dATP Deoxyadenosine triphosphate dCTP Deoxycytidine triphosphate dGTP Deoxyguanosine triphosphate dTTP Deoxythymidine triphosphate bP Base pair SDS Sodium dodecyl sulfate MW Molecular weight KD Kilodalton SD sequence Shine-Dalgarno sequence Then, the base sequence shown as a single strand is the positive strand (sense
 Strand) base sequence, the left end of which is the 5'-end,
The right end shows the 3'-end. The left end of the amino acid sequence is N-terminal
Edges and right edges indicate C-termini. The present invention provides an amino acid sequence represented by the formula [1] in Table 1.
The 16th, 31st to 34th, 36th, 48th,
73rd, 82nd, 85th, 89th, 94th,
97th, 98th, 103rd, 113th, 115th
Eyes, 117th, 118th, 131st, 132nd,
At least the 141-146th and 153rd amino acid residues
One has an amino acid sequence converted to another amino acid residue
Polypeptide (provided that the 115th amino acid residue is
If it has been converted to another amino acid residue, the 67th and
And the 1st or 99th amino acid residue is replaced with another amino acid residue.
It may have been converted. ) Or the N-terminus of the polypeptide
Polys with up to 8 deletions of amino acids from the N-terminal
Regarding peptides. More specifically, the human TNF polypeptide variant of the present invention
For example, (A) the amino acid sequence represented by the formula [I] in Table 1
The 16th Ala is Val, the 31st Ala is Thr, and the 32nd Asn is Ala, Cys, Asp, His, Ile, Arg, Ser, Thr, V
al, Tyr, 34th Leu to Ile, 36th Ala to Val, 48th Val to Met, 73rd Leu to Pro, 82nd Ala to Asp, The 85th Tyr is His, the 89th Val is Ile, the 94th Ala is Thr, the 97th Ser is Asn, the 98th Pro is His, Leu, and the 103rd. Thr is Pro, 113th Tyr is Cys, 115th Pro is Leu, His, Gln, Ser, Ala, Phe, Asn, Thr,
Gly, Tyr, Val, Gln, Met, Ile, Asp, Trp, Lys, Arg, Thr, 117th Tyr is His, 118th Leu is Gln, 131st Ser is Ile, The 132nd Ala is Thr, the 141st Asp is Tyr, the 143rd Ala is Val, the 144th Glu is Lys, the 145th Ser is Cys, and the 146th Gly. To Glu and the 153rd Ile to Leu.
Human TNF polypeptide, which has the amino acid sequence represented by the formula [I] in Table 1
The 67th and / or 99th Cys is converted to Ser.
And the 115th Pro is changed to another amino acid other than Pro.
A human TNF polypeptide variant having an altered amino acid sequence.
A variant, and (C) the human TNF polypeptide of (A) or (B) above
1 to 8 amino acids are sequentially deleted from the N-terminal side of the mutant
Human TNF polypeptide variants. Of the above polypeptide variants (A), (B), (C)
In vitro, excellent cytotoxic activity in vivo
Examples of polypeptide variants that show anti-tumor activity and antitumor activity
It is as follows. In the amino acid sequence represented by the formula [I], the 16th Ala is Val, the 36th Ala is Val, the 73rd Leu is Pro, the 98th Pro is His or Leu, and The 103rd Thr has Pro, the 115th Pro has His or Gln, the 131st Ser has Ile, or the 143rd Ala has Val.
Polypeptide. In vitro activity is low among the above mutants, but
Polypeptide showing excellent antitumor activity in vivo
The mutants are as follows. In the amino acid sequence represented by the formula [I], the 31st Ala is Thr and the 32nd Asn is Ala, Cys, Asp, His, Ile, Arg, Ser, Thr, V
Al, or Tyr, the 115th Pro is Ser, Ala, Phe, Asn, Thr, Gly, Tyr, Val,
Glu, Met, Ile, Asp, Trp, Leu, or Lys, or the 117th Tyr has an amino acid sequence converted to His.
Polypeptide. Particularly preferred mutants are those represented by the formula [I].
Amino acid in which the 32nd Asn is converted to Tyr, His, Asp, or Ser in the mino acid sequence, the 115th Pro is converted to Leu, Ser, Asp, or Gly, or the 117th Tyr is converted to His A polypeptide having a sequence
It is done. DNA encoding the human TNF polypeptide variant of the present invention
Publishes DNA encoding known human TNF or its precursors.
Known method, for example, one described in European Published Patent 155549
Or by total synthesis, and then
For example, the method of A. Wang et al. [Science,224, 1431 (198
4)] and prepare DNA with point mutation of the target base
Or isolate the DNA fragment with an appropriate restriction enzyme,
Synthetic oligos in which the base sequence of the target site is artificially changed
By using a deoxynucleotide adapter
D encoding the human TNF polypeptide variant of the present invention
Identification of the constituent amino acid sequences of NA, a human TNF polypeptide
To convert other amino acids (one or more) to other amino acids
DNA encoding the polypeptide variant of the present invention, or
In such a polypeptide variant, up to 8 nucleotides from the N-terminal
Producing DNA encoding a polypeptide lacking mino acid
can do. For example, the 115th amino acid (Pro) in formula [I] is Le
The DNA encoding the polypeptide variant converted to u is
Can be manufactured as follows. DNA containing the nucleotide sequence encoding the human TNF precursor was
Isolate according to the method described in European Published Patent. The nucleotide sequence of this human TNF precursor is shown in Table 8.
The salt from the 235th base to the 699th base.
The base sequence corresponds to the base sequence encoding human TNF. Hi
It is the 115th amino acid in the TNF amino acid sequence.
The codons encoding Pro are the 577th to 579th positions in Table 8.
Corresponds to the base (CCC) of. Therefore, the DNA fragment containing this codon should be digested with an appropriate restriction enzyme.
Cut out by combining. Separately, above this DNA fragment
Change the codon (CCC) of Pro to the codon (CTC) corresponding to Leu
Chemically synthesize a DNA fragment containing the altered nucleotide sequence,
Replace this synthetic DNA fragment with the previously cut out DNA fragment.
To encode the above polypeptide variant by
Can produce DNA. More specifically, for example, restriction enzymesDdeI andPvu
Using II, DNA fragments corresponding to the 555th to 603rd positions in Table 8
Cut out. Next, chemically synthesized oligodeoxyribonucleotides
The nucleotide sequence of
The base sequences of the two DNA fragments of
The DNA fragments corresponding to the 555th to 603rd DNA fragments in Table 8 which were extracted were placed.
Replace. 5'-TGAGGCCAAGCCCTGGTATGAGCTCAT-3 '3'-CCGGTTCGGGACCATACTCGA-5' and 5'-CTATCTGGGAGGGGTCTTCCAG-3 '3'-GTAGATAGACCCTCCCCAGAAGGTC-5' The DNA thus produced is known in the art.
Technology to obtain the appropriate sequence in the appropriate expression vector.
And insert it into the host
By culturing, the polypeptide variant of the present invention is produced.
It can be made to live. More specifically, for example, the present invention
It has a nucleotide sequence that encodes the polypeptide variant itself.
Start codon ATG at the 5'end and 3'end of the DNA
Creates a DNA fragment with a stop codon and
The promoter and SD sequences are subsequently attached and then
Of the polypeptide of the present invention
It is possible to produce a trait expression vector for production. Step
As a motor, for example,lac,trp,tac,phoS,pho
A， P_L, SV40 early promoter, etc. vector
For example, plasmids (pBR322, etc.), phages
(Λ phage derivative etc.), virus (SV40 etc.), runner
A way plasmid is mentioned. This polypep of the present invention
Using a gene expression vector for the production of tide mutants in an appropriate host,
For example, the method of Cohen et al. [Proc.Natl.Acad.Sc.
i., USA,69, 2110 (1972)]
A transformant can be obtained. Then this transformant
By culturing under appropriate culture conditions.
The target polypeptide variant or its Me
It is possible to produce a polypeptide variant in which t is bound.
Wear. This culture is for example digested with lysozyme and freeze thawed.
After crushing with ultrasonic wave, ultrasonic crushing, French press, etc.,
The polypeptide of the present invention by centrifugation or filtration
A mutant-containing extract can be obtained. This extract is
Common white matter purification methods (ultrafiltration, dialysis, ion exchange
Romanography, Gel filtration, Electrophoresis, Affinity
-Chromatography, etc.)
It is possible to obtain the polypeptide variants of the invention in pure form.
Wear. In addition, the polypeptide variant of the present invention contains no organic acid.
The present invention can be obtained by reacting an organic acid or base according to a conventional method.
It is possible to produce a salt of the polypeptide variant of. Examples of the polypeptide variants of the present invention
explain about. Human TNF used as a control was European.
Purification disclosed in Example (5) of Japanese Patent Application Publication No. 155549
Human TNF polypeptide was used. (1) Transformants produced in Examples and Reference Examples described below
By culturing the
SDS-PAGE [Laemmli, U.K., Nature]
(London)227, 680 (1970)] and EIA. That is, transformants for producing various polypeptide variants
Was cultured according to the method described in Example 1 below, and the
Each polypeptide variant produced in the intestinal fungus body was treated with 0.1%
50 mM Tris-HCl buffer containing zozyme and 30 mM Nacl (p
H8). The target polypeptide alteration recovered in the extract was
The amount of the foreign substances was measured by SDS-PAGE and EIA, and
The solubility of the Tide variant in the above medium was evaluated. In SDS-PAGE, calculate the target polypeptide variant
The presence or absence of protein detected at the position corresponding to the molecular weight is indicated.
It was judged as the mark. In addition, in EIA, a poiton that immunologically reacts with anti-human TNF antibody.
Quantification of the amount of the lipopeptide variants was performed by EIA.
The method for quantifying a lipopeptide variant is based on the following principle.
is there. That is, the human TNF polypeptide variant and β-gam
Anti-human T between human TNF labeled with lactosidase
A competitive binding reaction was performed with NF rabbit antiserum.
Then, anti-rabbit IgG bound to the bacterial cell wall and insolubilized
By adding goat antiserum, enzyme-labeled human TNF-
Anti-human TNF rabbit antibody-anti-rabbit IgG goat antibody conjugate
Formed. The reaction solution was centrifuged to obtain a solid phase. In solid phase
The amount of enzyme-labeled human TNF in the above-mentioned complex collected in
The enzyme activity was determined as an index. That is, 2-nitrophenyl-β-D-as an enzyme substrate
Substrate produced by enzymatic reaction with the addition of galactopyranoside
The amount of decomposed product (2-nitrophenol) was adjusted to 410 nm.
It was determined by the absorbance. Enzyme-labeled human in this complex
TNF amount reflects the amount of human TNF polypeptide variant in the sample
Do. Using a standard curve prepared separately using human TNF,
The amount of human TNF polypeptide variant in the body is
I asked for it. For the production of anti-human TNF rabbit antiserum, Yamada et al. [J. Biote
chnology,3, 141 (1985)].
TNF was used as the antigen. The results are shown in Table 3 below. Target polypeptide detected in each cell extract
When the mutant is almost comparable to human TNF as a control
Is displayed as (). This is because the polypeptide variant
It means that it is a soluble protein. On the other hand, when the detected amount is a little small, it is displayed as (+).
However, if it is extremely small or not recognized (-)
Was displayed. Polypeptide variants indicated by (-) are specific
Changes in the structure of the polypeptide due to the acid conversion
Therefore, the solubility was significantly reduced or it was degraded in E. coli.
It is thought to have been done. (2) Various polypepnes of the present invention obtained in the examples described below.
The isoelectric point and cytotoxic activity value of the Tide mutant are shown in Table 4 below.
Show. The cytotoxic activity is mouse LM cells (ATCC, CCL1.2)
Using the method of Yamada et al. [J. Biotechnology,3, 141 (198
5)]. Polypeptide used in this test
All mutants were confirmed to contain impure proteins by SDS-PAGE.
It is an unrefined product. (3) Of various polypeptide variants described in Examples below
The antitumor activity in vivo is shown in Table 5 below. The antitumor activity was evaluated as follows. Female A sarcoma cells 2 x 10^FiveBALB / C female mice (8 weeks
(Age) intradermally transplanted into the abdomen, and 7 days after transplantation, this polypepti
One mutant was administered intravenously. Tumor necrosis inducing action
Twenty-four hours after administration, the criteria of Carswell et al. [Proc. Nat
l.Acad.Sci.USA,72, 3666 (1975)]. As shown in Table 5, there are factors that selectively damage tumors.
In vitro cells characterized by TNF
Injury activity and antitumor activity against transplanted tumors in vivo
There is poor correlation with sex. For example, TNF-131I is human TNF
In Vitro Cytotoxicity and In Vivo Antitumor Activity
It has almost the same activity as sex. However, that
Other polypeptide variants, eg 32nd from the N-terminus,
Polypep in which the 115th and 143rd amino acids have been converted
Tide mutants were compared to their in vitro cytotoxic activity
With strong in vivo antitumor activity and low lethal toxicity
The features are shown. (4) Regarding some examples of the polypeptide variant of the present invention,
The fever-inducing effect was tested. The results are shown in Table 6 below.
I will. This febrile test was administered intravenously to rabbits and immediately after administration.
The change in intestinal temperature was observed. Observation is performed up to 4 hours after administration,
The result shows that the body temperature rise below 0.4 ℃ (-), 0.5 ~ 0.9 ℃
Increase body temperature (+) and increase body temperature above 1.0 ℃ ()
Was displayed. (5) The effect of TNF-115L on blood pressure was tested. blood pressure
SHR / NCrj male rats (weighing 264-304 g, daily
TNF-115L in the tail vein of this Charles River K.K.
Was administered to rats, and the tail artery pressure measurement device for rats (KN-209 type, Natsume
Systolic blood pressure was measured under no anesthesia. Conclusion
The results are shown in Table 7 below. The present invention will be described in more detail with reference to Examples and Reference Examples below.
However, the present invention is not limited to this embodiment.
There is no. Example 1 Production of human TNF polypeptide variant TNF-32Y (1) Construction of trait expression plasmid Corresponding to the sequence from amino acid number 1 to 155 in Table 9
A polypeptide consisting of 155 amino acid residues (hereinafter, TN
F-32Y) production expression plasmid (pHNY-32)
Were constructed as shown in Figs. A cloned cDNA encoding human TNF is available in Europe
A set made according to the method described in published patent 155549
Restriction enzyme from recombinant plasmid pHTNF 13PSTFor I
It was excised by digestion and isolated. This cloned cDNA is furtherAvaI andHindIII
Region that is digested with and encodes human TNF polypeptide
A DNA fragment containing most of the was isolated. this
The isolated DNA fragment is hereinafter referred to as TNF-DNA fragment. This TNF-
The DNA fragment is located at the downstream side from the 250th base number in Table 8.
It is a fragment of about 600 bp in length that contains the nucleotide sequence corresponding to the sequence.
The complete nucleotide sequence is reported by Yamada et al. [J. Biotechnology,3,14
1 (1985)]. This TNF-DNA fragment is a restriction enzymeHpaII andBg1Erase by II
DNA fragmentation corresponding to nucleotide numbers 250 to 321 in Table 8
Piece [referred to as DNA-1 fragment], based on base number 322 to 337
The corresponding DNA fragment [referred to as DNA-2 fragment] and the 338th
3 of DNA fragment [referred to as DNA-3 fragment] corresponding to the downstream side from
It is cleaved into two DNA fragments and the DNA-1 and DNA-3 fragments are separated.
Separated and purified. These DNA-1 fragment and DNA-3 fragment are
Via the oligonucleotide adapter represented by the following formula 5′-CGGGCCTATGCCCTCC-3 ′ [a] 3-CCGGATACGGG-5 ′ synthesized according to the method
Ligation was performed using 4DNA ligase. Got
The DNA fragment is called NY-DNA fragment. This NY-DNA fragment is
The following formula 5′-AACTAGTACGCAAGTTCACGTAAGGAGGTTATC-3 ′ synthesized by
 [B] 3'-TTGATCATGCGTTCAAGTGCATTCCTCCAATAGCTA-5 'and 5'-GATTATGTCATCTTCTCGAACC-3' [c] The oligonucleotide adapter represented by 3'-ATACAGTAGAAGAGCTTGGGGCT-5 'is T4DNA Riga.
Ligation was used to connect them sequentially. The obtained DNA fragment is
It is called the lower peptide region DNA fragment. On the other hand, plasmid pCT-1 [M.Ikehara et al., Proc.N
atl.Acad.Sci., USA,81, 5956 (1984)]HpaI
WhenAatAct IItrpAbout including a part of promoter region
380 bp DNA fragment [This DNA fragmenttrpPromoter region salt
The base sequence is reported by Bennett et al. (J. Mol. Boil.,121, 113,19
[Shown in 1978)] and cut it into the above pep
The tide region DNA fragment was ligated with T4 DNA ligase.
The obtained DNA fragment is referred to as promoter / peptide DNA fragment
It is called a piece. Separately, the restriction enzyme was added to the plasmid pBR322.AvaI andPvuII
Allow it to act on 0.7% of a large DNA fragment (about 3.7 kbp)
Separation was performed by gel electrophoresis. D on both ends of this DNA fragment
NA polymerase I (Klenow fragment) and dGTP, d
Use ATP, dCTP, and dTTP to create blunt ends, and then use T4 DNA at both ends.
Ligation was used to ligate. This plasmid vector
Is called pBRS6. Furthermore, this vector (pBRS6) was digested with restriction enzymes.
ElementaryAatII andHinA large DNA fragment (about 3.6 Kb
p) was isolated and purified. This DNA fragment was previously adjusted
The DNA fragment of the peptide peptide region using T4 DNA ligase
By ligation, the expression plasmid pHNY-32
It was constructed. (2) Production of TNF-32Y The expression plasmid pHNY-32 obtained in (1) is used in a conventional method.
[S.N.Cohen et al., Proc.Natl.Acad.Sci., USA,69, 2110
(1972)] and transformed into Escherichia coli HB101 to obtain transformants.
Was made. LB broth (composition; 1%
Totryptone, 0.5% yeast extract, 1% NaCl, pH 7.5)
After culturing at night, the culture broth contains 10 times the volume of modified M9 medium (composition;
0.45% casamino acid, 0.4% glycerol, 25 μg / ml ampi
Syringe), incubate at 37 ℃ for 1 hour, then in India
Acrylic acid to a final concentration of 20 μm / ml,
After culturing for another 24 hours, centrifuge to remove the bacterial cells.
collected. Add 1/10 volume of the culture medium to 0.1% lysozyme
And suspended in 50 mM Tris-HCl buffer (pH 8) containing 30 mM NaCl
And let stand for 30 minutes in ice water. Further dry ice / d
After repeated freezing in a thanol bath and thawing at 37 ° C,
An extract was obtained by removing the bacterial cell residue by heart separation. This extract was added to 20 mM Tris-HCl buffer (pH 7.8)
After dialysis, it was centrifuged and the supernatant was collected. this
The supernatant was previously equilibrated with the same buffer solution as DEAE-Sepharose C.
Load non-adsorbed components on L-6B (Pharmacia) column
It was thoroughly washed and removed with the same buffer solution. Then in the same buffer, N
Continuously increase the aCl concentration from 0 to 0.3 M to dissolve TNF-32Y.
I put it out. The elution position of the peptide is SDS-polyacrylic
Detected at the position of about 17KD by mid gel electrophoresis analysis.
The index was used as the index. This fraction was collected, and again 20 mM Tris-HCl buffer (pH 7.8)
After dialysis and desalting against DEAE-Sepharose CL-
6B column chromatography, using a mild NaCL concentration
Repeated elution by gradient. The peptide elution fractions were collected and subjected to ultrafiltration (molecular sieve membrane YM1
0, Amicon). This concentrated solution was applied to Bio-Gel P-6 (Bio-Rad)
At the ram, use 5 mM phosphate buffered saline as the eluent
Gel filtration to obtain purified TNF-32Y. The N-terminal amino acid sequence of purified TNF-32Y is protein
Sequencer (Applied Biosystems, 470A
Type) automatic Edman decomposition method. As a result, the N-terminal amino acid of TNF-32Y was Ser.
That is, the translation initiation codon (AT
Met derived from G) was excluded. Example 2 Production of human TNF polypeptide variant TNF-115L (1) Construction of trait expression plasmid Corresponds to the sequence from amino acid number 1 to 155 in Table 10.
A polypeptide consisting of 155 amino acid residues (hereinafter, TN
F-115L) trait expression plasmid for production pHPL-115)
Was constructed as shown in Fig. 4. TNF-prepared by the method described in Example 1- (1)
DNA fragment restriction enzymePvuII andTaqDigested with I, Table 8
DNA fragment [DNA-
4 fragments], DNA corresponding to base numbers 370 to 603
Fragment [referred to as DNA-5 fragment], base numbers 604-1653
Corresponding DNA fragment [referred to as DNA-6 fragment] and base number
DNA fragment corresponding to the downstream side from 654 [DNA-7 fragment
C] into four DNA fragments, and each is separated and purified.
The DNA-5 fragment was further digested with a restriction enzyme.DdeCut with I,
DNA fragment corresponding to base numbers 370 to 554 in 8 [DNA-8
Fragment]] was isolated and purified. DNA-4 fragment and DNA-8 fragment
The pieces were ligated using T4 DNA ligase, and then combined by a standard method.
An oligonucleotide adapter represented by the following formulas 5′-TGAGGCCAAGCCCTGGTATGAGCTCAT-3 ′ [d] 3′-CCGGTTCGGGACCATACTCAGA-5 ′ and 5′-CTATCTGGGAGGGGTCTTCCAG-3 ′ [e] 3′-GTAGATAGACCCTCCCCAGAAGGTC-5 ′ was used.
Ligation was used to connect them sequentially. The obtained DNA was further ligated with DNA-6 fragment and DNA-7 fragment.
I put them together. The obtained DNA fragment is called PL-DNA fragment. Hereinafter, according to the method described in Example 1- (1), except that N
Using the above PL-DNA fragment instead of the Y-DNA fragment,
The current plasmid pHPL-115 was constructed. (2) Production of TNF-115L Using the expression plasmid pHPL-115 constructed in (1)
The transformant according to the method described in Example 1- (2)
Were prepared and cultured. The method described in Example 1- (2) from the cell extract
Purification was performed according to the procedure described above to obtain purified TNF-115L. (3) Amino acid sequence determination Amino acid sequences of purified TNF-115L and its peptide fragment
For automatic Edman minutes using a protein sequencer
It was decided by the solution method. The peptide fragments were prepared as follows. That is, purified TNF-
115 L of 500 μg in 5 mM Tris-HCl buffer containing 4 M urea (pH
8) in 0.1 ml of 10 μg of lysyl endopeptidase (EC
3.4.21.50: Wako Pure Chemical Industries, Ltd.).
After digestion, the digestion product (peptide) is treated with Syn Chropak RP-P.
High-speed liquid using 300 columns (250 x 4.6 mm; Shinchrom)
Isolated by body chromatography. The elution is 0.1%
Acetate containing 0.07% trifluoroacetic acid in trifluoroacetic acid
A linear concentration gradient from 10% to 50% of trinitrile, 1
The flow rate was ml / min for 60 minutes. Figure 5 shows the elution diagram. No.1 to No.7 in Figure 5
Of the N-terminal part of the peptide fragment isolated from each fraction of
The acid sequence was determined by the automated Edman degradation method. That conclusion
The partial amino acid sequence of the peptide fragment of Fraction No. 6 is
-X-Tyr-Glu-Leu-Ile-Tyr-Leu-Gly-Gly-Val-Phe-Gln-
It was Leu-Glu. X in the sequence is an indeterminate amino acid
means. The above amino acid sequence is shown in Table 10 as amino acid No. 111.
It matched the ~ 125th sequence. From this result, the 115th amino acid from the N-terminal of TNF-115L was detected.
It was confirmed that the noic acid was Leu. In addition, N of TNF-115L
The terminal amino acid is Ser, which originates from the translation initiation codon (ATG).
The incoming Met was excluded. Example 3 Production of human TNF polypeptide variant TNF-115LΔN8 (1) Construction of trait expression plasmid The 9th amino acid (Ly
s) to 147th residue corresponding to the 155th amino acid (Leu)
Polypeptide consisting of amino acids (hereinafter TNF-115LΔN
8) for expression plasmid for production (pHPL-147)
Was constructed as shown in Fig. 6. The expression plasmid pHPL-1 obtained in Example 2- (1)
15 restriction enzymesClaI andBstDigested with EII, base number in Table 10
Downstream from No. 380 (coding the C-terminal part of TNF-115L
Region), tetracycline resistance gene, ampicillin
Resistance gene andtrpSize including part of promoter region
DNA fragments (hereinafter referred to as vector DNA fragments) and salts in Table 10
Small D containing the base sequence corresponding to base number 1 to 379
The NA fragment was separated and purified. This small DNA fragment is even more restricted
enzymeHgiAfter digested and cut with AI, base numbers 219 to 379
A DNA fragment corresponding to the eye was isolated. This DNA fragment is referred to below as H
It is called gi-DNA. Traits constructed separately according to the method described in Reference Example 2
Restriction enzyme for expression plasmid pHT147ClaI andHgiDigest with AI
The base sequence corresponding to the 25th to 218th base numbers in Table 10.
A DNA fragment of about 200 bp containing was isolated. This DNA fragment is
Below, it is called ΔN8-DNA fragment. This ΔN8-DNA fragment and the above Hgi-DNA fragment were combined with T4 DNA liger.
Ligate using the enzyme and prepare the resulting DNA fragment first.
It was. Expression by incorporating into an ecto DNA fragment
The plasmid pHPL-147 was constructed. (2) Production of TNF-115LΔN8 Example 1-using the expression plasmid pHPL-147
A transformant is prepared and cultured according to the method described in (2).
Nourished. From the cell extract, described in Example 1- (2)
Purification was carried out according to the method described in 1. to obtain purified TNF-115LΔN8. Translation initiation as N-terminal amino acid of purified TNF-115LΔN8
Met derived from codon (ATG) is detected by the automated Edman decomposition method.
Was detected. Example 4 Production of human TNF polypeptide variant TNF-115L-Ser67 (1) Construction of trait expression plasmid Corresponding to No. 1-115 of the amino acid sequence shown in Table 10.
Cys, which is the 67th amino acid in the amino acid sequence, is Ser
Replaced with TNF-115L-Ser67
U) The expression plasmid for production is shown in Fig. 7.
It was constructed. The trait expression plasmid constructed by the method described in Reference Example 3.
Restriction enzyme for pHTP392ClaI,HgiAI andHpaDigested with I, table
The base sequence corresponding to the base numbers 1 to 218 of 10
Nucleotide sequence in which the 200th and 201st nucleotides GC are replaced by CT
A DNA fragment of about 226 bp containing was isolated. This DNA fragment is
Below, it is called Ser67-DNA fragment. This Ser67-DNA fragment and the one described in Example 3- (1)
The Hgi-DNA fragment prepared by the method was ligated using T4 DNA ligase.
The obtained DNA fragments were combined and the obtained DNA fragment was described in Example 3- (1).
By incorporating into the vector DNA fragment obtained by the method of
The expression plasmid pHPL-Ser67 was constructed. (2) Production of TNF-115L-Ser67 Using the expression plasmid pHPL-Ser67, Example 1-
A transformant is prepared and cultured according to the method described in (2).
did. From the bacterial cell extract, the description of Example 1- (2)
Purification was carried out according to the method described above to obtain purified TNF-115L-Ser67. Automated N-terminal partial amino acid sequence of purified TNF-115L-Ser67
Ser, determined by Edman decomposition
Met derived from the start codon (ATG) was removed. Example 5 Preparation of Human TNF Polypeptide Variant TNF-115LΔN8-Ser67
Construction (1) Construction of trait expression plasmid 1 corresponding to the 9th to 115th amino acid sequences in Table 10
The 67th amino acid from the N-terminus in the 47-residue sequence
A polypeptide in which a certain Cys is replaced by Ser (hereinafter, TNF-11
5LΔN8-Ser67) expression plasmid (pH)
PL147S67) was constructed as shown in Fig. 8. Expression expression plus obtained by the method described in Example 4- (1)
Restriction enzyme for amide pHPL-Ser67ClaI andBstDigestion with EII
As two DNA fragments. For large DNA fragments in Example 3
It is the same fragment as the prepared vector DNA fragment. Small DNA
Fragment further restriction enzymeRsaAfter digestion with I, the bases in Table 10
DNA fragment containing the nucleotide sequence corresponding to the numbered 161-379
Isolated. Hereinafter, this fragment is referred to as Rsa-DNA fragment. On the other hand, the expression of a trait obtained by the method described in Example 3- (1)
Restriction enzyme for plasmid pHPL-147ClaI andRsaDigested with I
The base sequences corresponding to base numbers 25 to 160 in Table 10.
A DNA fragment of about 144 bp containing was isolated. Use this DNA fragment and the above Rsa-DNA fragment with T4 DNA ligase.
The resulting DNA fragment was ligated using
TNF-115LΔN8-Ser67
The expression plasmid for production pHPL147S67 was constructed. (2) Production of TNF-115LΔN8-Ser67 Using the expression plasmid pHPL147S67, Example 1-
A transformant is prepared and cultured according to the method described in (2).
did. The method described in Example 1- (2) from the bacterial cell extract
Purification was performed according to the method to obtain purified TNF-115LΔN8-Ser67. Spirit
As a N-terminal amino acid of TNF-115LΔN8-Ser67 manufactured by
Met derived from the start codon (ATG) was detected. Example 6 Production of other human TNF polypeptide variants-1 (1) Construction of trait expression plasmids 1 to 155 of the amino acid sequence represented by the formula [I] in Table 1
No. 32 from the N-terminal in the sequence consisting of 155 residues corresponding to No.
Asn, which is the amino acid of the eye, is a different amino acid, such as His, Asp
Alternatively, a phenotypic expression plasmid for producing a polypeptide converted into Ser.
Lasmid was prepared according to the method described in Example 1- (1), except that
Instead of the synthetic adapter [a], a chemical compound represented by the following formula
Use one of the synthetic oligonucleotide adapters
It was constructed by 5'-CGGGCCCACGCCCTCC-3 '3'-CCGGGTGCGGG-5' (for conversion to His) 5'-CGGGCCGATGCCCTCC-3 '3'-CCGGCTACGGG-5' (for conversion to Asp) or 5'-CGGGCCAGCGCCCTCC -3 '3'-CCGGTCGCGGG-5' (in the case of conversion to Ser) (2) Production of human TNF polypeptide variant Each of the expression plasmids prepared in (1) above is subjected to a conventional method.
Pursuant to E. coli HB101 strain according to
It was. The transformant was cultured by the method described in Example 1- (2).
The method described in Example 1- (2) from the cell extract obtained by culturing
Purified according to the method, the following human TNF polypeptide
A peptide mutant was obtained. TNF-32H: The 32nd amino acid in the amino acid sequence of formula [I]
Polypeptide in which Asn that is an acid is converted to His TNF-32D: The 32nd amino acid in the amino acid sequence of formula [I]
Polypeptide in which Asn that is an acid is converted to Asp: TNF-32S: The 32nd amino acid in the amino acid sequence of formula [I]
Polypeptide in which Asn that is an acid is converted to Ser Example 7 Production of other human TNF polypeptide variants-2 (1) Construction of expression plasmid First amino acid sequence represented by formula [I] in Table 1 ~ 155
No. 115 from the N-terminus in the sequence consisting of 155 residues corresponding to sequence No.
The amino acid Pro is another amino acid, for example, Ser, As
p or Gly-converted polypeptide expression plasmid for production
Lasmid was prepared according to the method described in Example 2- (1), except that
Instead of the synthetic adapter [d], a chemical compound represented by the following formula
Use any of the synthetic oligonucleotide adapters
It was constructed by and. 5'-TGAGGCCAAGCCCTGGTATGAGTCCAT-3 '3'-CCGGTTCGGGACCATACTCAG-5' (in the case of conversion to Ser) 5'-TGAGGCCAAGCCCTGGTATGAGGACAT-3 '3'-CCGGTTCGGGACCATACTCCT-5' (in the case of conversion to Asp) TG or GC 5'CT or AG 5'- -3 '3'-CCGGTTCGGGACCATACTCCC-5' (in the case of conversion to Gly) (2) Production of human TNF polypeptide variant Each transforming plasmid prepared in the above (1) is subjected to a conventional method.
According to the procedure, E. coli strain HB101 was introduced to prepare transformants.
It was. The transformant was prepared according to the method described in Example 1- (2).
And cultured, and described in Example 1- (2) from the cell extract.
Purification according to the method described in 1.
A lipotide mutant was obtained. TNS-115S: 115th amino acid in the amino acid sequence of the formula [I]
Polypeptide TNS-115D in which Pro, which is a noic acid, is converted to Ser: The 115th amino acid in the amino acid sequence of formula [I]
Polypeptide TNF-115G in which Pro which is a noic acid is converted to Asp: 115th amino acid in the amino acid sequence of the formula [I].
Polypeptide in which No, an acid, Pro is converted to Gly Example 8 Production of human TNF polypeptide variant TNF-117H (1) Construction of trait expression plasmid Tables 1 to 1 of the amino acid sequence shown by [I] No. 155
No. 117 from the N-terminal in the sequence consisting of 155 residues corresponding to
Amino acid of Tyr is converted to other amino acid, eg His
For the production of purified polypeptide (TNF-117H)
The quality expression plasmid was prepared according to the method described in Example 2- (1).
However, the following formula is used instead of the synthetic adapter [e].
Chemically synthesized oligonucleotide adapter
I built it. 5'-CCATCTGGGAGGGGTCTTCCAG-3 '3'-GTAGGTAGACCCTCCCCAGAAGGTC-5' (2) Production of TNF-117H Using the expression plasmid prepared in the above (1) according to a conventional method.
Therefore, it was introduced into E. coli HB101 strain to prepare a transformant.
It was. The transformant was prepared according to the method described in Example 1- (2).
And cultured, and described in Example 1- (2) from the cell extract.
TNF-117H was obtained by purification according to the method described in 1. Example 9 Production of Other Human TNF Polypeptide Variants-3 According to the method described in Example 1, the following polypeptide was produced.
Construct a gene expression plasmid for production and introduce it into E. coli.
A transformant was prepared. Culture the transformant and
The following polypeptides were isolated and purified from the extract. TNF-16V: 16th amino acid in the amino acid sequence of formula [I]
Polypeptide where acid Ala is converted to Val TNF-31T: The 31st amino acid in the amino acid sequence of formula [I]
Polypeptide in which acid Ala is converted to Thr TNF-32G: The 32nd amino acid in the amino acid sequence of formula [I]
A polypeptide in which the acid Asn is converted to Gly TNF-32L: The 32nd amino acid in the amino acid sequence of formula [I]
Polypeptide in which the acid Asn is converted to Leu TNF-36V: 36th amino acid in the amino acid sequence of formula [I]
Polypeptide in which the acid Ala is converted to Val TNF-73P: 73rd amino acid in the amino acid sequence of formula [I]
Polypeptide in which the acid Leu is converted to Pro TNF-82D: The 82nd amino acid in the amino acid sequence of formula [I]
A polypeptide in which the acid Ala is converted to Asp (isoelectric point 5.
3) TNF-85H: the 85th amino acid in the amino acid sequence of formula [I]
Polypeptide in which the acid Tyr is converted to His (isoelectric point 6.
4) TNF-98H: the 98th amino acid in the amino acid sequence of formula [I]
Polypeptide TNF-103P in which the acid Pro is converted to His: The 103rd amino acid in the amino acid sequence of the formula [I].
Polypeptide TNF-115T in which Thr, which is a non-acid, is converted to Pro: TNF-115T: the amino acid sequence at position 115 in the amino acid sequence of formula [I]
Polypeptide TNF-115H in which Pro, which is a non-acid, is converted to Thr: The 115th amino acid in the amino acid sequence of formula [I].
Polypeptide in which Pro, which is a non-acid, is converted to His TNF-115R: 115th amino acid in the amino acid sequence of formula [I]
Polypeptide TNF-131l in which Pro, a non-acid, is converted to Arg: The 131st amino acid in the amino acid sequence of formula [I].
Polypeptide TNF-141Y in which Ser which is a non-acid is converted to Ile: the 141st amino acid in the amino acid sequence of the formula [I].
Polypeptide TNF-143V in which Aso which is a noic acid is converted to Tyr: 143rd amino acid in the amino acid sequence of formula [I]
Polypeptide TNF-144K in which the amino acid Ala has been converted to Val: the 144th amino acid in the amino acid sequence of the formula [I].
Polypeptide TNF-146E in which Glu which is a noic acid is converted to Lys: The 146th amino acid in the amino acid sequence of the formula [I].
Polypeptide in which Gly which is a noic acid is converted to Glu Example 10 Production of Other Human TNF Polypeptide Variants-4 According to the method described in Example 1, the following polypeptide production was performed.
Construct a gene expression plasmid for production and introduce it into E. coli.
A transformant was prepared. Culture the transformant and
The following polypeptides were isolated and purified from the extract. TNF-32A: The 32nd Asn in the amino acid sequence of the formula [I] is A
Polypeptide converted into la TNF-32C: The 32nd Asn in the amino acid sequence of the formula [I] is C
Polypeptide converted into ys TNF-32I: The 32nd Asn in the amino acid sequence of the formula [I] is I
Polypeptide converted into le TNF-32R: The 32nd Asn in the amino acid sequence of formula [I] is A
Polypeptide converted into rg TNF-32T: The 32nd Asn in the amino acid sequence of formula [I] is T
Polypeptide converted into hr TNF-32V: The 32nd Asn in the amino acid sequence of formula [I] is V
Polypeptide TNF-34I converted into al: The 34th Leu in the amino acid sequence of formula [I] is I
Polypeptide converted into le TNF-48M: 48th Val in the amino acid sequence of formula [I] is M
Polypeptide TNF-89I converted into et: The 89th Val in the amino acid sequence of formula [I] is I
Polypeptide converted into le TNF-94T: 94th Ala in the amino acid sequence of formula [I] is T
Polypeptide converted into hr TNF-97N: The 97th Ser in the amino acid sequence of formula [I] is A
Polypeptide converted to sn TNF-98L: The 98th Pro in the amino acid sequence of formula [I] is L
eu-converted polypeptide TNF-113C: Tyr at position 113 in the amino acid sequence of formula [I]
Polypeptide in which is converted to Cys TNF-115Q: The 115th Pro in the amino acid sequence of formula [I]
GNF-converted polypeptide TNF-115A: the 115th Pro in the amino acid sequence of formula [I]
Is converted to Ala. TNF-115F: The 115th Pro in the amino acid sequence of formula [I]
Is converted into Phe TNF-115N: Proposition 115 of the amino acid sequence of formula [I]
Is converted to Asn TNF-115Y: Pron at position 115 in the amino acid sequence of formula [I]
Is converted to Tyr. TNF-115V: Pron at position 115 in the amino acid sequence of formula [I]
Is converted into Val TNF-115E: Pron at position 115 in the amino acid sequence of formula [I]
GNF-converted polypeptide TNF-115M: The 115th Pro in the amino acid sequence of formula [I]
Is converted to Met. TNF-115I: The 115th Pro in the amino acid sequence of formula [I]
Is converted to Ile TNF-115W: The 115th Pro in the amino acid sequence of formula [I]
In which TRP is converted to Trp: TNF-115K: The 115th Pro in the amino acid sequence of formula [I]
Polypeptide in which is converted to Lys TNF-118Q: The 118th Leu in the amino acid sequence of the formula [I]
Is converted to Gln TNF-132T: Ala at the 132nd position in the amino acid sequence of formula [I]
Is converted to Thr TNF-145C: Ser at the 145th position in the amino acid sequence of formula [I]
Is converted into Cys TNF-153L: 153rd Ile in the amino acid sequence of the formula [I]
Polypeptide in which is converted to Leu Reference Example 1 Construction of expression plasmid for human TNF production Cloned cDNA encoding human TNF was cloned into Europe.
Recombination prepared according to the method described in published patent 155549
Excised from the body plasmid pHTNF13 with the restriction enzyme PstI
And isolated. This cloned cDNA was further digested with restriction enzymes.EcoErase by RI
Cleavage cuts part of the untranslated region downstream of the TNF translation region
After removal, a DNA fragment of about 1.1 kbp was obtained. This DNA fragment is
Smid pBR322 restriction enzymeEcoRI andPSTFragment by I
(About 3.6 kbp), the TNF cDNA and tetra
A new recombinant plasmid containing a cyclin resistance gene.
I built the code. This recombinant plasmid was named pHT113.
Ok. This plasmid pHT113 has a restriction enzymeAvaI andSalAct I
3 types of DNA fragments (about 0.8 kbp, 1.3 kbp and 2.6 k, respectively)
bp). Among these DNA fragments, human TNF
Of the plasmid pBR322
Approximately 1.3 kbp DNA fragment containing part of the cleanse resistance gene was isolated.
It was purified by separation (this DNA fragment is called AvaI-SalI fragment). The AvaI-SalI fragment was added to the synthetic oligonucle
Cleotide adapter [f] was attached. 5′-CGATATGTCATCTTCTCGAACC-3 ′ [f] 3′-TATACAGTAGAAGAGCTTGGGGCT-5 ′ The obtained DNA fragment is hereinafter referred to as HTNF-adapter fragment.
U. Separately,trpIncludes part of the promoter region
DNA fragment (35 bp long) was used as plasmid pDR720 [PL Bio
chemicals; D.R.Russell, et al., Gene,20, 231 (198
3)] From restriction enzymesEcoRI andHpaCut out by I,
Released. The base sequence of this DNA fragment is as shown in the following formula. 5′-AATTCCCCTGTTGACAATTAATCATCGAACTAGTT 3′-GGGGACAACTGTTAATTAGTAGCTTGATCAA In addition to this, the following formula 5′-AACTAGTACGCAAGTTCACGTAAAAAGGGTAAT [g] 3′-TTGATCATGCGTTCAAGTGCATTTTTCCCATTAG4 adapter represented by the following formula is used.
Ligation was performed using DNA ligase. DNA fragment obtained here
One piece belowtrpIt is called a promoter fragment. On the other hand, the restriction enzyme was added to the plasmid pBR322.EcoRI andSalI
Cut out a large DNA fragment (about 3.7 kbp long)
It was. In addition to this, the HTNF-adapter fragment prepared earliertrp
Ligating promoter fragments with T4 DNA ligase
Therefore, in the amino acid sequence of Table 8, amino acid number 79
Human TNF consisting of 155 amino acids corresponding to ~ 233
A trait expression plasmid for production was constructed (see Fig. 9).
See). This expression plasmid was named pHTR91. Reference Example 2 Transfection for production of human TNF polypeptide derivative TNF (147)
Construction of the current plasmid In the amino acid sequences shown in Table 8, the sequences 84 to 233
Human TNF derivative consisting of the corresponding 147 amino acid residues [below
Below, TNF (147)] for the expression plasmid for production (p
HT147) was constructed (see Fig. 10). Recombinant plasmid obtained by the method described in Reference Example 1
pHTR91 restriction enzymeClaI andBalDigested with I, 4 kinds of DN
This is the A fragment. Two of those small pieces (each one
113bp and 0.6kbp) 5% polyacrylamide gel electrophoresis
Separated and purified in. Of these, a small fragment (113BP)
Further restriction enzymeDdeTwo DNA fragments (47 bp and 66 bp) in I
It was cleaved and a 47 bp DNA fragment was isolated. This DNA fragment is
It is called the lower 47 bp fragment. This fragment is represented by the following formulas 5'-CGATTATGAAGCCTGTAG-3 '[h] 3'-TAATACTTCGGACATCGGG-5' and 5'-CCCATGTTGTAGCAAACCCTCAAGC-3 '[i] 3'-TACAACATCGTTTGGGAGTTCGACT-5', each synthesized by a conventional method. Two types of oligonucleotide adapters
Combine [h] and [i] with T4 DNA ligase
It was. Further, a chemically synthesized oligonucleotide adapter [j] represented by the following formula 5′-AACTAGTACGCAAGTTCACGTAAGGAGGTTAT-3 ′ [j] 3′-TTGATCATGCGTTCAAGTGCATTCCTCCAATAGC-5 ′ was connected.
I put them together. The DNA fragment obtained here is hereinafter referred to as a 5'-DNA fragment.
Say. On the other hand, plasmid pCT-1 [M.Ikehara et al., Proc.N
atl.Acad.Sci., USA,81, 5956 (1984)]Hpa
I andAatLet II act,trpPart of the promoter region
A DNA fragment of about 380 bp containing it was cut out and the 5'-
The DNA fragments were ligated. The obtained DNA fragment is referred to as
It is called a motor 5'-DNA fragment. Separately, the recombinant plasmid prepared by the method described in Reference Example 1 was used.
Restriction enzyme for rasmid pHTR91BalI andHinacted dIII
The region encoding the C-terminal portion of human TNF polypeptide.
A DNA fragment (487 bp) containing the region was excised and separated and purified. This
The promoter 5'-DNA fragment previously adjusted to the DNA fragment of
Ligation was performed using T4 DNA ligase. The obtained DNA fragment
Is hereinafter referred to as promoter TNF (147) -DNA fragment. On the other hand, the plasmid pBRS described in Example 1- (1)
Restriction enzyme to 6AatII andHinCut out dIII
A large DNA fragment (about 3.6 kbp) was isolated. Promoter TNF (147) -D previously adjusted to this DNA fragment
By ligation of NA fragment, expression plasmid pH
We constructed T147. Reference Example 3 Human TNF Polypeptide Variant TNF-67S Production Expression Expression
Construction of Lasmid In the amino acid sequence shown in formula [I] of Table 1,
Polypep with the 67th amino acid Cys converted to Ser
Tide (hereinafter TNF-67S) production expression plasmid
(PHTP392) was constructed (see Fig. 11). Control recombinant pHTR91 obtained by the method described in Reference Example 1.
The salts shown in Table 8 were digested with the restriction enzymes AvaI and HindIII.
Corresponds to the region downstream from base number 250 in the base sequence
A DNA fragment of about 600 bp in length was isolated. This DNA fragment is furtherAvaII andHgi3 in AI
Cut into two DNA fragments (about 162 bp, 41 bp and 375 bp)
162 bp and 375 bp DNA by acrylamide gel electrophoresis
The fragment was isolated. These DNA fragments are converted into chemically synthesized oligos represented by the following formula.
Nucleotide adapters [k] and [l], 5'-GTCCTCTTCAAGGGCCAA-3 '3'-GAGAAGTTCCCGGTTCCGA-5' [k] and 5'-GGCTCTCCCTCCACCCATGTGCT-3 '[l] 3'-GAGGGAGGTGGGTAC-5' using T4 DNA ligase. And combined. Furthermore, the obtained DNA fragment is chemically synthesized by the following formula.
Oligonucleotide adapters [j] and [m], 5′-AACTAGTACGCAAGTTCACGTAAGGAGGTTAT-3 ′ [j] 3′-TTGATCATGCGTTCAAGTGCATTCCTCCAATAGC-5 ′ and 5′-CGATTATGTCATCTTCTCGAACC-3 ′ [m] 3′-TAATACAGTAGTAADNAGCGGCT, 5′-TAATACAGTAGAGAGCTTGGCT Were connected sequentially using. Got here
The obtained DNA fragment is hereinafter referred to as TNF (Ser67) -DNA fragment. on the other hand,trpDNA fragment containing the promoter region (approximately 380b
p) is the plasmid pCT-1 as described in Reference Example 2.
Restriction enzymeHpaI andAatIt was cut out by II and isolated.
This DNA fragment was ligated with the previously prepared TNF (Ser67) -DNA fragment.
I put them together. This DNA fragment is referred to as the promoter TNF (Ser6
7) -It is called a DNA fragment. Separately, obtained by the method described in Example 1- (1)
Plasmid vector pBRS6AatII andHindII
It was cut with I and a large DNA fragment (about 3.6 kbp) was isolated.
This DNA fragment contains the above promoter TNF (Ser67) -DNA fragment
By joining the pieces together using T4 DNA ligase,
A quality expression plasmid pHTP392 was constructed. This trait expression plasmid is described in Example 1- (2).
Introduced into E. coli and culturing the transformant according to the method of
TNF-67S was produced by feeding. Reference Example 4 The following expression plasmid for producing the polypeptide was prepared.
It was constructed according to the method described in Example 1. Culture E. coli transformed with the expression plasmid
By doing so, the following polypeptide was produced. TNF-70Y: The 70th Thr in the amino acid sequence of formula [I] is T
Polypeptide TNF-99S converted into yr: 99th Cys in the amino acid sequence of formula [I] is S
er-converted polypeptide Reference Example 5 The following expression plasmid for polypeptide production was prepared.
It was constructed according to the method described in Example 1. E. coli transformed with these expression plasmids
The following polypeptides were produced by culturing
However, most of these polypeptides are soluble proteins.
It was not extracted at all. TNF-12T: 12th Ala in the amino acid sequence of formula [I] is T
Polypeptide TNF-13Y converted into hr: 13th His in the amino acid sequence of formula [I] is T
Polypeptide TNF-14A converted to yr: 14th Val in the amino acid sequence of formula [I] is A
Polypeptide converted to la TNF-17T: In the amino acid sequence of formula [I], the 17th Asn is T
Polypeptide TNF-24F converted into hr: In the amino acid sequence of formula [I], the 24th Leu is P
Polypeptide TNF-26R converted to he: Trp at the 26th position in the amino acid sequence of formula [I] is A
Polypeptide TNF-35P converted to rg: In the amino acid sequence of the formula [I], the 35th Leu is P
Polypeptide converted into ro TNF-44D: Asn at position 44 in the amino acid sequence of formula [I] is A
Polypeptide TNF-45P converted into sp: 45th Gln in the amino acid sequence of formula [I] is P
Polypeptide converted into ro TNF-50P: The 50th Ser in the amino acid sequence of formula [I] is P
Polypeptide converted into ro TNF-54C: Tyr at position 54 in the amino acid sequence of formula [I] is C
Polypeptide converted to ys TNF-58P: The 58th Ser in the amino acid sequence of the formula [I] is P
Polypeptide TNF-59L converted into ro: Gln at the 59th position in the amino acid sequence of formula [I] is L
eu-converted polypeptide TNF-60D: Val at the 60th position in the amino acid sequence of formula [I] is A
Polypeptide TNF-60G converted into sp: 60th Val in the amino acid sequence of formula [I] is G
ly converted polypeptide TNF-62S: Phe at the 62nd position in the amino acid sequence of the formula [I] is S
er-converted polypeptide TNF-93P: In the amino acid sequence of formula [I], the 93rd Ser is P
Polypeptide converted into ro TNF-121G: Val at the 121st position in the amino acid sequence of formula [I]
In which Gly was converted to Gly TNF-124Q: Leu at position 124 in the amino acid sequence of formula [I]
GNF-converted polypeptide TNF-128A: Asp at position 128 in the amino acid sequence of formula [I]
Is converted to Ala TNF-128N: Asp at position 128 in the amino acid sequence of formula [I]
Is converted to Asn TNF-135D: Asn at the 135th position in the amino acid sequence of formula [I]
Converted into Asp TNF-138Y: Asp at position 138 in the amino acid sequence of formula [I]
Is converted to Tyr. TNF-148D: Val at position 148 in the amino acid sequence of formula [I]
Polypeptide converted from Asp to TNF-148G: Val at position 148 in the amino acid sequence of formula [I]
Is converted to Gly. TNF-150L: Phe at position 150 in the amino acid sequence of formula [I]
Is converted into Leu TNF-151E: 151st Gly in the amino acid sequence of formula [I]
Polypeptide converted to Glu

[Brief description of drawings]

Fig. 1 shows an example of restriction enzyme cleavage recognition sites of DNA encoding human TNF polypeptide. In the figure Indicates the region encoding human TNF. Figures 2-3 show the steps for constructing a trait expression plasmid for the production of TNF-32Y. In the figure, synthetic oligonucleotide [a],
[B] and [c] mean the synthetic DNA adapters described in Example 1- (1), respectively. FIG. 4 shows the steps for preparing the PL-DNA fragment used for constructing the expression plasmid for TNF-115L production. In the figure, synthetic oligonucleotides [d] and [e] mean the synthetic DNA adapters described in Example 2- (1). FIG. 5 shows the elution pattern of the digestion product (peptide fragment) of lysyl endopeptidase of TNF-115L by high performance liquid chromatography. FIG. 6 shows the steps for constructing the expression plasmid pHPL-147 (Example 3). FIG. 7 shows the steps for constructing the trait expression plasmid pHPL-Ser67 (Example 4). FIG. 8 shows the steps for constructing the trait expression plasmid pHPL147S67 (Example 5). Figure 9 shows the expression plasmid pHTR9 for the production of mature human TNF.
1 shows the construction process. In the figure, synthetic oligonucleotides [f] and [g] are synthetic DNAs described in Reference Example 1, respectively.
Means adapter. FIG. 10 shows the steps for constructing the expression plasmid pHT147 for producing a polypeptide having an amino acid sequence of 147 residues.
In the figure, synthetic oligonucleotides [h], [i] and [j] mean the synthetic DNA adapters described in Reference Example 2, respectively. Figure 11 shows the expression plasmid pHTP for TNF (Ser67) production.
The construction process of 392 is shown. In the figure, synthetic oligonucleotides [k], [l], [j] and [m] are respectively referred to in Reference Example 3.
The synthetic DNA adapter described in 1.

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Claims (3)

[Claims] 1. In the amino acid sequence represented by the following formula [I], the 16th Ala is Val, the 31st Ala is Thr, and the 32nd Asn is Ala, Cys, Asp, His, Ile. , Arg, Ser, Thr, Val
Or at Tyr, the 34th Leu to Ile, the 36th Ala to Val, the 48th Val to Met, the 73rd Leu to Pro, the 82nd Ala to Asp, the The 85th Tyr is His, the 89th Val is Ile, the 94th Ala is Thr, the 97th Ser is Asn, the 98th Pro is His or Leu, and the 103rd is Thr to Pro, 113th Tyr to Cys, 115th Pro to Leu, His, Gln, Ser, Ala, Phe, Asn, Thr, Gl
y, Tyr, Val, Gln, Met, Ile, Asp, Trp, Lys, Arg or Thr
The 117th Tyr to His, the 118th Leu to Gln, the 131st Ser to Ile, the 132nd Ala to Thr, the 141st Asp to Tyr, and the 143rd. Of Ala to Val, 144th Glu to Lys, 145th Ser to Cys, 146th Gly to Glu or 153rd Ile to Leu, or a polypeptide having an amino acid sequence A polypeptide in which a maximum of 8 amino acid residues on the N-terminal side of the polypeptide are sequentially deleted from the N-terminal. 2. The polypeptide according to claim 1, wherein Asn at the 32nd position in the amino acid sequence represented by the formula [I] in claim 1 is converted to Tyr. 3. The polypeptide according to claim 1, wherein the 115th Pro in the amino acid sequence represented by the formula [I] of claim 1 is converted to Leu.