JPS6357440B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to the general field of recombinant DNA technology. More particularly, the present invention relates to mutationally modified biologically active proteins that differ from their parent analogs by one or more substitutions/deletions of cysteine residues. . (Prior art and problems to be solved by the invention) Biologically active proteins produced microbiologically using recombinant DNA (rDNA) technology contain cysteine residues, Free to form undesired inter- or intra-molecular bonds, although not essential to the One such protein is human beta interferon (IFN-β), which is produced microbiologically. In the process of producing IFN-β using rDNA technology, high concentrations of IFN
It has been found that microbiologically produced IFN-β dimers and oligomers are formed in E. coli extracts containing -β. This multimer formation makes the purification and isolation of IFN-β very laborious and time-consuming, requiring the protein to be reduced during purification and then re-oxidized to return it to its original conformation. , requires several additional steps in the purification isolation procedure, thereby increasing the possibility of incorrect disulfide bond formation. Furthermore, it has been found that microbiologically produced IFN-β consistently exhibits low specific activity, possibly due to multitamer formation or random intramolecular disulfide bridge formation. Therefore, IFN
- Microbiologically produced and biologically active proteins such as β can be treated with undesirable tertiary structures that do not adversely affect their activity (e.g., conformations that reduce protein activity).
It may be desirable to modify the polymer in a manner that reduces or eliminates its ability to form intermolecular crosslinks or intramolecular bonds that would result in the formation of intermolecular crosslinks or intramolecular bonds. (Means for Solving the Problems) The present invention provides directed mutation (directed mutation).
mutagenesis), the parent structural analogue (parent)
Concerning the production of mutationally modified biologically active proteins that retain the activity of analogs) but lack intermolecular disulfide bonds or the ability to form undesired intramolecular disulfide bonds. ” (mutein). “Glossary of Genetics and Cytogenetics”
Genetics and Cytogenetice) 4th edition, p. 381, Springier-Berlag (1976)]. In this regard, Shepard, H.
M.) et al. [Nature (1981) Vol. 294, pp. 563-565]
describes a mutein in which cysteine at position 141 of the amino acid sequence of IFN-β is replaced with tyrosine [human IFN-β has cysteine at positions 17, 31, and 141]. Gene (1980)
Volume 10, pages 11-15 and Nature (1980) Volume 285, 542.
547 pages]. This mutein is a partial IFN-β gene with a G→A transition at nucleotide 485 of the IFN-β gene.
Produced by bacterial expression of hybrid genes constructed from cDNA clones. This mutein has lost its original biological activity of IFN-β,
The authors concluded that the replaced cysteine was essential for activity. Directed mutagenesis techniques are well known and have been described by Lather, RF and Lecoq, JP, Genetic Engineering, Academic Press (1983). (2013), pp. 31-50. Oligonucleotide-directed mutations are
Smith, M. and Gillam, S., Genetic Engineering: Principles and Methods, Plenum Press, 1981.
A detailed discussion is given in Volume 3, pp. 1-32. The present invention provides natural interleukin-2 in which the cysteine residue at position 125 of natural interleukin-2 is replaced with another amino acid residue, and natural interleukin-2
The present invention relates to a modified interleukin-2 having the biological activity of, and a method for producing the same. This modified interleukin-2 can generally be produced as follows. (a) Single-stranded DNA consisting of a single strand of the structural gene encoding the parent protein is hybridized with a mutant oligonucleotide primer (this primer is paired with the codon for cysteine that is deleted or to be replaced, or, if necessary, with this codon). complementary to the region containing the antisense triplet that makes the combination, but mismatched with the codon or antisense triplet that defines the deletion of the codon or the triplet encoding another amino acid. ), (b) extend the primer with a DNA polymerase to form a mutant heteroduplex, and (c) replicate the mutant heteroduplex. The mutant oligonucleotide primers used in this method are another aspect of the invention. In the present invention, cysteine residues that are not essential for the biological activity of biologically active proteins are intentionally deleted to eliminate sites for forming intermolecular crosslinks or erroneous intramolecular disulfide bonds. or muteins in which other amino acids are substituted, mutant genes encoding such muteins, and means for producing such muteins. Proteins that can be mutationally modified according to the present invention include cysteine content of biologically active proteins;
and the role that cysteine residues play in terms of activity and tertiary structure can be ascertained from available information. For proteins for which such information is not available in the literature, systematically alter each cysteine residue in the protein by the procedures described herein;
This information can be determined by testing the resulting muteins for biological activity and their tendency to form undesirable intermolecular or intramolecular disulfide bonds. Therefore, the present invention
As specifically described and exemplified below with respect to IFN-β and IL-2 muteins, functionally non-essential cysteine residues that may sensitize the protein to the formation of undesired disulfide bonds are included. It will be appreciated that the following teachings apply to any other biologically active protein contained therein. In addition to IFN-β and IL-2, examples of proteins that are candidates for mutagenic modification according to the present invention include lymphotoxin (tumor necrosis factor) and colony-stimulating factor.
1, and IFN-α1. Candidate proteins usually have an odd number of cysteine residues. In the case of IFN-β, both glycosylated and unglycosylated IFNs show quantitatively similar specific activities; therefore, the glycosyl moiety
It was reported that it did not participate or contribute to the biological activity of However, non-glycosylated IFN-β produced by bacteria consistently exhibits a quantitatively lower specific activity than glycosylated native IFN-β. IFN-β is known to have cysteine residues at positions 17, 31, and 141. Cysteine 141 was demonstrated by Shepard (supra) to be essential for biological activity. In IFN-α, which contains four cysteine residues, there are two intermolecular -S-S- bonds, one with cys29.
Some are between cys138, others are between cys1 and cys98. IFN
Based on the homology between -β and IFN-α, IFN-β
cys141 has an intramolecular-SS bond with cys31, and cys17 can freely form an intermolecular cross-link. By deleting cys17 or substituting it with another amino acid, one can determine whether cys17 is essential for biological activity and its role in -S-S- bond formation. If cys17 is not essential for the biological activity of the protein, the resulting cys17-deficient or cys17-substituted proteins exhibit a specific activity close to that of native IFN-β;
It would also probably facilitate protein isolation and purification. An oligonucleotide-directed mutagenesis procedure using synthetic oligonucleotide primers that are complementary to the region of the cys17 codon of the IFN-β gene but contain one or more base changes in this codon. is used to create a designer gene, thus replacing cys17 with any other amino acid of choice. If a deletion is desired, use an oligonucleotide primer lacking the codon for cys17. cys17 to glycine, valine, alanine, leucine, isoleucine, tyrosine, phenylalanine, histidine,
Conversion to neutral amino acids such as tryptophan, serine, threonine and methionine is a preferred method. Serine and threonine are the most preferred alternatives due to their chemical similarity to cysteine. When cysteine is deleted, the mature mutein is one amino acid shorter than the natural parent protein or microbially produced IFN-β. Human IL-2 is reported to have cysteine residues at positions 58, 105, and 125 of the protein. IFN-
As in the case of β, IL-2 is in aggregated oligomeric form when isolated from bacterial cells, and in order to obtain good yields from bacterial extracts,
Must be reduced with a reducing agent. Moreover, purified reduced IL-2 is unstable and easily reoxidized to oligomeric inactive forms upon storage.
The presence of three cysteines means that upon reoxidation, the protein can randomly form one of three possible intramolecular disulfide bridges, whereas only one is the correct bridge as found in natural molecules. It means. Since the disulfide structure of natural IL-2 protein is unknown, IL-2
Create mutations in codons 58, 105, and 125 of the gene to determine which cysteine residues are required for activity and are therefore also most likely involved in natural disulfide bridge formation. The present invention can be used for Similarly, modification of cysteine residues not required for activity prevents the formation of erroneous intramolecular disulfide bridges, and removal or replacement of free cysteine residues reduces the opportunity for intramolecular disulfide bridges. can be minimized. The size of the oligonucleotide primer is determined by the conditions necessary for stable hybridization of the primer to the gene region to be mutated and by the limitations of currently available oligonucleotide synthesis methods. In designing oligonucleotides for use in oligonucleotide-directed mutagenesis, factors to be considered (e.g., overall size, size of portion bypassing the mutation site) are described by M. Smith and Described by S. Gillam (cited above). In general, the overall length of the oligonucleotide is such that it optimizes stable and unique hybridization at the mutation site, and extensions less than 5' and 3' from the mutation site are DNA It should be large enough to avoid the effects of mutation due to the exonuclease activity of the polymerase. The oligonucleotides used for mutagenesis according to the invention are:
Usually about 12 to about 24 bases, preferably about
It contains 14 to about 20 bases, more preferably about 14 to 18 bases. These usually contain at least about 3 bases 3' of the codon that is altered or deleted. Methods for creating modified IFN-β genes generally involve using synthetic nucleotide primers that either eliminate codon 17 or change it so that it encodes a different amino acid. (TGT) to induce site-specific mutations. When changing cysteine to threonine and hybridizing the primer to the antisense strand of the IFN-β gene, the preferred nucleotide primer is GCAATTTC
ACTCAG (underlined indicates changed codon). When it is desirable to delete cysteine,
The preferred primer is
AGCAATTTTCAGCAGAGAGCTCCTG, which has lost the TGT codon for cys. When replacing cysteine with serine, the 17-nucleotide primer GCAATTTTCAG AGT CAG containing the AGT codon for serine is selected.
A change from cysteine to serine occurs at cys17 by transposition of the first base T→A. When introducing deletions, it must be recognized that the proper reading frame of the DNA sequence must be maintained for expression of the desired protein. The primer is a primer such as M13, fd, or φ×174, in which the single strand of the IFN-β gene has been cloned.
Hybridization occurs to the double-stranded phage. It will be appreciated that phages can carry either the sense or antisense strand of a gene. If the phage carries an antisense strand, the primer is directed to a region of the sense strand that does not match this codon but defines a codon deletion or triplet encoding another amino acid, but that contains the codon to be mutated. are the same. If the phage carries a sense strand, it is suitably mismatched in the triplet that pairs with the codon to be deleted, but complementary to the region of the sense strand that contains the codon to be mutated. The conditions used for hybrid formation are described by M. Smith and S. Gillam (supra). The temperature is usually around 0℃ or
70℃, but generally ranges from about 10℃ to 50℃. After hybridization, the primers
DNA polymerase, T4DNA polymerase,
It is extended on the phage DNA by reaction with reverse transcriptase or other suitable DNA polymerase.
The resulting dsDNA is processed using enzymes such as T4 DNA ligase.
It is converted to closed circular dsDNA by treatment with DAN ligase. DNA molecules containing single-stranded regions are
Can be destroyed by S1 endonuclease treatment. Oligonuclease-directed mutagenesis generates a mutant IL-2 encoding a mutein with IL-2 activity but with a cys125 to serine 125 change.
It can be similarly used to create two genes. If the phage carries the sense strand of the gene, the preferred oligonucleotide primers used to create this mutant IL-2 gene are
GATGATGCTTCTG AGA AAAGGTAATC. This oligonucleotide has a C→G change at the base in the middle of the torpette that pairs with codon 125 of the IL-2 gene. The resulting mutant heterodiplex is
Used to transform competent host organisms or cells. Replication of the heteroduplex by the host produces progeny strands from both strands. Following replication, the mutant gene is isolated from the progeny of the mutant strand, inserted into a suitable vector, and this vector is used to transform a suitable host organism or cell. Preferred vectors are plasmids
pBR322, pCR1 and their variants, synthetic vectors, etc. A suitable host organism is E. coli (E.
Coli), Pseudomonas, Bacillus subtilis, Bacillus thuringiensis
thuringiensis), various yeast strains, Bacillus thermophilus, animal cells such as mouse, rat, and Chinese hamster ovary (CHO) cells, plant cells, and animal and plant hosts. It should be appreciated that when transforming the host of choice with the vector, appropriate promoter-operator sequences are also introduced in order for the mutein to be expressed. The host may be a prokaryote or a eukaryote. (Methods for inserting DNA into eukaryotic cells are described in PCT application numbers US81/00239 and US81/00240, published September 3, 1981). E. coli and CHO cells are preferred hosts. Muteins obtained according to the invention may or may not be glycosylated depending on the glycosylation that occurs in the natural parent protein and the host organism used for mutein preparation. As desired,
The unglycosylated muteins obtained when using E. coli and Bacillus as hosts can be produced in vivo by chemical, enzymatic and other modifications known in the art. It may optionally be glycosylated ex situ. In a preferred embodiment of the present invention regarding IFN-β, as shown in FIG. 1, the cysteine residue at position 17 in the amino acid sequence of IFN-β is
- Codons on the sense strand of the DNA sequence encoding β
The first base of 17 is converted to serine by T→A rearrangement. This site-directed mutagenesis is performed using synthetic 17-nucleotide primers.
GCAATTTTCAG AGT CAG, this primer is identical to the 17 nucleotide sequence on the sense strand of IFN-β in the region of codon 17, except for a single base mismatch at the first base of codon 17. It is. This mismatch is at nucleotide 12 of the primer. It must be recognized that the genetic code (codons) is degenerate and many amino acids are encoded by more than one codon. For example, the base code for serine is degenerate in six ways, such that the codons TCT, TCG, TCC, TCA, AGT, and ACG all code for serine. For convenience, the AGT codon was chosen as the preferred embodiment. Similarly,
Threonine is encoded by any one of the ACT, ACA, ACC and ACG codons. When one codon is specified for a particular amino acid, it is intended that this includes all degenerate codons encoding that amino acid. 17-mer is
Hybridized to single-stranded M13 phage DNA carrying the antisense strand of the IFN-β gene.
The oligonucleotides are then extended onto the DNA using DNA polymerase Klenow fragment, and the resulting dsDNA is used with T4 ligase to form closed circular DNA.
Convert to Replication of the resulting mutagenic heteroduplex yields a clone from the DNA strand containing the mismatch. Mutant clones are
Confirmed and screened for the presence or absence of specific restriction positions, antibiotic resistance or sensitivity, or other methods known in the art. When cysteine is replaced by serine, a new Hinf restriction site is created in the structural gene by the T→A rearrangement shown in FIG. Mutant clones are identified using oligonucleotide primers as probes in hybridization screens for mutated phage plaques. As shown in Figure 2, the primers will have only one mismatch when hybridized to the parent, but will have a perfect match when hybridized to the mutated phage DNA. Hybridization conditions can be devised so that the oligonucleotide primer hybridizes preferentially to the mutant DNA rather than the parent DNA. newly occurred
The Hinf site also serves as a means of identifying single base mutations in the IFN-β gene. M13 phages carrying mutated genes
The DNA is isolated and incorporated into a suitable expression vector, such as plasmid pTrp3, and this vector is transformed into E. coli strain MM294. Suitable growth media for culturing transformants and their progeny are known to those skilled in the art. The expressed muteins of IFN-β are isolated, purified, and characterized. The following examples are presented to aid in a further understanding of the invention and for illustrative purposes only. They should not be considered as limiting the scope of the invention in any way. Examples 1 to 9
The preparation of muteins for IFN-β is described. Examples 10-15 describe the preparation of muteins of IL-2. Example 1 (Reference example) Cloning of IFN-β gene into M13 vector The use of M13 phage vector as a source of single-stranded DNA template is described by GFTemple et al., Nature (1982). Volume 296 537-
Proven on page 540. Plasmid containing the IFN-β gene under the control of the E. coli trp promoter
pβ1trp (Figure 3) was digested with restriction enzymes Hind and Xho. M13mp8〔G.Metzing (J.
Messing). "Third Cleveland Symposium on Macromolecules: Recombinant DNA" edited by A. Walton, Elsavier Press, pp. 143-153 (1981)] Replicative (RF) DNA (Fig. 4) is extracted with restriction enzyme Hind. and BamH and pre-Hind
and pβ1trpDNA that had been digested with Xho. The mixture was then ligated with T4 DNA ligase,
The ligated DNA was transformed into competent cells of Escherichia coli strain JM103 and plated on Xgal indicator plates [J. Metzing et al.
Nucleic Acids Res (1981) Vol. 9, pp. 309-321].
Plaques containing recombinant phages (white plaques) were picked and inoculated into fresh cultures of JM103, and minipreps of RF molecules were prepared from infected cells (HDBirnboim and J.D. .
Doly), Nucleic Acid Res (1979) Vol. 7, 1513.
1523 pages). To identify clones containing IFN-β inserts, RF molecules were digested with various restriction enzymes. The restriction map of one such clone (M13-β1) is shown in FIG. Single-stranded (ss) phage DNA was generated from the M13-β1 clone and used as a template for site-directed mutagenesis using synthetic oligonucleotides. Example 2 (Reference example) Site-directed mutagenesis 0.1mM adenosine triphosphate (ATP), 50mM hydroxymethylaminomethane hydrochloride (Tris-HCl) PH8.0, 10mM magnesium chloride, 5mM dithiothreitol (DDT) and Synthetic oligonucleotides in 50 μl in the presence of 9 units of T4 kinase
40 pmol of GCAATTTTCAGAGTCAG (primer) was treated with T4 kinase for 1 hour at 37°C. 50mM sodium chloride, 10mM Tris-HCl, PH8.0, 10mM magnesium chloride and 10mM β
- 5 μg of single-stranded (ss) M13-β1 DNA by heating this kinased primer (12 pmoles) at 67°C for 5 minutes and at 42°C for 25 minutes in 50 μl of a mixture containing mercaptoethanol. A hybrid was formed. The annealed mixture was then cooled on ice and supplemented with 0.5mM each deoxynucleotide triphosphate (dNTP), 80mM Tris-HCl,
Add to 50 μl of a reaction mixture containing pH 7.4, 8 mM magnesium chloride, 100 mM sodium chloride, 9 units of DNA polymerase Klenow fragment, 0.5 mM MATP and 2 units of T4 DNA ligase and incubate at 37°C for 3 hours.
and incubated for 2 hours at 25°C. The reaction was then stopped by phenol extraction and ethanol precipitation. DNA in 10mM Tris-HCl, PH
8.0, 10mM ethylenediaminetetraacetic acid (EDTA),
It was dissolved in 50% sucrose and 0.05% bromophenyl blue and subjected to electrophoresis on a 0.8% agarose gel in the presence of 2 μg/ml ethidium bromide. M13-β1
percolate the DNA band corresponding to the RF type of [RWDavis]
3. “Advanced Bacterial Genetics”
Genetics), Cold Spring Harbor Laboratory, NY 178-179 (1980)]. The eluted DNA was used to transform competent JM103 cells, the bacteria were grown overnight, and ssDNA was isolated from the culture supernatant. This ssDNA was used as a template in the second cycle of primer extension, and the gel-purified RF form was
The DNA is transformed into competent JM103 cells, plated on agar plates, and incubated overnight to obtain phage plaques. Example 3 (Reference example) Site-directed mutagenesis The experiment of Example 2 above is repeated. However, as a synthetic oligonucleotide primer, it is possible to change codon 17 of the IFN-β gene from one encoding cysteine to one encoding threonine.
Use GCAATTTTCAGACTCAG. Example 4 (Reference example) Site-specific deletion The experiment of Example 2 above is repeated. However, as a synthetic oligonucleotide primer, it is possible to delete codon 17 of the IFN-β gene.
Use AGCAATTTTCAGCAGAGCTCCTG. Example 5 (Reference example) Screening and identification of mutagenized plaques Plates containing mutated M13-β plaques (Example 1) and non-mutated M13-
The two plates containing β1 phage plaques were cooled to 4°C and the plaques from each plate were placed onto two nitrocellulose round filters, and in the case of the first filter, the dry filter was placed onto an agar plate for 5 minutes. In the case of the second filter, transfer was carried out in layers for 15 minutes. Next, the filters were placed on thick filter paper soaked in 0.2NNaOH, 1.5M sodium chloride, and 0.2% Triton X-100 for 5 minutes, and then
Neutralization was achieved by layering on filter paper soaked in 0.5M Tris-HCl, PH7.5, and 1.5M sodium chloride for an additional 5 minutes. The filters were washed in a similar manner twice on filters soaked in 2×SSC (standard citrate), dried, and dried in a vacuum drying oven at 80° C. for 2 hours. 10ml per filter with duplicate filters
DNA hybridization buffer (5x SSC), PH
7.0, 4x Denhard solution (polyvinylpyrrolidone,
Ficoll and bovine serum albumin, 1× = 0.02 each
%), 0.1% sodium dodecyl sulfate (SDS),
50mM sodium phosphate buffer, PH7.0 and 100μ
Prehybridization was performed with denatured salmon sperm DNA at 55° C. for 4 hours. A ^32P -labeled probe was created by underkinase treatment of the oligonucleotide primer with ^32P -labeled ATP. 5ml DNA per filter
Filters were hybridized to 3.5 x 10 ⁵ cpm/ml of ³² P-labeled primer in hybridization buffer for 24 hours at 55°C. Filters were washed at 55° C. for 30 minutes each in wash buffer containing 0.1% SDS and decreasing amounts of SSC. First, wash the filters with a buffer containing 2x SSC.
Control filters containing unmutated M13-β1 plaques were then tested for the presence of radioactivity using a Geiger counter. The SSC concentration was reduced stepwise and filters were washed until no detectable radioactivity remained on control filters with unmutated M13-β plaques. The minimum concentration of SSC used was 0.1×SSC. Filters were air dried and autoradiographed at -70°C for 2-3 days. 480 mutated M13-β1 plaques and 100 nonmutated control plaques were screened with a kinased oligonucleotide probe. In control plaques, there was no hybridization with the probe;
On the other hand, five mutated M13-β1 plaques formed hybrids with the probe. One of the five mutant M13-β1 plaques (M13-SY2501) was picked and inoculated onto JM103 culture medium. ssDNA was prepared from the supernatant and double-stranded (ds) DNA from the cell pellet.
ssDNA as a template for dideoxy sequencing of clones using the M13 universal primer
It was used. The results of the sequencing analysis are shown in FIG. This result confirms that the TGTcys codon was converted to an AGTser codon. Example 6 (Reference example) Expression of mutant IFN-β in E. coli RF DNA from M13-SY2501 was extracted with restriction enzymes.
Digested with Hind and Xho and the 520 bp insert was purified on a 1% agarose gel. Plasmid pTrp3 containing the E. coli trp promoter (Figure 7)
was digested with the enzymes Hind and BamH and purified.
It was mixed with the M13-SY2501 fragment and ligated in the presence of T4 DNA ligase. Concatenate DNA with E. coli MM294
transformed into a strain. Ampicillin-resistant transformants were screened for sensitivity to the drug tetracycline. Plasmids from 5 ampicillin-resistant and tetracycline-sensitive clones
DNA was digested with Hinf to screen for the presence of the M13-SY2501 insert. 8th
Figure a shows Hinf of one of the clones (pSY2501).
The restriction pattern is shown in the figure, which is compared to the original IFN-
The Hinf patterns of the β clone, pβ1trp, are compared. As expected, pSY2501 has additional
There is a Hinf site, and the 197bp IFN-β internal fragment is cut into a 169bp fragment and a 28bp fragment (8th
Figure b). The restriction map of clone pSY2501 is shown in FIG. The complete DNA sequence of the mutant IFN-β gene is shown in FIG. 10 along with the predicted amino acid sequence. The plasmid named clone pSY2501 is
Deposited in the Agricultural Research Culture Collection (NRRL), Fermentation Research Institute, Northern Research Center, U.S. Department of Agriculture, Service of Science and Education, 1815 North University Street, Peoria, Illinois 60604. The designated deposit numbers are CMCC No. 1533 and NRRLB-15356 (deposit under the Budapest Treaty). Cultures of pSY2501 and pβ1trp (including progeny)
The cells were grown to an optical density of 1.0 (0D ₆₀₀ ). Cell-free extracts were prepared and the amount of antiviral activity of IFN-β was assayed using GM2767 cells using a microtiter assay. The extract of clone pSY2501 is from pβ1trp to
The clone showed high activity in 10 cultures (Table 1).
It is shown that either pSY2501 synthesizes a larger amount of a protein exhibiting IFN-β activity, or that the protein produced has a higher specific activity. Table 1 Extract antiviral activity (units/ml) pSY2501 6×10 ⁵ pβ1trp 1×10 ⁵ ptrp3 (control) 30 Extraction of both clones to determine whether clone pSY2501 was synthesizing several times more active protein. The samples were subjected to electrophoresis on SDS polyacrylamide gels along with control extracts, and the gels were stained with Coomassie blue to develop the proteins. As shown in Figure 11, approximately
There was only one protein band corresponding to the 18,000 Dalton protein. Although this protein has a molecular weight of approximately 20,000 daltons, it exhibits a gel migration pattern of a protein of 18,000 daltons, and purification of this protein from extracts of pβ1trp revealed that IFN-
It was known in advance that β. Since the amount of this protein is lower in the extract of pSY2501 than in the extract of pβ1trp, the specific activity of the protein in the extract of clone pSY2501 is higher than that of clone pβ1trp. Example 7 (Reference Example) Purification of IFM-βser17 E.
IFN-βser17 was recovered from E. coli. E. coli was grown in the following growth medium to an optical density of 10-11 at 680 nm (8.4 g/dry weight). Ingredient concentration Ammonium chloride 20mM Potassium sulfate 16.1mM Monopotassium phosphate 7.8mM Disodium phosphate 12.2mM Magnesium sulfate heptahydrate 3mM Trisodium citrate dihydrate 1.5mM Manganese sulfate tetrahydrate 30μM Zinc sulfate - Heptahydrate 30μM Copper sulfate pentahydrate 3μM L-tryptophan 70mg / Ferrous sulfate heptahydrate 72μM Thiamine hydrochloride 20mg / Glucose 40g / PH adjusted with ammonia Harvest of transformed E. coli 9.9 (9.9Kg)
Cooled to 20° C., the crop was concentrated by passing through a cross-flow filter with an average pressure drop of −110 kpa and a constant filtrate flow rate of 260 ml per minute until the filtrate weight was 8.8 Kg. The concentrate (approximately 1) was poured into a container and cooled to 15°C. Heat the concentrate at 5℃, approx.
The bacterial cells in the concentrate were disrupted by passing it through a Manton-Gorlin homogenizer at 69,000 kpa.
Phosphate buffered saline, PH7.4 (PBS) 1
The homogenizer was washed with water and the wash was added to the crush to give a final volume of 2. This volume was subjected to continuous centrifugation at 12000 xg with a flow rate of 50 ml per minute. The solid was separated from the supernatant and resuspended in PBS 4 containing 2% by weight SDS. After stirring this suspension for 15 minutes at room temperature, there was no visible suspension. The solution was then extracted with 2-butanol at a 1:1 volume ratio of 2-butanol:solution. liquid-
Extraction was carried out in a liquid phase separator using a flow rate of 200 ml per minute. The organic phase was then separated and evaporated to dryness, yielding 21.3 g of protein. This was resuspended in distilled water at a volume ratio of 1:10. The recovered product was assayed for human IFN-β activity using a protection against viral cytopathic effect (CPE)-based assay. This assay was performed in microtiter plates. Fill each container with a minimum of 50 μl of basic medium, and add the materials to the first container.
25 μl was added, and subsequent dilutions of 1:3 were carried out successively. Virus (vesicular stomatitis), cells (human fibroblast GM-2767 line) and standard IFN-β control group were loaded onto each plate. Standard IFN-β at 100 units per ml was used.
The plate was then irradiated with ultraviolet light for 10 minutes. After irradiation, 100 μl of cell suspension (1.2 x 10 ⁵ cells per ml)
was added to each container and the trays were incubated for 18-24 hours. Virus solution was added at 1 plaque forming unit per cell to each vessel except the cell control plate. Trays were incubated until the virus control showed 100% CPE. This usually occurred 18-24 hours after adding the virus solution. Standard IFN-
The assay results were interpreted in terms of the location of the β control 50% CPE container. From this point, interferon titers were determined for all samples on the plate. The specific activity of the recovered product was determined to be 5×10 ⁷ U/ml. Example 8 (Reference example) Purification by acid precipitation and chromatography The method of Example 7 was repeated, except that the extraction,
After separation of the aqueous and organic phases and mixing of the organic phase with PBS in a volume ratio of 3:1, the PH of the mixture was lowered to about 5 by addition of glacial acetic acid. The resulting precipitate
Separate by centrifugation at 10,000-17,000 x g for 15 min and pellet in 10% W/V SDS, 10mM
DTT was redissolved in 50mM sodium acetate buffer, PH5.5 and heated to 80°C for 5 minutes. The solution is then converted using a Beckman gradient system to
Brownlee RP-300, 10 μM, was applied to an “Aquapore” column. Buffer A was 0.1% trifluoroacetic acid (TFA) in water and buffer B was 0.1% TFA in acetonitrile. Detection was based on ultraviolet absorbance at 280 nm. Solvent program is 0 in 3 hours
The gradient was linear from % Buffer B to 100% Buffer B. The fractions containing the highest interferon activity were pooled, and the specific activity of this pooled interferon preparation was approximately 2 x 10 ⁸ international units per mg of protein in native IFN-β.
It was determined to be between 9.0×10 ⁷ and 3.8×10 ⁸ U/ml. Example 9 (Reference Example) Biochemical characterization of IFN-βser17 Amino acid composition was determined after 40 μg of sample was subjected to hydrolysis at 108°C for 24-72 hours in 200 μl of 5.7N HCl, 0.1% phenol. . Proline and cysteine were determined in the same manner after performic acid oxidation. In this case, phenol was omitted from the hydrolysis. For tryptophan, prepare 400 μl of sample in 5.7N hydrochloric acid, 10% mercaptoacetic acid (no phenol).
It was analyzed after 24 hours of hydrolysis. The analysis is
Beckmann using a single column of AA10 resin
The analysis was carried out using a 121MB amino acid analyzer. Purified IFN
-The amino acid composition calculated from representative 24-, 48-, and 72-hour acid hydrolysis of βser17 is predicted by the DNA sequence of the cloned IFN gene, except that the N-terminal methionine is missing. It matches well. The amino acid sequence of the first 58 residues from the amino acid terminus of purified IFN was determined from 0.7 mg of sample using a Beckman 890C sequencer using 0.1 M Quadrol buffer. PTH amino acids were purified by reverse-phase high-pressure liquid chromatography (HPLC) on an Artex Ultrasphere 0DS column (4.6 x 250 mm) at 45°C for 1.3 min with 40% buffer B and 40-70% buffer B.
Determined by elution at 8.4 minutes. Here buffer A was 0.0115M sodium acetate, 5% tetrahydrofuran (THF), pH 5.11 and buffer B was 10% THF in acetonitrile. The determined N-terminal amino acid sequence of IFN-βser17 corresponds to the predicted sequence predicted from the DNA sequence, except for the absence of the N-terminal methionine. As shown above, IFN-β ser17 preparations exhibit activity very close to or even superior to the specific activity level of native IFN-β. IFN-βser17 has no free sulfhydryl groups, but exhibits one -SS- bond between the remaining cysteines only at positions 31 and 141. Proteins do not easily form oligomers and are considered to be essentially in monomeric form. The IFN-βser17 obtained according to the invention, as a single product or as a mixture of various types, is inert, non-toxic, non-allergenic and can be prepared into pharmaceutically acceptable formulations in a physiologically acceptable carrier medium. Can be prescribed for clinical and therapeutic use in cancer therapy or in conditions where interferon therapy is indicated.
It can also be used for viral infections. Such vehicles include, but are not limited to, distilled water, saline, Ringer's solution, Hank's solution, and the like. Other non-toxic stabilizing and solubilizing additives such as dextrose, HSA (human serum albumin), etc. may also be optimally included. Therapeutic formulations can be administered orally or parenterally, such as by intravenous, intramuscular, intraperitoneal and subcutaneous administration. The modified IFN-β formulations of the invention can also be used for topical application in suitable vehicles commonly utilized for topical applications. The main advantage of the above-mentioned IFN-β muteins is that IFN-
The free sulfhydryl group (-SH group) at position 17 of β is eliminated, which allows the mutein to form the correct disulfide bond between cys31 and cys141, clearly resulting in full biological activity. I am getting the necessary conformation. IFN-
Due to the increased specific activity of βser17, lower doses can be used for treatment. By deleting the cysteine at position 17 and eliminating the free -SH group,
IFN-βser17 protein is an IFN produced by microorganisms-
It does not form dimers or oligomers as easily as β. This facilitates purification of the protein and enhances its stability. Example 10 The nucleotide sequence of a cDNA clone encoding human IL-2, the procedure for constructing an IL-2 cDNA library, and the procedure for screening it for IL-2 were described by Taniguchi (Taniguchi).
T.), Nature (1983), pages 302 , 24, and 305 onwards. A cDNA library enriched for potential IL-2 cDNA clones was created from the enriched IL-2 mRNA fraction obtained from induced peripheral blood lymphocytes (PBL) and Jyakkat cells by conventional procedures. To enrich mRNA for IL-2, we fractionated the mRAN, injected the fraction into immature oocytes of Xenopus laevis, and measured the IL-2 activity of the oocyte lysate into HT-2 cells. This was done by confirming the fraction with IL-2 mRNA activity [J.Watson, J.Exp.Med.
(1979) Vol. 150, pp. 1570-1519 and S. Gillis et al., J. Immun. (1978) Vol. 120, pp. 2027-
2032 pages]. Example 11 Screening and identification of IL-2 cDNA clones IL-2 cDNA clones were isolated using colony hybridization method.
2 cDNA libraries were screened. Each microtiter plate was copied by a replica method onto a layered nitrocellulose filter paper (S&S type BA-85), and grown on L agar containing 50 μg/ml ampicillin at 37° C. for 14 to 16 hours. By lysing the colonies and sequentially treating them with 500mM sodium hydroxide, 1.5M sodium chloride for 5 minutes.
DNA was fixed on the filter and washed twice with 5x standard citrate solution (SSC) for 5 minutes each. The filter was air dried and oven dried at 80°C for 2 hours. Add 10 ml of DNA hybridization buffer (50% formamide, 5x
SSC, PH7.0, 5x Denhard's solution (polyvinylpyrrolidine, Ficoll and bovine serum albumin; 1
x = 0.2% each), 50mM sodium phosphate buffer, pH 7.0, 0.2% SDS, 20μg/ml polyU and 50μg/ml modified salmon sperm DNA for 6-8 hours at 42°C. A 20-mer oligonucleotide probe labeled with ³² P was created based on the IL-2 gene sequence reported by Taniguchi (supra). The nucleotide sequence of the probe is
It was GTGGCCTTCTTGGGCATGTA. Samples were hybridized for 24-36 hours at 42°C with 5 ml of DNA hybridization buffer per filter containing the ³² PcDNA probe. The filters were washed twice for 30 min each at 50°C with 2x SSC, 0.1% SDS, then washed with 1x SSC and 0.1% SDS at 90°C.
Wash at 50°C for 2 min each, air dry, and dry at -70°C for 2 min.
Autoradiographed for ~3 days. Positive clones were identified and rescreened with the probe. Clones of sufficient length were identified by restriction enzyme mapping and the IL-
This was confirmed by comparison with the sequence of 2 cDNA clones. Example 12 Cloning of IL-2 gene into M13 vector IL-2 gene was cloned in the same manner as described in Example 1 using plasmid pLW1 (Fig. 12) containing the IL-2 gene under the control of the E. coli trp promoter. Two genes were cloned into M13mp9. The pLW1 sample was dated August 4, 1983, 12301 Parklawn Drive, Lockeville, Maryland, USA 20852.
It was deposited with the American Type Culture Collection and designated as ATCC No. 39,405 (International Deposit under the Budapest Treaty). One clone containing the IL-2 insert (referred to as M13-IL-2)
The restriction map is shown in Figure 13. single strand phage
DNA was generated from clone M13-IL-2 and used as a template for oligonucleotide-directed mutagenesis. Example 13 Oligonucleotide-Directed Mutagenesis As described above, IL-2 was isolated at amino acid positions 58,105.
and contains a cysteine residue at 125. IL-2
Based on the nucleotide sequence of the portion of the gene containing codons for these three cysteine residues, three oligonucleotide primers were designed and synthesized to mutate the codons for these residues into codons for serine. did. These oligonucleotides have the following sequences. CTTCTAGAGACTGC AGA TGTTTC changing cys58
(DM27) CATCAGCATACTC AGA CATGAATG changing cys105
(DM28) GATGATGCTCTG AGA AAAGGTAATC changing cys125
(DM29) 0.1mM ATD, 50mM Tris-HCl, PH8.0,
Forty pmoles of each oligonucleotide were separately kinased in 50 μl for 1 hour at 37° C. in the presence of 10 mM magnesium chloride, 5 mM DTT, and 9 units of T4 kinase. Each of the kinase-treated primers (10 pmol) was added to 100 mM sodium chloride;
5 min at 67°C and 25 min at 42°C in 15 μl of a mixture containing 20mM Tris-HCl, PH 7.9, 20mM magnesium chloride and 20mM β-mercaptoethanol.
ssM13-IL- by heating for minutes
Hybridization was performed on 2.6 μg of 2DNA. Cool the annealed mixture on ice and then add 0.5mM
Each dNTP, 17mM Tris-HCl (PH7.9), 17mM
Magnesium chloride, 83mM sodium chloride,
Reaction mixture containing 17mM β-mercaptoethanol, 5 units of DNA polymerase Klenow fragment, 0.5mMATP, and 2 units of T4 DNA ligase.
A final volume of 25 μl was prepared and incubated at 37° C. for 5 hours. Stop the reaction by heating to 80℃,
The reaction mixture was used to transform competent JM103 cells, plated on agar plates, and cultured overnight to obtain phage plaques. Example 14 Screening and identification of mutagenized phage plaques Plates containing mutagenized M13-IL-2 plaques and non-mutagenized M13-
Cool the two plates containing IL-2 phage plaques to 4°C, transfer the phage plaques from each plate onto two nitrocellulose circular filters, and in the case of the first filter, transfer the dried filter to an agar plate. Transfer for 5 minutes on top and 15 minutes in the case of the second filter. The filter was then placed on thick filter paper soaked in 0.2N sodium hydroxide, 15M sodium chloride and 0.2% Triton for 5 minutes, then soaked in 0.5M Tris-HCl (PH7.5).
Neutralization was achieved by layering on filter paper soaked in 1.5M NaCl for an additional 5 minutes. Filter in the same way, 2x
It was washed twice on a filter soaked in SSC, dried, and then dried in a vacuum drying oven at 80°C for 2 hours. Add a layered filter to 10 ml per filter of DNA hybridization buffer (5x SSC), pH 7.0, 4x Denhard's solution (polyvinylpyrrolidine, Ficoll and bovine serum albumin, 1x = 0.02% each), 0.1%.
Prehybridization was performed with SDS, 50mM sodium phosphate buffer (PH7.0) and 100μg/ml denatured salmon sperm DNA for 4 hours at 42°C. A ³² P-labeled probe was created by kinase treatment of an oligonucleotide primer with labeled ATP. 5ml per filter
8 hours at 42°C in DNA hybridization buffer.
The filter was hybridized to 0.1×10 ⁵ cpm/ml of ³² P-labeled primer. 0.1%
50°C in wash buffer containing SDS and 2x SSC
twice for 30 minutes each, then 0.1% SDS and 0.2
The filters were washed twice in ×SSC at 50°C for 30 minutes each. Air dry the filter at -70°C for 2 hours.
Autoradiography was performed for ~3 days. Oligonucleotide primer DM28 and
DM29 is new to the mutagenized clone
RF-DNA from several clones hybridized with these kinased primers was digested with the restriction enzyme Dde, as it was designed to create a Dde restriction site (Table 14). hybridized with primer DM28 and mutagenized with a new Dde restriction site.
One of the M13-IL-2 plaques (M13-LW44) was picked up and inoculated into a JM103 culture, ssDNA was prepared from the culture supernatant, and dsRF-
DNA was prepared. Similarly, plaques hybridized with primer DM29 (M13-
LW46), and from now on ssDNA and RF-
DNA was prepared. Oligonucleotide primer DM27 is designed to create a new Pst restriction site in place of the Dde position. Plaques hybridized to this primer were therefore screened for the presence of new Pst sites. One such phage plaque was identified (M13-LW42) and ssDNA and RF-
DNA was prepared from this. For target cysteine
To confirm that the TGT codon was converted to a TCT codon for serine, the DNA obtained from all three clones was sequenced. Example 15 Mutagenic IL for expression in E. coli
Recloning of 2 genes M13-LW42, M13-LW44, and M13-
Restriction enzyme Hind for each RF-DNA from LW46
and Ban and the insert was purified from a 1% agarose gel. Similarly, plasmid pTrp3
(Fig. 7) was digested with Hind and Ban, and the large plasmid fragment containing the trp promoter was purified on an agarose gel.
Each of the inserts isolated from LW44 and M13-LW46 were ligated. The ligated plasmid was transformed into competent E. coli K-12MM294 strain.
Plasmid DNA from these transformants was analyzed by restriction enzyme mapping and plasmid
The existence of pLW42, PLW44, and pLW46 was confirmed. Figure 14 is a restriction map of pLW46. To induce the trp promoter, each of these individual clones was grown in the absence of tryptophan, the cell-free extracts were analyzed on an SDS-polyacrylamide gel, and the three clones pLW42, pLW44 and
All of pLW46 was shown to synthesize a 14.5 Kd protein similar to that found in the positive control pLW21, which was demonstrated to synthesize a 14.4 Kd IL-2 protein. When these same extracts were assayed for IL-2 activity in mouse HT-2 cells, clonal
Only pLW21 (positive control) and pLW46 were found in significant amounts.
It shows IL-2 activity (Table 2 below), and cys58 and
cys105 is required for biological activity, and converting it to serine (pLW42 and
This means that biological activity is lost due to pLW44). while cys125 uses this as ser125
(pLW46) does not affect biological activity, so it must be unnecessary for activity. Table 2 Clone IL-2 Activity (μ/ml) PIL2-7 (negative control) 1 PLW21 (positive control) 113000 PLW42 660 PLW44 1990 PLW46 123000 Figure 15a shows the nucleotide sequence of the coding strand of clone pLW46. Compared to the coding strand of the natural human IL-2 gene, clone pLW46 has a single base change from G to C at nucleotide 374.
Figure 15b shows the corresponding amino acid sequence of the IL-2 mutein encoded by pLW46. This mutein is called IL-2ser125. Compared to natural IL-2, muteins have serine instead of cysteine at position 125. E. coli K-12MM294 transformed with pLW46
Strain samples were sent to the American Type Culture Collection, 12301 Burklawn Drive, Lockeville, Maryland 20852, United States on September 26, 1983.
Collection) and directed to ATCC No. 39452 (International Deposit under the Budapest Treaty). Cysteine at position 125 is deleted or replaced with another amino acid like mutein IL-2ser125
IL-2 muteins retain IL-2 activity. Therefore, they can be formulated and used in the same way as natural IL-2. Therefore, such muteins can be used in the diagnosis and treatment of bacterial, viral, parasitic, protozoan, and fungal infections, in the development of lymphokines or immunodeficiency, and in the reconstitution of normal immune function in older humans and animals. In addition, the development of diagnostic assays, such as enzyme amplification, radiolabeling, radioimaging, and other methods known in the art to monitor IL-2 levels in pathological conditions, will utilize receptor sites for lymphokines. It is useful for the promotion of T cell growth in vitro for therapeutic and diagnostic purposes, as well as for various other therapeutic, diagnostic, and research applications. human IL
-2 for various therapeutic and diagnostic applications, see S.A. Rosenberg,
E.A. Grimm et al., A. Mazumder et al., and E.A. Grimm and S.A. Rosenberg have reported research. IL-2 can be used by itself or in combination with other immunologically relevant B or T cells or other therapeutic agents. For therapeutic or diagnostic use, they can be formulated in non-toxic, non-allergenic, physiologically acceptable carrier media such as distilled water, Ringer's solution, Hank's solution, physiological saline and the like. Administration of IL-2 muteins to humans or animals can be oral, or intraperitoneal or intramuscular or subcutaneous, as deemed appropriate by the physician. Examples of relevant cells are B or T cells, natural killer cells, etc. Examples of therapeutic reagents that can be used in combination with the polypeptides of the invention are various interferons,
Especially gamma interferon, B cell growth factor,
It is IL-1 grade.

[Brief explanation of the drawing]

FIG. 1 is a diagram of the amino acid sequence of IFN-β.
FIG. 2 is a schematic diagram showing the preparation of mutant IFN-β genes by oligonuclease-directed mutagenesis. FIG. 3 is a diagram of plasmid pβ1trp containing the IFN-β gene. FIG. 4 is a diagram of the cloning vector M13mp8 phage.
Figure 5 shows the restriction map of clone M13-β1.
FIG. 6 shows a sequencing gel pattern of a mutant IFN-βser17 gene showing a single base change in the coding region. Figure 7 shows the expression plasmid
FIG. 3 is a diagram of pTrp3. Figure 8a shows the clone
Showing the Hinf restriction pattern of pSY2501, section 8b
The figure shows the two resulting 169bp and 28bp fragments. FIG. 9 is a restriction map of clone pSY2501. FIG. 10 shows the DNA sequence encoding the mutein IFN-βser17 and the corresponding amino acid sequence. Figure 11 shows clones pSY2501 and
A single 18,000 Dalton protein band corresponding to IFN-βser17 in the extract of pβ1trp is shown. FIG. 12 is a diagram of plasmid pLW1 containing the human interleukin-2 (IL-2) gene under the control of the E. coli trp promoter. Figure 13 shows
This is a restriction map of Phage clone M13-IL2.
Figure 14 is a restriction map of plasmid pLW46. Figures 15a and 15b show clone pLW46
The nucleotide sequence of the coding strand of and IL-
The corresponding amino acid sequences of the IL-2 mutei designated 2ser125 are shown, respectively.

Claims (1)

[Scope of Claims] 1. A modified interleukin in which the cysteine residue at position 125 of natural interleukin-2 is replaced by another amino acid residue, and which has the biological activity of natural interleukin-2.
2. 2 The cysteine residue is serine, threonine,
Modified interleukin-2 according to claim 1, characterized in that it is replaced with glycine, alanine, valine, leucine, isoleucine, histidine, tyrosine, phenylalanine, tryptophan or methionine. 3. The modified interleukin-2 according to claim 1, characterized in that interleukin-2 is not glycosylated. 4. Modified interleukin-2 according to claim 1, characterized in that the cysteine residue is replaced by a serine residue and the interleukin-2 is not glycosylated. 5. A modified interleukin-2 in which the cysteine residue at position 125 of natural interleukin-2 is replaced by another amino acid residue and which has the biological activity of natural interleukin-2.
A method for producing, comprising: (1) encoding natural interleukin-2;
Prepare a DNA fragment; (2) An oligonucleotide primer in which the codon encoding the amino acid at position 125 of interleukin-2 is replaced with a codon encoding an amino acid other than cysteine, or an oligonucleotide primer complementary to this primer. By mutating the DNA encoding natural interleukin-2 through a chain extension reaction using a nucleotide primer and DNA polymerase, the codon encoding the amino acid at position 125 of interleukin-2 is changed to a codon for an amino acid other than cysteine. A certain DNA fragment is obtained, (3) an expression vector is obtained by inserting the mutated DNA fragment into a vector, a transformant is obtained by transforming a host using this, and (4) the transformation is performed. A method characterized by producing the modified interleukin-2 by culturing the body, and collecting the modified interleukin-2.