JPH01503038A - Novel polypeptide with gamma interferon activity - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] New polypeptide technology field with gamma interferon activity The present invention has a molecular weight significantly greater than that of known gamma interferon (γ-IFN) species. The present invention relates to novel polypeptides with low gamma interferon activity.

Background technology Interferons (IFNs) are a broad spectrum of viral and non-viral agents. a genus of inducible, antiviral proteins secreted by most animal cells in response to It is. These IFHs play a direct role in the antiviral defenses of mammals, including humans. have a role. Of course, these possess immunomodulatory activity and influence cell growth. It shows the characteristics of Because of these properties, these IFNs are currently used in cancer and immunodeficiency disorders. All treatments are being tested.

Three major species of IFN have been characterized. a- Or leukocyte IFN is It is the main species produced by culturing Rus with cells of lymphoid origin.

β- or fibroblast IFN induces non-lymphoid cells to be exposed to viral or non-viral agents. This is the main species produced when used and cultured. γ- or immune or II base I FN stimulates lymphocytes, especially T cells, when treated with mitogens, or sensitization when treated with specific antigens. It is the main species produced by lymphocytes.

These three types of IFN have very different amino acid sequences and different physicochemical properties and can be distinguished using specific antiserum. For example, anti-a serum inhibits β- or γ-IFN activity. Not suppressed.

One of the problems in the clinical application of IFN is that it is in limited supply. be. Purification of natural IFN requires complex and expensive procedures, and resolution is often poor. The yield is also low. Furthermore, IFN does not respond well to manipulations such as those required for purification; May be easily inactivated.

Synthesis techniques using recombinant DNA methods or solid-phase peptide synthesis methods can produce γ-IFN products. increased the availability of However, recombinant DNA γ-IFN products are Peptide synthesis products require purification with similar problems to those encountered during purification of peptides. The amino acid chain is extremely long (146 amino acids for γ-IFN), making it difficult to produce. It is worth it.

If there is a γ-IFN peptide with a lower molecular weight than the existing γ-IFN species, , appears to be preferred as it is easy to synthesize using solid phase synthesis techniques. .

Regarding the existence of biologically active peptide fragments of human γ-IFN with a fairly low molecular weight, nothing is known. Aldrock's U.S. Patent No. 4,599,306 The amino acid sequence consists of the last 19 amino acid residues at the carboxyl group end of 1-IFH. Discloses a replicated nonadecapeptide. However, this peptide was used to demonstrate and quantify the binding of monoclonal antibodies to N. There is no disclosure regarding physical activity.

Paper by Linderknecht et al. (J, Biol, Cbtm, 259:679 0-7697.1984) is the 16th carboxyl group from the end of γ-IFN. discloses that removal of amino acids up to no effect on biological activity. deer However, Mr. Linderknecht proposed that the remaining number of γ-IFNs was smaller than the 130th one. A peptide that has amino acids but still retains the biological activity associated with this protein. It does not disclose or suggest that there is a problem.

Kung et al., U.S. Pat. The Luton fragment is disclosed. Kung et al. showed that the purified γ-IFN preparation was homogeneous. However, it was observed that it contained various fragments. Kung et al.'s 15 kilodalton γ -The IFN fragment is generated at the C-terminus by a protease present in the IFN-containing upper groove. Obtained by removing amino acid residues 132-146. But it's 131 pieces. Active peptides containing fewer amino acid residues are neither disclosed nor suggested.

Surprisingly, a novel peptide with a sequence of less than about 100 amino acids and γ-IFN activity has been discovered. Chido was discovered. This discovery suggests that the γ-IFN peptide has a higher molecular weight than conventional γ-I This was particularly surprising since it was recovered from the supernatant material containing the FN peptide. This new page Peptide has a molecular weight of approximately 6000-8000 Daltons and has a high resistance to γ-IFN antibodies. It had immunochemical reactivity and exhibited antiviral and cytolytic activity. New page peptides can be separated into two populations of glycosylated and non-glycosylated molecules. Ru. Glycosylated γ-IFN peptide is derived from dodecyl sulfate polya By sodium crylamide gel electrophoresis (SDS-PAGE), i.e. could not be detected under sexual conditions. In 5DS-PAGE, only two types of glycosylation γ-IFN polypeptides detected with apparent molecular weights of 52KD and 28KD and the non-glycosylated form can be separated into 6KD and 8KD components.

Novel γ-IFN peptides can achieve high resolution and speed in the separation of protein molecules. A novel method that achieves a high recovery rate of biologically active IFN molecules. Produced using purification methods.

The aim of the present invention is to isolate novel, relatively small polypeptides with γ-IFN activity. Characterization and purification of this novel peptide with little loss of biological activity and γ, which may be suitably produced by recombinant DNA or synthetic organic chemistry techniques. - Isolation and characterization of novel polypeptides with IFN activity, etc. Not limited to these.

Demonstration of invention The present invention significantly improves γ-IFN or known polypeptides exhibiting γ-IFN activity. Provides peptides with low molecular weight. The novel peptide of the present invention is a γ-IFN antibody It has antiviral and antiviral properties similar to known γ-IFN. and cytolytic activity.

In another aspect, the invention provides methods for purifying both novel and known γ-IFN peptides. provide. This method involves preparations containing known γ-IFN and the novel polypeptide of the present invention. Continuous chromatography with silicic acid as medium, treated with anion exchange resin, Runs under conditions where new and known γ-IFN species are excluded and is highly resolved under non-denaturing conditions. Exposed to rate molecular sieve chromatography and purified through a cation exchange resin column. The process consists of passing the product through it.

Brief description of the drawing Embodiments of the present invention will be described below with reference to the drawings.

Figure 1 shows a graph of IFN activity and protein elution profile from a high separation rate molecular sieve column. Rough diagram, Figure 2 shows known (A) and new (B) γ-IFN from ConA column. A graph of the elution profile, Figure 3 shows the novel γ-IFN 5DS-P purified in four steps. AGE gel electropherogram, Figure 4 shows 5DS-PAGE electrophoresis (A) and Novel (B) Graphical diagram of active profile of γ-IFN, Figure 5 shows mixed glycosidase treatment 5DS-PAGE electrophoresis of novel γ-IFN before (A) and after (B) Figure 6 shows the elution profile of known γ-IFN from a cation exchange column. Graph diagram, Figure 7 is a graph of the elution profile of the new y-IFN from the cation exchange column. Figure 8 shows the elution profile of a γ-IFN-containing solution from a conventional molecular sieve column. The graph diagram, FIG. 9, shows the γ-IFN-containing solution isolated from the middle region A of FIG. Graphical diagram of elution profile when re-chromatographed using LC, 1st Figure 0 shows the γ-IFN-containing solution isolated from the middle region B in Figure 8 using HPLC. FIG. 2 is a graph of the elution profile when the sample is subjected to chromatography again.

BEST MODE FOR CARRYING OUT THE INVENTION A novel biologically active peptide exhibiting γ-IFN activity was unexpectedly isolated and identified. It was done. This peptide (hereinafter referred to as “novel γ-IFNJ”) is a peptide of γ-IFN. It is a subspecies. This classification is based on the well-established human γ-IFN (hereinafter known γ-IF The antiviral activity of the peptide was fully demonstrated by a monoclonal antibody specific for It is based on neutralization.

Natural human γ-IFN is believed to be encoded by a single gene; This protein is believed to exist in the form of a dimer in its natural state. ing. This human γ-IFN was analyzed using conventional molecular sieve chromatography under non-denaturing conditions. an apparent molecule of approximately 58,000 Daltons (58 KD), as determined by have a quantity. Dodecyl sulfate polyacrylamide sodium ge The molecular weight determined by electrophoresis (SDS-PAGE) is that human γ-IFN is 2. It can be separated into two main components with apparent molecular weights of 5KD and 20KD. It shows. The difference in molecular weight between previously known human γ-IFN subspecies is due to glycosylation. difference in degree of glycosylation, i.e., complete or partial glycosylation at two N-linked glycosylation sites. It is believed that this shows the difference in

The feature that distinguishes the new γ-IFN from the known 7-IFN is its size. Main departure Ming's new γ-IFN is much smaller than known γ-IFN, that is, it has a significantly lower molecular weight. amount, and either 25KD or 20KD components. as used here In other words, the expression “much smaller than γ-IFN” refers to a sequence of less than 100 amino acids. Defined as a molecular weight less than about 10,000 Daltons corresponding to the column length. unchanging Size evaluation by molecular sieve chromatography under normal conditions shows that the new γ-IFN The apparent molecular weight is shown to be between 6000 and 8000 Daltons. a Calculated using 115 as the average molecular weight of amino acid residues, the novel γ-IFN is It will contain less than 70 amino acids, which means that the peptide will contain carbohydrates. Therefore, this is the upper limit evaluation value (see the third embodiment described later). The amazing thing is the dodecyl monkey Phate polyacrylamide sodium gel electrophoresis (SDS-PAG) In E), the glycosylated novel γ-IFN is approximately 52KD and 28KD, respectively. The appearance is broken down into major and minor components with molecular weight. However, non-glycosylated new γ-IFN is separated into two species, 6KD and 8KD.

This new γ-IFN was produced using high performance liquid chromatograph 4 (H PLC), especially high-speed protein solution from Pharmacia, Bislitowei, New Shashi. In molecular sieve chromatography (FPLC), molecular sieve chromatography is performed under non-denaturing conditions. It was first identified using graphics. This compound, classified as a γ-IFN species, Consistent with the above-mentioned classification of peptides, both novel γ-IFNs also have the characteristics of γ-IFN. as well as the ability to activate monocytes to express cytotoxic activity in tumor cells. was discovered to exhibit cytotoxic activity directly against tumor cells.

This previously unidentified natural form of γ-IFN is 12-0-tetradeconylforpo - Diterpene phorbol esthetics such as 13-acetate (TPA) Stimulated with a combination of 1-cell and T-cell mitogen phytohemagglutinin (PHA) containing γ-IFN, including but not limited to culture medium for human peripheral blood leukocytes. Can be obtained from solution.

Purification of the present γ-IFN and its separation from media containing larger γ-IFN subspecies and components A preferred continuous chromatographic procedure for separation involves the following steps: ■Adsorption of 1-IFN to silicic acid, ■ Adsorption and removal of some contaminant proteins with anion exchange residues, ■ High separation rate molecular sieve chromatography, and cation exchange chromatography It is equipped with

The first step in the purification procedure is to prepare γ-IFN containing known and novel γ-IFNs on silicic acid. It consists of adsorption of a solution, such as a biological medium. This can be done in column format or preferably This can be accomplished using batch methods. After adsorption, the silicic acid was dissolved in phosphate buffered saline (PBS). Remove unbound protein by washing with a neutral buffer such as New and known γ-IF If the binding substance containing both N and N contains 50% (volume/volume) of a suitable solvent as an eluent, eluted using PBS.

Suitable solvents include glycerol, propylene glycol, and polyethylene glycol. Recall or preferably non-ionic hydroxylation such as ethylene glycol including, but not limited to, compounds.

The silicic acid eluate is further treated with DEA using a column format or preferably a batch process. Cation exchange residue such as E (DEAE-5EPHACEL from Pharmacia) It is purified by adsorbing and removing contaminant proteins on the substrate. Cation exchanger at pH 7.7 Equilibrated and binds non-γ-IFN contaminants, but the isoelectric point of known γ-IFN (8,5 (PI)) and a novel γ-IFN with an expected isoelectric point greater than 8.5. Binding remains (excluded) in the eluent.

The anion exchange unbound supernatant is then subjected to high separation rate molecular sieve chromatography under non-denaturing conditions. undergo matography. The undenatured state can be removed by heating, which destroys non-covalent interactions. As a denaturing treatment or the absence of denaturing agents such as sodium dodecyl sulfate defined. High speed separation techniques are used in conjunction with molecular sieve columns.

This high speed applies increased pressure to the column and allows the chromatograph to withstand this pressure. This is accomplished using a fimed medium. Preferably, for example, a fast protein liquid chromatograph High performance liquid chromatograph compatible with biological materials such as FPLC (HPLC) is used. Most preferably, files operated under the conditions described below. A 5UPERO5E12 column from Macia is used.

The properties required for this method are as follows: be able to separate the boards and operate under the pressure necessary to ensure rapid completion of this process. That's all. The sample is subjected to thin membrane dialysis or preferably as known to those skilled in the art. Standards such as dialysis using carboxymethyl cellulose sodium salt powder It is concentrated into a small volume using technology. These samples are added to the column and The separated and γ-IFN-containing fractions were identified by their antiviral activity as described below. The two novel γ-IFNs were separated into fractions corresponding to the 6000-8000 Dalton region. while the known γ-IFN is separated into the so,ooo Dalton region (which is This is probably due to the assembly of the subunits under the non-denaturing conditions used. ).

The new γ-IFN-containing eluate from the molecular sieve chromatography step is then Purified by cation exchange chromatography. The preferred column is New Jersey Carboxymethyl cellulose, a product of Whatman Laboratories, California CM-3EPHADEX from Macia Fine Chemicals and preferably Fal. Contains Macia's MONOS. 8000 Dalt containing new γ-IFN The fraction corresponding to and consisting of a buffer plus a suitable salt, preferably 1 mol of sodium chloride. It is eluted using a linear concentration gradient. Novel γ-IFN-containing fraction has antiviral activity Identified by analysis or radioimmunoassay. the latter The method is described in the fourth example below.

The experimental method that gave a clue that enabled the identification of the novel γ-IFN of the present invention is preferable. was the use of high separation rate molecular sieve chromatography using HPLC. H Chromatography in PLC systems provides high resolution and speed for protein separation. I will provide a. HPLC/molecular sieve chromatography experiments completed in less than 1 hour However, on the other hand, using conventional techniques of flow control, e.g. by gravity or by a centrifugal pump, A similar experiment would take more than 10 hours to complete. Heavy waste in the refining process A key determining factor is the speed at which the molecular mesh chromatography step is performed. This process In a preferred embodiment, highly cross-linked agarose, ram (SUPERO) 5E　1　2) is used to separate proteins. Used under HPLC conditions - at least about 125 bonds per square inch, preferably per square inch. A constant pressure of about 225 bonds per inch is maintained. Maximum pressure that can be used with this method is a function of the materials and equipment used. For example, the crosslinked atom of the preferred embodiment Garose can withstand up to about 430 pounds per square inch. Therefore, The entire chromatographic separation can be completed in about 2 hours, preferably less than 1 hour. Can be completed.

Following the discovery of novel γ-IFN, conventional molecular sieve columns (Calif. Conventional portion on Bioland Research new BIO-GEL P-200) The elution profile of known γ-IFN by small sieve chromatography was re-examined. public The elution profile of Chiγ-IFN, as shown in Figure 8, is consistent with that previously reported. , the antiviral activity is present in an essentially broad symmetrical elution peak with an estimated molecular weight of 60 KD. (estimated molecular weight of 58 KD was published in the 1981 Journal of the National Academy of Sciences 7). 8, pp. 1601-1605 Y). HPLC /A sample of the same γ-IFN preparation chromatographed on a molecular sieve column was As shown in FIG. 1, it was separated into 50KD, 14.5KD and 8KD.

The peak portion marked A in Figure 8 eluted in a conventional molecular sieve chromatography experiment. and the base part marked B in Figure 8 are pooled separately, and carboxymethyl Sodium salt of AQUAC I DE n) from Calbiochem in Ndiego Each is concentrated by dialysis using a powder, and then the highly cross-linked aphrodisiac HPLC/molecular sieve chromatography on Garose (SUPEROSE12) column Dialyzed by Rafi. The results summarized in Figures 9 and 10 show that the peak (Pool A in Figure 8) consists essentially of known γ-IFN, while the tail The pool from the field part (pool B in Figure 8) consists essentially of novel γ-IFN. It shows that there is.

The control results shown in FIGS. 1, 8, 9 and 10 are based on the known results of γ-IFH. Conventional molecular sieve column chromatography is used to separate new species from seeds. It clearly shows that you are incompetent. Furthermore, conventional molecular sieve chromatography Obviously, the diffusion of IFN molecules over long periods of time when performing This further reduces the already poor resolution obtained.

The four-step chromatography procedure described above can be performed using any known γ-IF obtained from any source. It is also suitable for purifying N. The only difference from the above procedure is the third step above. The public equivalent to the soooo Dalton region obtained from molecular sieve chromatography Both components containing γ-IFN are applied to a cation exchange resin and eluted. Ru.

According to the 1-IFN purification procedure of the present invention, both known and new γ-FN are almost purified. Manufactured. Using this method, antiviral activity is almost inactivated or inactivated. Not possible. Accurate determination of purity requires quantification due to the small amount of protein present in the sample. It seems like it can't be done. However, analysis of this material by SDS-PAGE (Fig. (see lane 4) is a protein that can be stained by the novel γ-IFN and its aggregate of the present invention. This indicates that it is a major component of quality (more than 95%).

Of course, the new γ-IFN has two components with molecular weights of 6000 Daltons (6KD) and 8KD. It was discovered that it could be separated in minutes. Two types of novel γ-IFN are the following second example. was discovered using a variation of the four-step continuous purification process shown in . This step ■ Adsorption of new γ-IFN to silicic acid, ■ Adsorption and removal of some contaminant proteins with anion exchange residues, ■ ConA-senchyme Adsorption and removal of dylated proteins on Sepharose, ■High separation rate molecular sieve chromatography, ■Cation exchange chromatography, and ■ Using a C4 column, acetonitrile concentration gradient (0-95%) and Reverse phase high performance liquid chromatography in fluoroacetic acid (0.1%) (R　P　H Consists of PL C).

The fourth step of the continuous purification procedure includes a C on A Sepharose chromatography step. The advantage of introducing the new γ-IFN peptide, which is not glycosylated, is that the amino acid sequence is It should be possible to clearly separate the two distinct species for column analysis.

The non-glycosylated novel γ-IFN peptide obtained after the 6th step (RPHPLC) is , biological activity (i.e. antiviral properties) due to the interaction of organic solvents and peptides. However, it is suitable for determining the amino acid sequence of proteins.

The partial amino acid sequence shown in the 5th Example below is different from that of known γ-IFN. Shows no homology.

Although the invention will be described with reference to specific examples below, it will be appreciated that the scope of the invention is It's not something you can do.

First example Induction of human γ-IFN production in cell culture Morphology of soft layer or platelet peresis A human blood cell concentrate obtained in 2008 was used to seed the production culture. child These cell concentrates are manufactured by Sigma, St. Louis, and are manufactured by Heparin 40USpHi. Serum-free RPMI 16 manufactured by Gibco, Grand Island, New York, containing It was diluted with 40% medium but was not further processed. ox, california 150mm diameter Petri manufactured by Nerd's Becton Dickinson Falcon branch. Cultures seeded with approximately 6 x 10' cells/1001 cells per dish were subjected to LC service. was treated with 5+D/ml TPA from North Carolina and 2 hours later with PHA (North Carolina). Burroughs Wellcome, Research Triangle Burke, 5μg/l) (see Yip et al., US Pat. No. 4,460,685). child These culture media were collected after incubation for 48-72 hours at 37°C in a humidified CO5 incubator. and stored at 4°C until use. used for isolation of novel γ-1FN Some culture media were stored at 4° C. for long periods of 12 months.

The results of the four experiments are shown in Table 1 below.

Table 1 IFN activity of medium of TPA/PHA stimulated PBL culture Experiment number IFN titer (Unit/1) 1 10.240 2 20.430 3 15.360 4 20.480 As previously described, IFN yields from PBL cultures stimulated with TPA/PHA were already The results were consistent with the reported results (Y, I+fect, lmmm of Yip et al. m, 34: HI-1N, 1911).

Second example Purification of novel γ-IFN Efficiency of each chromatographic step in the purification scheme to recover antiviral activity protein recovery was not measured except for the anion exchange step. .

The first step of the adsorption purification procedure of 1-IFN on silicic acid is carried out by a batch method. Supplied by Mallinckrodt of St. Louis, it is a new silicate bead. and adsorption of a solution containing known γ-IFN. These silicate beads are 1:40 These “mixtures” are added to the culture fluid containing γ-IFN in the ratio (volume/volume). The mixture was heated at 4°C on a roller apparatus commercially available from Belco, Philadelphia. Rotated at rpm.

- After overnight adsorption, γ-IFN-bound silicate beads are collected by centrifugation. phosphate buffered saline (PBS, pH7, 4/ Washed 3 times with 0, 15 M-Na C1) to remove unbound proteins. It was. γ-IFN was then added to 3 volumes of PB550% (vol/vol) ethylene glycol. It is eluted from the silicate beads by Cole PBS solution. Eluted fraction The presence of γ-IFN in human foreskin connective tissue forming cells (FS-4) The well-known IFN-mediated inhibition of the cytopathic effects of ring myocarditis virus (hereinafter referred to as antiviral Determined by biological analysis based on E, mu, Mr. Havel et al.'s nt+-+atcrob, MU L Cbsmotlsr, 4H: (See 4H, N8 years). Example of recovery of IFN activity from silicic acid chromatography are shown in Table 2 below.

Table 2 Recovery experiment of IFN activity in silicic acid adsorption chromatography Summer FN unit XIO ” Number At start During flow After elution 1 10.240 Hg (1,25%)*s, 2Ill (8%) 2! 0,4 10 256 (1,25%)! o, yii (to1%) 3 1s, 3!11 06 (1,66%) If, till (11@%) 4 20.410 1N (0 , H%’) 11. ! 44 (93%) *Recovery of IFN activity is based on starting titer. Expressed as a percentage.

The data presented in Table 2 above indicate that γ-IFN activity was quantitatively recovered after this purification step. This shows that it is possible to

second step, Adsorption and removal of contaminated proteins using anion exchange beads from the silicic acid process 1-I The FN-containing eluate was purified by extensive dialysis with 25mM sodium phosphate to pH 7.7. Equilibrated with .

The obtained γ-IFN sample was then treated with anion exchange beads by a batch method. Remove contaminated proteins on beads (DEAE-5EPHACEL beads from Pharmacia). It was further purified by adsorption removal. These beads were made with 25mM sodium phosphate. 7-IFN sample equilibrated at pH 7°7 and at a ratio of 1:100 (vol/vol). and mixed overnight as described for the silica step. greater than 8.5 Known and human new @7-IFNs with isoelectric point (PI) are anion exchangers at pH 7.7. Most other beads known to have a PI less than or equal to 7.7 do not bind to exchange beads. Which proteins are removed from the sample solution by adsorption to the beads. These bits The beads were then removed by centrifugation, and the γ-IFN-containing supernatant was AQUACIDEn carboxymethyl cellulose sodium salt powder The molecular weight is cut to 2000 daltons by bed dialysis and placed in a dialysis bag. It is concentrated to about one-tenth of its starting volume. The knee recovered from this process Examples of FN activity are shown in Table 3 below.

Table 3 Anion exchange chromatography? Recovery of protein and IFN activity from 1 25.600 19,200 (75%) b 1.194 0.144 (1, 2%) L2 30, NO311, No (1611%) 0425 0.0H (1 4.2%) 3 3.14 @ 1. Hlt (Sl1%) 0. @37 0.110 5 (13,5%) L The IFN samples used in these experiments were Partially purified by on-glass chromatography.

The above data indicate that although the majority of antiviral activity can be recovered in the unbound fraction, 8 It is shown that more than 5% of non-IFN contaminating proteins are removed in this step.

The third step, molecular sieve chromatography, is a non-denaturing It was carried out under sexual conditions.

Highly cross-linked Taagaro with a resolution range of molecular weights from 103 to 3 x 10” Daltons Base column, 10 x 300mm (SUPEROSE 12) has 5 columns Washed and equilibrated with 1 volume of PBS. This column contains bovine serum albumin. (6LOOOD), ovalbumin (45, GOOD), chymotrypsinogen A (25,0OOD) and cytochrome C (12,7 (IcD)) calibrated. Purified by chromatography on silicate beads and anion exchange The γ-IFN samples were concentrated by dehydration. 200/7M volume sump was added to the column and eluted with PBS. The elution buffer flow rate was 225 points. by HPLC controller (Pharmacia FPLC) under pressure of It was adjusted to 0.5 ml/min. Fractions of 0.25 ml each were collected. this An example of IFN activity recovered from the process is shown in Table 4.

Table 4 Recovery experiment of IFN activity from HPLC molecular sieve chromatography IFN unit 2 H, Ha 71,4H (76%) 25,440 (H%) 103,920 (126%) 3 10.140 116. ? 40Q4%) 22,340 (16 %) 139,080 (15%) 4 163.140 140,540 (l1% ) H, 400 (H%) 17!440 (106%) L Known (50KD) and The recovery of IFN activity in the elution volume corresponding to the new (8KD) species is 100% of the overall recovery. Expressed as a percentage.

b Overall recovery of IFN activity is expressed as a percentage of the starting titer.

4th step, ion exchange chromatography cation exchange column (Pharmacia Co., Ltd. MONOS/HR515) is 20I + 1M Tris HCI (buffer A) It is equilibrated to pH 7.5. 50KD from molecular sieve column (known γ-I Each fraction corresponding to the FN) and 8KD (novel γ-IFN) regions was pooled separately. and equilibrated with buffer A by extensive dialysis. Purification of these IFN species , from loading buffer (buffer A) and final buffer (1,0 M NaCl buffer A). This salt concentration gradient was carried out with a NaCl concentration gradient of 0.5+nl/min. was generated by an HPLC controller with a flow rate of .

Both portions of 0.25 ml each were collected. of IFN activity recovered from this step. Examples are shown in Table 5 (known γ-IFNs) and Table 6 (new γ-IFNs).

Table 5 Experiment for recovery of known IFN activity from ion exchange chromatography IFN unit Number: At the start, during flow, elution, overall recovery rate I II, 920 21.4600 3%) Mutual To, 0111 (77%) Mutual 91,540 (112%)! 2 40 ．． 960　14.92fl(34%)! 4,740 (66%) i7,660 ( H%)3 1114.4011 19. ! 00 (19%) Hj311 (11% ) 99,430 (97%) 4, 307.200 10L400 (40%) 105,890 (60%) 253,290 (82%)a IFN activity is 50 HPLC/5UPERO5E (from Pharmacia) corresponding to KD elution capacity 1 Two column fractions were pooled.

b The recovery rate of IFN activity during flow is expressed as a percentage of the total recovery rate.

Table 6 Experiment to recover new IFN activity from ion exchange chromatography IFN unit Number: At the start, in flow, eluting species, overall recovery rate 1 12.100 0 11.4 0・(100%) mutual Is, 40G (144%) standing 2 51, fall 1,6 00 (4%) mutual 33,90 (H%) 35,5H (70%) 3 25.600 0 36,370 (100%) 36,370 (It2%) 4 1N, 401 1 2. ...(5%) 53. No (95%) 56,700 (55%) a IF N activity was determined by HPL C/molecular sieve column fraction corresponding to 8KD elution volume. were pooled.

Σ The recovery rate of IFN activity during flow is expressed as a percentage of the total recovery rate.

c Overall recovery of IFN activity is expressed as a percentage of the starting titer.

As can be seen from the above results, novel γ-IFN activity can be quantitatively recovered from each of the above steps. It was collected. Using the biological assay of the invention (suppression of viral cytopathic effects) It is noted that there is a two-fold deviation inherent in the determination of IFN titers. Therefore, above Some of the recoveries listed are over 100%.

Furthermore, the new γ-IFN was added to the ion exchange color as shown in Table 6.

However, as shown in Table 5, known γ-IFN binds quantitatively to 19% of the sample. ~40% indicates no binding. This is because known γ-IFN starting materials are This reflects the heterogeneity of the 50 KD starting material into the 8 KD novel γ-IF. It is more heterogeneous in overall net loading than N.

3rd Example Physicochemical properties A of novel γ-IFN, determination of molecular size under non-denaturing conditions fixed Natural human γ-IFN can be purified by HPLC/molecular sieve chromatography under non-denaturing conditions. It was separated into three different molecular sizes by Highly cross-linked agarose A typical elution profile of protein and IFN activity on the column is shown in FIG. No. One active ingredient corresponds to an apparent molecular size of approximately 50 KD, and the second active ingredient corresponds to an apparent molecular size of approximately 1 KD. 4.5 KD, and the third active ingredient has a capacity corresponding to an apparent molecular weight of approximately 8 KD. eluted at The antiviral activity of all these three molecular fish species is shown in the fourth example below. As shown in Figure 2, the antibody against γ-IFN rather than the antibody against σ-IFN was specifically neutralized.

The 50KD component was expected and reported in literature 1 by the inventors ( Y., Yip et al., Journal of the American Academy of Sciences, Vol. 78, pp. 1601-1605, 198 (see 1st year). The 14.5 KD γ-IFN is probably the known non-glycosylated γ- It is probably a monomer of IFN. The novel 8KD γ-IFN of the present invention can be used under non-denaturing conditions or is natural or recombinant human γ-IFN under denaturing conditions! It has not been identified or reported as a II product. It wasn't.

B. Determination of the presence of carbohydrates in the novel γ-IFN 50KD (7) Known 7-I FN was dissociated into 25KD and 20KD subunits on 5DS-PAGE. It will be done. The difference in molecular size between the 25KD and 20KD subunits is due to the difference in molecular size of the polypeptide chain. indicated by differences in the degree of glycosylation. Therefore, the new γ−■FN is abnormally small. Determine whether the small molecular size is at least partially due to lack of glycosylation. That's interesting. The presence or absence of carbohydrates is determined by Determined by chromatography on a Canavalin A (ConA) column. summarize and known and novel compounds isolated by HPLC/molecular sieve chromatography. γ-IFN was prepared using a ConA column (Pharmacia's ConA-5EPHAROS E) respectively. After sample loading is complete, each column has 4 column volumes of P Washed with BS. specifically binds to proteins, i.e. carbohydrate-containing proteins was then added with 0.15 M NaCl and 0.2 M σ-methyl-D-mannoside. Eluted using 50mM sodium phosphate (PI (7°4) containing Con Typical elution profiles of known and new γ-IFN on Anfucolumn are shown in Figure 2. .

Recovery of IFN activity from ConA column chromatography is summarized in Table 7 below. C.

Table 7 Recovery of IFN activity from ConA column chromatography (IFN) IFN unit Species Starting Unbound Combined Overall recovery rate known 204,800 H,560 ( 10%) Sat 263,040 (90%) Seen 291,600 (142%) Mutual new 143,360 37,280 (25%) 110. He (75%) 147 , 360 (102%) complete recovery of antiviral activity was obtained with both species of IFN.

Regarding known γ-IFH, 28.560 units out of 291,600 units were recovered. (10%) was recovered in the unbound fraction (in flow and PBS washes), and the remaining 26 3,040 units (90%) were dissolved in σ-methyl-〇-mannoside containing buffer. Recovered by separation. Regarding the new γ-IFN, 147,360 units were recovered. Of the total, 37,280 units (25%) were recovered in the unbound reservoir and 110,08 0 units (75%) were recovered from the bound fraction. Therefore, the size of the new γ-IFN molecule Many are known γ-! It was found that it is glycosylated similarly to that of FN. both The unbound activity in the case of It remained as it was, so it was not due to overload. Molecular size under C0 denaturing conditions Unexpected discovery confirms molecular weight and four-step purification of novel γ-IFN was observed while examining the purity of the protein by 5DS-PAGE under reduced conditions.

Electrophoresis was performed using Laemmli's procedure (Lxesmli, UK +-227=680 ．． 12.5% (w/v) acrylamide slab gel according to 1970) The presence of protein bands was detected using a highly sensitive silver staining procedure (C, R, (see Lil et al., Anxl, Biockum, 105:361, 1980). Therefore, it was decided. The molecular weight markers used in these experiments were Lylase B (9L5110D), bovine serum albumin (6!, HllD), Barbumin (45, GOOD), carbonic anhydrase (31, GOOD), soybean chicken zucin inhibitory factor (21,5fiOD) and lysozyme (14,400D). .

The silver-stained protein outline of the novel γ-IFN after four-step purification is shown in the fourth lane of Figure 3. , the first and fifth lanes are molecular weight markers, and the second and third lanes are 50KD markers. used as an additional molecular weight marker in the region (immunoglobulin heavyweights). This is a replication of a purified monoclonal antibody. Equivalent to over 95% of stainable proteins The major protein band (band b), which is believed to be 2KD) was observed. One of the two small protein bands (band a) is 92.5KD and 68KD, and the other (band C) is 31KD and 2KD. It is between 1.5KD. Less than 14.4 KD corresponding to 8 KD of novel γ-IFN No protein band is observed. The absence of a stainable protein band around the 8KD region is due to this The amount of the novel γ-IFN protein used in the experiment was below the detection limit of silver staining technology. This may be due to the fact that this new protein exhibits irregular behavior in migration on 5DS-PAGE. It may be by showing that

In order to reliably identify novel γ-IFN proteins on 5DS-PAGE, anti-viral Identification based on rus activity was attempted. This method was developed by Yip et al. - As shown in 1982, Vol. 79, pp. 1820-1824, known γ-IFN was described to outline the subunit composition of Figure 4 shows the 5DS-PA Out-activity of known γ-IFN (panel A) and novel γ-IFN (panel B) after GE Showing a comparison of shapes. The active profiles for these two IFN species are different from each other. It is clear that Known γ-IFN typically contains 25KD and It shows 20KD activity. The novel γ-IFN has an estimated molecular weight of 52 KD. The activity peak and the secondary activity peak at 28KD are shown. Therefore, the new γ-I For FN, the active outline in Figure 4 and the silver-stained protein outline in Figure 3 agree well with each other. There is.

Overestimation of the molecular weight of glycoproteins by 5DS-PAGE is a well-established phenomenon. Ru. The presence of carbohydrates reduces the amount of bound SDS per unit mass of polypeptide body. Make it less. The reduced surface charge of the molecular complex accounts for the slower electrophoretic migration speed. Become.

For example, the apparent molecule of blood purification estimated to be 31 KD by ultracentrifugation studies (Biocem, Biopbys, J. P. Segrest et al. Rls, Commun, 44:390-395.1971).

8KD and 5DS-PAG determined by the above abnormalities and molecular sieve chromatography To correct the discrepancy in the estimated molecular weight of 52KD determined in E. γ-IFN is a mixed glycosidase (FDA, 0rfice of Bio From Mr. L2oon of logies R*5earcb and R1vit The 5DS-PAGE experiment was repeated after treatment with (as a gift). These mixed groups Similar preparations of lycosidase are available from KIlilN & Fancy Q, Int. erferon bs, 2:421-429゜1982) and Mr. Llksr. (J, Biol, Cbem, 251:8HOJOI3.1983). Thus, carbohydrates from β-IFN and γ-IFN, respectively, are was achieved. Figure 5: Before treatment with mixed glycosidase (panel The active profile of the novel γ-IFN is shown in A) and after treatment (panel B). of treatment The result is that the main component shifts from 52KD to 40KD, and the minor component shifts from 28KD to It showed a shift to 17KD. New as determined by 5DS-PAGE Estimated molecular weights for γ-IFN varied between 50 and 53 KD. 52 for simplicity Referenced as KD. Used in this experiment based on experiments using known γ-IFN. the amount of enzyme required to completely remove carbohydrates from the novel γ-IFN. It was more than the amount.

The activity peak of gOKD is clearly a mixed glycosidase component and is induced by The results were not based on IFN activity from the culture.

The mixed glycosidase used in these experiments was approximately 256C11/+nl It was discovered to contain contaminants exhibiting viral activity.

This resolved into a single antiviral activity of approximately 80 KD on 5DS-PAGE. It was.

D. Comparison of electrostatic charge by ion exchange chromatography with cation exchange column Representative elution profiles of known and new γ-IFN are shown in Figures 6 and 7, respectively. There is. Known γ-IFN is separated into two main active components, one at 0.20M NaC1i::, the other eluted in 0.27M NaCl (see Figure 6). one On the other hand, the novel γ-IFN is essentially a single active component eluted at 0.35 M NaCI. (See Figure 7). The requirement for high salt concentrations in elution is due to ion exchange At pH 7.5, where the chromatography was performed, the net electrostatic charge of the novel γ-IFN was This means that it is higher than that of known γ-IFN. Therefore, the new γ-IFN The isoelectric point (PI) is necessarily higher than that of known γ-IFN. Known γ-IFN PI was isoelectrically focused on a polyacryamide gel with reference to the reference cited above. Therefore, it was determined to be 8°6.

Fourth Example: Activity and reaction A of novel γ-IFH, cytotoxic activity In addition to its antiviral activity, known γ-IFN also acts directly on tumor cells and mediates mononuclear cells. It is known to exhibit cytotoxic activity through Novel γ-IFN also affects these cells. Tested for the expression of cytotoxic activity, known antiviral units are compared with cytotoxic activity.

MusicmτypeC++It++rsC of Rockville, Maryland Human colonic gland obtained from o11sction under the variety name ATCCHTB 38 Cancer cell line HT-29 was used as target cells. Culture of HT-29 cells 5 μC in the presence of 3 μM 5-fluorodeoxyuridine (Sigma) 12″ of i (micro cucumber)!-iododeoxyuridine (”t U dR , ICN R*diocl+5m1ca1, located in Irvin, California). Be enlightened, 1! Improved uptake of $IUdR. These cells are moistened with CO After labeling at 37°C for 24 hours in an x incubator, complete medium [fetal bovine serum CF] Is it subsidized by B?S)? : RPMl 1640 medium, 2mM L-glutamine, 2 Washed three times with 0mM HEPES and 50pH 7ml gentamicin].

These labeled cells were then plated at 1x10 in each well of a 96-well flat bottom plate. 4 were distributed at the rate of 0.21 complete medium wells.

For analysis of direct cytotoxic activity, 1-IFN samples at the indicated concentrations were A volume of 50 μl was added per bottle. These plates were placed in a humidified CO2 incubator. After 72 hours of incubation at 37°C, centrifugation was performed and 100 μl of supernatant from each well was collected. The radioactivity was determined in a gamma counter.

For analysis of mononuclear cell-mediated cytotoxic activity, human mononuclear cells were plated as follows. It was isolated by sticking to a stick culture container.

Peripheral blood mononuclear cells were collected using a concentration gradient on Ficoll-Hybac (Pharmacia). isolated from normal donor soft tissue cells by imaging. These cells in interphase collect FBS-precoated plasti were washed three times with complete medium and incubated for 1 hour at 4°C. Distribute approximately 3X10' cells/1 in complete medium in a tissue culture flask. It was. After incubation at 37°C for 2 hours, non-adherent cells were removed by discarding the medium. Ta. Adsorbed mononuclear cells were washed briefly three times with complete medium, followed by 0.2% ETD. A and PBS supplemented with 5% FBS were incubated for 30 min at 4°C. .

For purified mononuclear cells, add 10:1 in a 200 μm bag of complete medium per well. Radiolabeled HT-29 was added at a ratio of . The rest of the process is done directly The method is similar to the analysis method for cytotoxic activity.

The cytotoxic activity (%”’I　U　d　R) is determined by the following equation: Calculated from the average count of 7 minutes.

Total value - spontaneous emission value.

However, the total incorporated radioactivity is determined by labeling with 1% dodecyl sulfate. Determined by lysis of HT-29 cells.

Spontaneous release of radioactivity occurs after treatment with γ-IFN or when cultured in complete medium without mononuclear cells. was determined from the supernatant of cultured labeled IT-29 cells. These results are shown in Section 8 below. Shown in the table.

Table 8 Direct and monocyte-mediated cytotoxic activity of human γ-IFN Cytotoxic activity (%”’IUdR release) None 1.2±0.9 24.2±2.650KD (1,12,6±1.9 211.0±2.4 Known 1.0 6.0±0.6 31.8±3.710 1 1.5±L6 310±2.8100 15.0±3.4 31.0±2.88 KD 0.1 1.4±1.2 27.4±1.3 New 1.0 4.2±0. 4 25.2 ± 2.410 1.2 ± 0.9 31.3 ± 1.9100 H, l :I:1.3 3S, 412.1 As understood from the results listed in Table 8, The novel γ-IFN, like known γ-IFN, has a direct effect on human colon adenocarcinoma cells. and hemisphere-mediated cytotoxic activity. Both types of γ-IFN are antiviral. These almost similar capacities in cytotoxic activity when compared to the basic unit of activity showed that.

B. Antigen specificity IF isolated by HPLC/Molecular Sieve Chromatography (see Figure 1) The three molecular weight species of N activity are (i) neutralizing antiviral activity and (ii) radioactive. Antigen specificity was tested by immunoassay (RIA).

(i) Determination of neutralization of antiviral activity The antiviral activity of a predetermined amount from a test sample is Supplied by srferon 5sciences;) Against human γ-IFN monoclonal antibody (M antibody) or polyclonal sheep anti-human IFNa (P antibody) ) was cultured in excess amount of t;.

The residual antiviral activity was assayed at 37°C for 1 hour. The results are shown in Table 9 below. It is guaranteed.

Table 9 IFN activity separated by HPLC/Molecular Oven Chromatography under non-denaturing conditions IFN (units/+1) sample in the presence of neutralizing antibody @L P antibody M antibody 50KDγ-IFN 256 192 <414.5KDγ-IFN 96 1 28　＜48KDγ-IFN　128　128　＜4σ-IFN　128　＜4 96 The results in Table 9 show that the antiviral activity of all three molecular weight forms of IFN is It is completely neutralized by antibodies against IFN, but it is completely neutralized by antibodies against a-IFN. This indicates that it will not be neutralized.

(il) determined by radioimmunoassay (RIA) used in these experiments; The γ-IFN-RIA kit (supplied by Centocor, Malvern) is a human γ- It was shown that it is specific to IFN (T., W., Chuck et al. (See Me Magazine, Vol. 81, pp. 5219-5222, 1984). Analysis is performed by the kit manufacturer. It was carried out in accordance with the guidelines provided. In summary, the test sample is immobilized with the first antibody. Cultured on coated polystyrene beads. After incubation for 2 hours at room temperature, these The beads were washed three times with distilled water. These washed beads are made of radioactive iodine (+ 2J)-conjugated secondary antibody (reagent) for 2 hours at room temperature. child The beads were washed again three times with distilled water to remove unbound second reagent and to remove the condensate. The combined second antibody was measured in a gamma counter. The results are summarized in Table 10 below. ing.

Table 10 Isolated IFN-based binding sample for γ-IFN-specific monoclonal antibody IFN Unit Number of bonds γ-IFN calibration standard 0 276 0.4 490 4 1.920 24 6.070 45 10.305 50KD 20 26,035 14.5KD 25 1g, 115 8KD 12 16,360 The above results again indicate that all three species isolated from the molecular sieve column are of the γ form. It shows.

Fifth Example: Amino acid sequence of novel γ-IFN The results of the third example show that a new low molecular weight The concentration of γ-IFN was determined by ConA-5EPHARO5E chromatography as a two-component, immediate This shows that it can be separated into a bound fraction and a non-bound fraction. Of course, these results , the unbound fraction is a carbohydrate-free or non-glycosylated component, and the bound fraction is a carbohydrate-free or non-glycosylated component. This indicates that it contains chemical residues.

To obtain samples for amino acid sequencing, the non-glycosylated form of the novel γ-IF N was used.

The novel γ-1FN present in the culture fluid is described in the first example above; It undergoes a five-step purification procedure and is contained in the eluent of reversed-phase high-performance liquid chromatography. The material obtained was used as a sample for sequencing. FPLC/5uperose Molecular sieve chromatography on 12 and cationic chromatography on FPLC/MONOS The results already shown from ion-exchange chromatography indicate that the new γ in non-glycosylated form - IFN has a molecular weight of approximately 8 KD in its native state and a novel γ-IFN in glycosylated form. It was shown to have an isoelectric point of 8,5 or more, similar to the value reported for t; .

However, as discussed below, the non-glycosylated form of novel γ-IFN is 5DS-P Separates on AGE into two molecular size components, with the - component having an estimated molecular weight of approximately 6 KD. , the other is about 8KD.

These results indicate that a protein with an apparent molecular weight of 52 KD in 5DS-PAGE This is different from that of the new γ-IFN in glycosylated form, which was considered as a quality.

A variation of the sequential chromatography procedure described above is to synthesize the novel non-glycosylated γ-IFN The next step is ■ adsorption of γ-IFN with silicic acid, ■ Adsorption and removal of some contaminant proteins with anion exchange residues, ■ Con A Sepharo Glycosylated protein, glycosylated novel γ-IFN and glycosylated protein on the column adsorption and loss of known γ-IFN, ■High separation rate molecular sieve chroma of unbound fraction from ConA Sepharose column tography, and ■Cation exchange chromatography It is equipped with

A novel low molecular weight γ-IFN protein that has been sufficiently purified to allow determination of the amino acid sequence. To obtain white matter samples, the non-glycosylated new γ-I obtained in step 5 was FN protein was further purified by reversed phase high performance liquid chromatography (RPHPLC). receive.

6th step: Purification of novel γ-IFN by RPHPLC. The minutes were pooled and placed on a reverse-phase Vydac cm Sot, Cropond, NY. Chromatography Co., Ltd.) column. Novel γ-IFN protein The quality was measured using three cycles of 0.1% (V/V) proceeding for 60 minutes at a flow rate of 1+al/min. Column using a linear concentration gradient (0-95%) of acetonitrile in fluorinated acetic acid. eluted from. The external shape of this eluted protein is monitored by ultraviolet absorption at a wavelength of 214I. I was nitpicked. The new γ-IFN is separated into two components on the 04 column, with the - component being 2 Eluted at 8% acetonitrile, other components eluted at 30% acetonitrile. It was.

5DS-PAGE analysis of RPHPLC-purified γ-IFN protein obtained from RPHPLC. The fractions containing the novel γ-IFN were obtained from Farmingdale, New York. 5avxnt fstrc+cents 5petd Vae Conee acetonitrile and trifluoride by vacuum centrifugation using a nLrator; Acetic acid was removed. These dried protein samples were redissolved in distilled water; These protein samples were analyzed by 5DS-PAGE. 28% acetoni The fraction eluted with tolyl migrated as a protein of about 6 KD and was eluted with 30% acetonitrile. The fraction eluted with RIL was found to migrate as a protein of approximately 8 KD.

Determination of amino acid sequence Determination of the amino acid sequences of these 6KD and 8KD novel γ-IFN species has been published in the art. Made using known standard techniques. Sequencing was performed using an automated sequential Edman decomposition procedure. Applied Biosystems, 7F City, California Formed with Applied Biosys L*m5470A gas phase array device . The 6KD and BKD fractions obtained from the RPHPLC step were Conce+tr! The dried protein was dried by vacuum centrifugation using a tor. The white matter sample is dissolved in a minimum volume (0.1 ml) of 0.1% (V/v) = 7 fluoroacetic acid. understood. The lysed protein samples (i.e. 6KD and 8KD) were It was added to the filter and dried in an oven at 120°C for 5 minutes. Sample containing fi The router was assembled into a cartridge that was attached to a 47OA array for analysis. . Each sample was analyzed 2-5 times and contained 2-5 μg of protein.

The chemical mechanism of automated Edman degradation comprises the following steps.

■ Forms a phenylthiocarbonyl group with the a-amino group of the polypeptide. Bonding with phenylthiocyanate, ■Amino acid residues modified by acid anhydride cleavage of and ■anilinothiazolinone derived from hydrolyzed amino acid residues Conversion of ATZ) to more stable phenylthiohydantoin (PTH) derivatives.

Solvent extraction, washing of excess reagents and by-products at each step, separation of PTH-derived amino acids The analysis is performed by a microprocessor using standard programs supplied by the manufacturer. controlled by a fully automatic procedure.

Amino acid sequence of 6KDγ-IFN Using 115 as the average amino acid residue weight, a 6KD protein has approximately 52 residues. It was calculated as having amino acid residues. In the array shown below, use The abbreviation is as follows. A■Alanine, C-cystine, D-asparagi acid, E-1 rutamic acid, F-phenylalanine, G-glycine, H-glycine N, I-inleucine, K-fusujin, L-leucine, M-methionine, N-as Paragine, P mist proline, Q-glutamine, R arginine, S-serine ’　m-threonine, v-valine, Y-kasumi tyrosine.

The following is a partial sequence of the 6KD polypeptide.

[AELRXsMXzDIKTXsSGIHPX*NNQXsLV　G　G] The sequence of 28 amino acid residues present at the amino terminus of the novel 7-IFN species is <No sequence homologous to that of established known γ-IFN. Position indicated by Xl , various amino acid residues are identified at these positions, and the amino acid at that position is This illustrates the ambiguity in acid identification. This phenomenon is probably caused by these positions. This is an illustration of possible structural polymorphisms. Amino acids identified at these positions is as follows.

X, -01G, E, T%F, D or AlX, proverb or 1, Xl-Ll I, %P%T or Q.

X, -P or 1 X, -5, Q or T.

--Blank, background signal or noise is too high to identify the amino acid at this position Show what you can't do.

Amino acid sequence of 8KDγ-IFN Using 115 as the average residue weight, an 8KD protein has approximately 69 amino acid residues. It was calculated as having a group. The following is a partial sequence of the 8KD polypeptide.

[5AKELRCQCIKTYSKPFHPKF IKEKRVIESGPH- ANA] Similar to 6KDγ-IFN, the 37 The sequence of these amino acid residues is homologous to that of well-established known γ-IFN. Not shown.

The amino acid sequences of these 6KD and 8KD novel γ-IFN species are known in the art. As such, we have cloned the genes encoding these peptides and Messenger mRNA. To synthesize oligonucleotide probes to clone and identify Can be used.

Sixth Example Sequence Comparison Box 2 between New and Known γ-IFN and New γ of Fifth Example - IFN and 25KD/20KD known γ-I FN (50KDγ-IFN support) The structural relationship between these protein units can be determined by comparing the amino acid sequences of these proteins. can be established quickly.

Digestion with trypsin was performed using 0.5% heavy charcoal overnight at room temperature using 2% enzyme by weight. Formed in ammonium acid, pH 8.5 (Sigma, TPCK Trypsin) , with the addition of a second oxide after a further 4 hours. Digestion is a variety of things that should be analyzed. It was performed on 30-40 μg.

The trypsinized peptides were analyzed using an Altex ODS reverse-phase high-performance liquid chromatograph. Laffy (HPLC) column (product of Beckman, Berkeley, 0.46x 25 cm) in 0.1% aqueous trifluoroacetic acid (TFA) solution (pH 2). and a concentration gradient of 0.1% TFA aqueous solution (pH 2) and 0.1% TFA in acetonitrile. Eluted with 1% TFA. Peaks were collected and analyzed by amino acid analysis and sequencing. was analyzed.

For amino acid composition analysis, samples were evacuated using 5.7N HCl. Hydrolyzed in a Pyrex tube at 110°C for 22 hours, Beckman 630D instrument.

Continuous Edman decomposition CP, Edman et al.'s E++r, J-Bi@chsm.

1:8G~91.1967) is Peckman's sequencer model 890. It is formed using B. Carbohydrate-containing peptides are isolated using the procedure described above. Although it could not be separated into peaks, it was analyzed using the method of Linderknecht et al. (See J. Biel, Chem. 259:6790-6797.1984 ).

Industrial applicability As explained above, the novel γ-IFN peptide of the present invention is useful for separating protein molecules. High resolution and high speed are achieved in the biologically active IFN fraction. It is produced using a novel purification method in which a high recovery of filtrate is also achieved.

Therefore, it is possible to exhibit a high degree of therapeutic effect in the medical field.

Figure 1 Figure 2 fluxicon number Figure 3 Figure 4 gel section number Figure 5 Figure 6 111・-Sodium chloride concentration gradient (M)A 2130tstn- Figure 7 -0-0-Sodium chloride concentration gradient (N1)A2tK)nrn- Figure 8 Incferon activity x 104 units/m I---Figure 9 42130nrn- Figure 10 international search report

Claims (31)

[Claims] consisting of a peptide having a molecular weight of less than 1.8,000 Daltons; has immunochemical reactivity with antibodies against γ-interferon, and γ-interferon biologically active products exhibiting antiviral properties and cytotoxic activity. 2. The product according to claim 1, characterized in that the isoelectric point of the peptide is higher than pH 8.5. product. 3. Claim 1 wherein said peptide consists of a sequence of less than about 100 amino acids. products of. 4. The peptide has a molecular weight of about 6,000 Daltons measured under non-denaturing conditions. The product of claim 1 having a molecular weight in the range between 8,000 Daltons. . 5. Isolated from human cells under γ-interferon production conditions and essentially purified. The product of claim 1. 6. human peripheral blood in which the human cells are induced to produce γ-interferon; 6. The product of claim 5, comprising white blood cells. 7. has an amino acid sequence consisting of less than 100 amino acids and has a γ-interface It has immunochemical reactivity with antibodies against γ-interferon and is substantially similar to γ-interferon. A peptide that exhibits antiviral properties and cytolytic activity is injected into a γ-interface with the peptide. It is a method of purification from an unpurified sample consisting of Elon and other proteins as impurities. So, The γ-interferon and the peptide are absorbed into the silicic acid by adsorbing the specimen to silicic acid. binding to acid and desorbing the γ-interferon and peptide from silicic acid; The peptides are purified by subjecting the desorbed product to anion exchange chromatography. The desorption occurs while the dehydrogen and γ-interferon are excreted into the chromatography effluent. Impurities in the kimono are bound to a chromatography column, and the chromatography The effluent was subjected to high separation rate molecular sieve chromatography under non-denaturing protein conditions. , whereby the γ-interferon and the peptide are separated, and the peptide containing the peptide is separated. Collect the fraction containing the peptide, and add the peptide-containing fraction to the cationic peptide. The peptide is bound to a cation exchanger by chromatography on a ion exchange resin. In addition to matching, A purification method comprising steps of desorbing and recovering the peptide from the cation exchanger. Law. 8. The unpurified specimen containing the peptide is induced under γ-interferon production conditions. 8. The purification method according to claim 7, wherein the purification method is obtained from derived human white blood cells. 9. Has an amino acid sequence consisting of less than 100 amino acids, and has a γ-interferon It has immunochemical reactivity with antibodies specific to 8. A peptide which exhibits cytolytic activity and cytolytic activity and is purified by the method of claim 7. 10. The unpurified standard containing the γ-interferon and other proteins as impurities A method for purifying γ-interferon from books, the method comprising: adsorbing the specimen containing the γ-interferon on silicic acid, Desorbing the γ-interferon, By subjecting the desorbed product to anion exchange chromatography, chromatography is performed. γ-interferon is excreted in the outflow of Raffi, The effluent from the chromatography was subjected to high separation rate molecular sieve chromatography under non-denaturing conditions. The desorbed product of γ-interferon is thereby separated from the γ-interferon. isolate taferon, The fraction containing γ-interferon was collected, and the fraction containing γ-interferon was collected. The above fraction was subjected to chromatography using a cation exchanger to obtain the above γ-ion. While binding interferon to a cation exchanger, the γ-interferon A purification method comprising steps of desorbing and recovering from the cation exchanger. 11. The unpurified specimen containing γ-interferon was subjected to γ-interferon production conditions. The purification method according to claim 10, which is obtained from human leukocytes induced under conditions. . 12. 8. The chromatography according to claim 7, wherein said chromatography is carried out using a highly cross-linked agarose medium. Purification method. 13. The high separation rate molecular sieve chromatography produces approximately 125 points per square inch. 8. The method of claim 7, wherein the process is carried out under a pressure ranging between about 430 pounds and about 430 pounds. 14. 14. The method of claim 13, wherein said pressure is about 225 pounds per square inch. Law. 15. 10. The chromatography is performed using a highly cross-linked agarose medium. Purification method. 16. The high separation rate molecular sieve chromatography produces approximately 125 points per square inch. 11. The method of claim 10, wherein the method is carried out under a pressure ranging between about 430 pounds and about 430 pounds. 17. 17. The method of claim 16, wherein said pressure is about 225 pounds per square inch. Law. 18. has an amino acid sequence consisting of less than 100 amino acids, and has a γ-interface It has immunochemical reactivity with antibodies against γ-interferon and is substantially the same as γ-interferon. A peptide that exhibits similar antiviral properties and cytotoxic activity is combined with the γ-interactive peptide. A method of purification from crude specimens consisting of feron and other proteins as impurities. There it is, The γ-interferon and the peptide are absorbed into the silicic acid by adsorbing the specimen to silicic acid. binding to acid and desorbing the peptide from silicic acid; The peptides are purified by subjecting the desorbed product to anion exchange chromatography. discharge into the chromatography effluent; The effluent from the chromatography was chromatographed with concanavalin A. to form bound and unbound fractions; The unbound fraction was subjected to high separation rate molecular sieve chromatography under protein non-denaturing conditions. over it The fraction containing the peptide is collected, and the fraction containing the peptide is subjected to cation exchange. The peptide is bound to a cation exchange resin by chromatography on a cation exchange resin. let me, desorbing and recovering the peptide from the cation exchange resin; The desorbed peptide is subjected to reversed-phase high performance liquid chromatography. Each step of desorbing and recovering the peptide from the reversed phase high performance liquid chromatography A purification method consisting of 19. The amino acid partial sequence is [There is an array] (However, if the above X1 is Q, G, E, T, F, D or A1, said X2 is D or I, said X3 is I, K, P, T or Q, the X4 is P or K, and the X5 is S, Q or A peptide that is T). 20. The amino acid partial sequence is [There is an array] in A certain peptide. 21. An isolated and purified DNA encoding the peptide of claim 19. 22. An isolated and purified DNA encoding the peptide of claim 20. 23. having a molecular weight of less than 8,000 daltons and relative to γ-inkferon It has immunochemical reactivity with antibodies against γ-interferon and is an antiviral agent substantially similar to exhibits viral and cytotoxic activity, The amino acid sequence is [There is an array] (However, if the above X1 is Q, G, E, T, F, D or A1, X2 is D or I, X3 is L, I, K, P, T or Q, the above X4 is P or K, and the above Xs is S, Q or or T). 24. has a molecular weight of less than 8,000 Daltons and is resistant to γ-interferon. It has immunochemical reactivity with antibodies against γ-interferon and is an antiviral agent substantially similar to exhibits viral and cytotoxic activity, The amino acid sequence is [There is an array] A peptide that is 25. 24. The polypeptide of claim 23, having a sequence of less than about 100 amino acids. 26. 25. The peptide of claim 24, having a sequence of less than about 100 amino acids. 27. 24. The peptide of claim 23, wherein the peptide has an isoelectric point higher than 8.5. Petite. 28. 25. The peptide of claim 24, wherein the peptide has an isoelectric point higher than 8.5. Chido. 29. Isolated from human cells under γ-interferon production conditions and essentially purified. 24. The peptide of claim 23. 30. Isolated from human cells under γ-interferon production conditions and essentially purified. 25. The peptide of claim 24. 31. Antibiotics having less than 100 amino acids and substantially similar to γ-interferon A biologically active polypeptide that has viral and cytotoxic activity.