Title:  Polypeptide having human HIV inhibitory activity, a gene encoding the polypeptide, a method to produce the polypeptide

United States Patent:  6,482,412

Issued:  November 19, 2002

Inventors:  Tanaka; Haruo (Machida, JP); Ohmura; Satoshi (Tokyo, JP)

Assignee:  Gakkou Houjin Kitasato Gakuen (Tokyo, JP); Japan Society for the Promotion of Science (Tokyo, JP)

Appl. No.:  674608

Filed:  January 24, 2001

PCT Filed:  September 8, 2000

PCT NO:  PCT/JP99/05199

371 Date:  September 22, 1999

A novel compound, which is effective for treatment of AIDS and has inhibitory activity on human immunodeficiency viruses (HIV), was examined. The K97-0003 peptide, which has anti-HIV activity caused by inhibition of syncytium formation by fusion of envelope glycoprotein of HIV and the host cells expressing the receptor to said virus, was provided by the present invention. Furthermore, the base sequence of the gene coding for said polypeptide, and the method for preparing said polypeptide using strain K97-0003 were provided.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The inventors have isolated and purified a compound, that inhibits syncytium formation by fusion of HeLa cells expressing envelope glycoprotein and HeLa cells expressing CD4 and CXCR4 from culture fluid of K97-0003 strain. Consequently, the inventors obtained a polypeptide having the amino acid sequence of SEQ.ID.NO.1 in a sequence list. As this compound is novel, the inventors have designated it as K97-0003 peptide. The present invention was performed according to such knowledge and includes substantially the amino acid sequence of SEQ.ID.NO. 1 in a sequence list. Moreover, this invention provides K97-0003 peptide having anti-HIV activity by inhibiting fusion of the HeLa cells expressing envelope glycoprotein and the HeLa cells expressing CD4 and CXCR4. The amino acid sequence of SEQ.ID.NO. 1 corresponds to an amino acid sequence that is down stream from the 47th alanine in SEQ.ID.NO.2, the precursor peptide.

The present invention provides DNA coding for K97-0003 peptide including substantially the amino acid sequence shown in SEQ.ID.NO. 1. Such DNA includes a DNA containing the base sequence shown, for example, in SEQ.ID.NO. 3. The present invention further provides DNA coding for the precursor of K97-0003 peptide including substantially the amino acid sequence shown in SEQ.ID.NO. 2. Such DNA includes a DNA containing the base sequence shown, for example, in SEQ.ID.NO. 4. The present invention also provides strain K97-0003 which belongs to actinomycetes and having the ability to produce K97-0003 peptide. Furthermore, the present invention provides the method for preparing K97-0003 peptide characterized by culturing strain K97-0003 in a medium, accumulating K97-0003 peptide in the culture broth and obtaining K97-0003 peptide from said cultured broth.

Herein, the wording "substantial" means that one or more modifications, such as a substitution, addition, deletion, or insertion may occur in the amino acid sequence of said protein, so far as the K97-0003 peptide of the present invention has the biological activity of native activated form K97-0003 peptide. In particular, the K97-0003 peptide of the present invention has the activity to inhibit fusion of HeLa cells expressing envelope glycoprotein and HeLa cells expressing CD4 and CXCR4. It also means that modifications such as a substitution, addition, deletion, insertion, etc. of one or more codons corresponding to the modification of these amino acid sequences may occur in the base sequence of said gene, so far as the DNA according to the present invention retains the function to express said K97-0003 peptide. Therefore, for example, the peptide in which the sequence was deleted from the 1 st (methionine) to the 46th (phenylalanine) of the amino acid sequence of K97-0003 peptide precursor (SEQ.ID.NO. 2) of the present invention is included in the modified amino acid sequence of this protein.

Accordingly, in the present invention, a polypeptide of K97-0003 peptide, a part of which is deleted, substituted by an amino acid sequence, or to which an amino acid sequence is added means the polypeptide for the amino acid sequence having at least 20%, preferably 30% and more preferably 50% of homology with the amino acid sequence of SEQ.ID.NO.1 in a sequence list. Similarly, a precursor polypeptide of K97-0003 peptide, a part of which is deleted, substituted by an amino acid sequence, or to which an amino acid sequence is added means the polypeptide for the amino acid sequence having at least 20%, preferably 30% and more preferably 50% of homology with the amino acid sequence of SEQ.ID.NO.2 in a sequence list.

Moreover, the gene that hybridizes with the base sequence of the gene coding for the amino acid sequence of K97-0003 peptide under a stringent condition means the gene for the base sequence having at least 20%, preferably 30% and more preferably 50% of homology with the base sequence of SEQ.ID.NO.3 in a sequence list. Similarly, the gene that hybridizes with the base sequence of the gene coding for the amino acid sequence of K97-0003 peptide precursor under a stringent condition means the gene for the base sequence having at least 20%, preferably 30% and more preferably 50% of homology with the base sequence of SEQ.ID.NO.4 in a sequence list.

In addition, the DNA according to the present invention includes the degenerate isomer coding for the same polypeptide which differs only in a degenerate codon, not only the base sequence encoding for the amino acid included in K97-0003 peptide according to the present invention. The activated-form K97-0003 peptide mentioned above means so-called mature form K97-0003 peptide. The K97-0003 peptide precursor means the peptide which has the signal peptide sequence region available for secretion of the peptide out of microorganism body, at the N terminal of so-called matured-form K97-0003 peptide.

A fragment of the polypeptide comprising the amino acid sequence of SEQ.ID.NO. 1 means at least 10 amino acids, and preferably at least 15 amino acids, for example, 20, 25, 30, 40, 50, and 60 amino acid portion of the polypeptide. The above-mentioned microorganism capable of producing the compound K97-0003 peptide, that inhibits fusion of HeLa cells expressing envelope and HeLa cells expressing CD4 and CXCR4, belongs to actinomycetes. It may be a microorganism without any particular limitation, so far as having the ability to produce said K97-0003 peptide compound. For example, strain K 97-0003 separated by the inventors is an example of such strain, which may be used the most effectively in the present invention, and the mycological properties of this strain is as follows.

Strain K97-0003 isolated by the inventors is the microorganism having the ability to produce K97-0003 peptide, and said strain exhibits following mycological properties. The vegetative hypha of said strain grows up moderately on oatmeal agar medium or nutrient agar medium, and the segmentation was observed. However, it does not grow in synthetic mediums, such as glucose and nitrate agar medium, or sucrose and nitrate agar medium. Although the aerial mycelium of said strain was not observed on almost all mediums, only on V8 juice agar medium of 1/10 concentration, the aerial mycelium was slightly observed. In the observation under a microscope, the shape of the aerial mycelium is a straight line, and chains of 20 or more of spores were observed. The shape of the spore is cylindrical and the size is 1.0.times.0.5 .mu.m. The surface of the spore is smooth and a sclerotium and a sporangium were not found out.

The cultural properties of the strain K97-0003, examined by E. B. Shirling-D. Gottlieb method (International Journal of Systematic Bacteriology, vol. 16, p.313, 1966) is shown in Table 1. The color tone was determined using Color Harmony Manual, the 4th edition (Container Corporation of America Chicago, 1958) as a standard color, and the code was described into the parenthesis, together with the color name. Unless otherwise specified, the detail data indicated below is the result of observation, performed on each medium at 27^o C. after 2 weeks.

 

The physiological properties of strain K 97-0003 are as follows.

(1) Formation of melanin pigment

(i) Tyrosine agar Negative

(ii) Peptone and yeast and iron agar Negative

(iii) Glucose and peptone and gelatin agar Negative

(iv) Trypton and yeast solution Negative

(2) Tyrosinase reaction Negative

(3) Production of hydrogen sulfide Negative

(4) Reduction of nitrate Positive

(5) Liquefaction of gelatin (21-23^oC.) Positive

(Glucose and peptone and gelatin medium)

(6) Hydrolysis of starch No growth

(7) Solidification (37^oC.) of skimmilk Positive

(8) Peptonization (37^oC.) of skimmilk Positive

(9) Growth temperature range 12-37^oC.

(10) Availability of Carbon Source (Priedhum-Gottlieb Agar medium)

Strain K 97-0003 utilizes glucose as carbon source. Said strain does not utilize arabinose, xylose, raffinose, melibiose, mannitol, fructose, rhamnose, inositol, and sucrose as carbon source.

(11) Decomposition of Cellulose Negative

Briefly, the faxonomic properties on strain K 97-0003 is as follows. The diaminopimelic acid in the cell wall of said strain is meso form. As for the selectivity of the growth medium, although the vegetative hypha of said strain grows up well on oatmeal agar medium and nutrient agar medium and the fission was observed, it does not grow on synthetic mediums, such as glucose and nitrate agar, or sucrose and nitrate agar. On V8 sap agar medium of concentration of 1/10, adhesion of the aerial hypha of above-mentioned strain was also found out slightly. The shape of the aerial hypha is a straight line and forms a long spore chain. The surface of the spore is smooth. As properties on culture, the vegetative hypha of said strain shows the color tone of beige, and the aerial hypha shows the color tone of white. From these results, it is estimated that said strain belongs to actinomycetes.

Although K97-0003 peptide-producing strain was explained above, the faxonomic properties of the above-mentioned strain is extremely easy to alter and not consistent, as general property observed in microorganisms. Said strain mutates naturally or by the artificial means for mutation, using ultraviolet irradiation or mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine or ethyl methane sulfonate, etc. All the strains capable of producing K97-0003 peptide that belongs to actinomyces, including such artificial variants and natural variants, can be used for the present invention. The strains mutated by the cell technological procedures, such as cell fusion or genetic manipulation, are also included in the range of K97-0003 peptide-producing strains of the present invention.

For the culture method of K97-0003 peptide-producing strains of the present invention, a conventional method utilized the incubation of actinomycetes can be adopted. As for the culture medium, either a natural medium or a synthetic medium, containing catabolizable carbon source, nitrogen source, inorganic substance, etc. utilizable by a microorganism at a proper amount, can be used. As a carbon source, carbohydrates, such as glucose, mannose, maltose and molasses, organic acids, such as citric acid, malic acid, acetic acid and fumaric acid, alcohols, such as methanol and ethanol, hydrocarbons, such as methane, ethane, propane and n-paraffin, amino acids, such as glutamic acid, or glycerol can be used.

As for a nitrogen source, ammonium salts, such as ammonium chloride, ammonium sulfate, ammonium nitrate and ammonium phosphate, amino acids, such as aspartic acid, glutamine, cystine and alanine, urea, peptone, meat extract, yeast extract, dry yeasts, cone steep liquor, soybean flour, soluble vegetable protein, cottonseed oil, soybean casein, casamino acids, Pharmamedia, etc. can be used. As an inorganic substance, potassium monohydrogenphosphate, potassium dihydrogenphosphate, sodium dihydrogenphosphate, magnesium phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, copper sulfate, cobalt sulfate, zinc sulfate, calcium pantothenate, ammonium molybdate, aluminum potassium sulfate, barium carbonate, calcium carbonate, cobaltous chloride, salt, etc. can be used. In addition, compounds effective for promoting proliferation of said strain or production of K97-0003 peptide, microelements such as metal salt, vitamin, thiamin, etc., may be added to the medium if needed. Furthermore, when a specific compound is required for the growth of the microorganism, it is needed to add such compound to the medium. As for such compound, any compound useful for K97-0003 peptide production can be used, and all the compounds known to be used for cultivation of actinomycetes can be used.

The shaking culture, submerged culture with aerating and stirring, etc. using a liquid medium are preferable for large scale culture of K97-0003 peptide-producing strains. The incubation temperature can be set in the range that the K97-0003 peptide-producing strains can grow and produce K97-0003 peptide. According to the properties of K97-0003 peptide-producing strains, the incubation conditions can be selected property for performance of microbial incubation. When K97-0003 peptide, which is the cultured product, exists in the culture fluid, the culture fluid containing the microbial cells may also be obtained as the status, and used. However, generally, according to the conventional method, the filtrate of supernatant containing K97-0003 peptide can be used for isolation of the peptide after filtration or centrifugation of the cultured broth. When K97-0003 peptide exists in the body of microbial cells, K97-0003 peptide can be obtained by separating microbial cells from the cultured broth and collecting cells using means, such as a filtration or centrifugation, from the obtained cultured product. Subsequently, the cells were disrupted using a mechanical method, or an enzymatic method using lysozyme etc., and adding a chelating agent such as EDTA and /or a surfactant for solubilization of K97-0003 peptide, if needed.

The solution containing K97-0003 peptide thus obtained can be concentrated by reduced pressure concentration, membrane concentration or precipitated by salting-out using ammonium sulfate, or sodium sulfate, or by fractional precipitation using a hydrophilic organic solvent such as methanol, ethanol, acetone, etc. Then, the low molecular weight impurities can be removed by dissolving this precipitate in water and dialyzing it by a semi-permeable membrane. It can be also purified by an adsorbent, by gel filtration using a gel-filtration agent by adsorption chromatography, by ion exchange chromatography or by reversed phase chromatography. The K97-0003 peptide-containing solution obtained by these means can be further purified by treatments, such as vacuum concentration or lyophilization. The K97-0003 peptide may be a synthetic peptide.

Claim 1 of 9 Claims

What is claimed is:

1. An isolated polypeptide comprising an amino acid sequence shown in following (a) or (b);

(a) a polypeptide referred to as amino acid numbers from 1 to 114 in SEQ.ID.NO. 1 in a sequence list;

(b) the polypeptide (a), a part of which is deleted, substituted by an amino acid sequence, and/or to which amino acid sequence is added, having inhibitory activity on syncytium formation.
 

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