CA1101842A - THYMOSIN .alpha.1 - Google Patents
THYMOSIN .alpha.1Info
- Publication number
- CA1101842A CA1101842A CA289,510A CA289510A CA1101842A CA 1101842 A CA1101842 A CA 1101842A CA 289510 A CA289510 A CA 289510A CA 1101842 A CA1101842 A CA 1101842A
- Authority
- CA
- Canada
- Prior art keywords
- buffer
- thymosin
- column
- tris
- protein peak
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/57581—Thymosin; Related peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
ABSTRACT
Thymosin .alpha.1 having the amino acid sequence
Thymosin .alpha.1 having the amino acid sequence
Description
T~YMO -1 ;~ The importance of the thymus gland in the development and senescence of immunological competence in animals and man is now generally accepted. Although there is little knowledge of the molecular events by which the thymus gland exerts control ovex T cell development, it appears that a vital part of the process occurs via a hormonal mechanism. The thymus gland produces a family of polypeptides termed thymosin and perhaps several other thymic hoxmones and/or factors which play an important role in the maturation, differentiation and function of T cells.
Thymosin has been found to induce T cell differentiation and to ;~
enhance immunological functions in genetically athymic mice, in adult thymectomized mice, in NZB mice with severe autoimmune reactions, in tumor bearing mice and in mice with casein-induced amyloidosis. ~ -It is known that thymosin fraction 5 is a potent immuno-potentiating preparation and can act in lieu of the thymus gland to xeconstitute immune functions in thymic deprived and/or immunodeprived individuals. Ongoing clinical trials with fraction 5 suggest that thymosin is effecti~e in increasing T cell num-bers and normalizing immune function in children with thymic dependent primary immunodeficiency diseases and can increase T
cell numbers in immunodepressed cancer patients.
Mez/5.9.1977 ~
.
' ~ '~ , . ' ,'' : ' ~
84~
. :
Analytical polyacrylamide yel electrophoresis and iso-electric focusing have demonstrated that fraction 5 consists of 10-15 m~jor components and 20 or more minor components with molecular weights ranging form 1,000 to 15,000.
The present invention rela~es to the isolation and first complete structural determination of an acidic polypeptide isolated form thymosin fraction 5. Thi~; peptide has been termed :
thymosin 1 Thymosin ~1 has been ound to be 10 to 1,000 times more active than fraction S in several in vltro and in vivo assay systems designed to measure T cell differentiation and :~
function.
Thus the present invention provides a method of pre~
paring thymosin ~1 from thymosin fraction 5. The inventive method comprises the :Eollowing steps in combination: - ;~
(a) chromatographing lyophilized thymosin fraction 5 on a ~:
column of carboxymsthyl-cellulose in a sodium acetatej2-mer-captoethanol buffer of p~l 5.0, washing the column first with , :~
this buffer and then eluting with a linear gradient of said buffer and said buffer plus 1.0 M NaCl;
~b) fractionating the first protein peak of step (a) on a .
column of a dextran gel (Sephadex G-25); :
(c) Chromatographing the second protein peak from step (b) on a 2-diethylaminoethyl-cellulose column equilibrated with tris/2-mercaptoethanol buffer of pH 8.0, eluating with said buffer alone followed by a l.inear gradient of said buffer and :~
said buffer plus 0.8 M NaCl;
* Trade Mark.
, :
:
` - 2(a) - ~ `
(d) passing the first sixth of the eluted protein peak from step (c) through a column o a dextran gel (Sephadex G-7S) in quanidine-hydrochlor.ide/tris bufer of pH 7.5 and (e) taking a single narrow cut from the protein peak of step (d) and desalting on a column of a dextran gel (Sephadex G-10).
Thymosin al has a molecular weight of 3,108 and a pI in the range of 4.0-4.3 as determined by gel isoelectric focusing at a pH range o~ 3-5. The compound has the following amino acid sequence~
~N-acetyl)-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-. Thr-Thr-Lys~Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH.
Thymosin al was isolated from fraction 5 by a combination ~.
of ion-exchange chromatography and gel filtration in the follo~
wing way:
, ,.' ~:
Lyophylized thymosin fraction 5 was chromatographed on ~ .
.~ a column of carboxymethyl-cellulose in 10 mM sodium acetate buffer, pH 5.0, containing 1.0 mM 2 mercaptoethanol. The column * Trade Mark :~:"
~.......................................................................... .
~'.-. .
.
. - , .
~.~1318~
was washed first with the buffer followed by a linear gradient of 2 liters each of starting buffer ancl the same buffer con-taining 1.0 M NaCl. The first protein i-raction was filtered on a oolumn of a dextran gel ~Sephadex G-25) in sterile water.
The second protein peak from the dextran gel colu~n was applied on a 2-diethylaminoethyl-cellulose col~ (DE-3~) equilibrated with 50 mM tris-buffer containing 1.0 mM 2-mercaptoethanol ~pH 800). The colu~n was eluted with the starting buffer ~;
followed by a gradient of 1.3 liters each of starting buffer and the same buffer containing 0 8 M NaCl. The first sixth of the protein peak from the DE-32 calumn was further purified by being passed through a column of a dextran gel (Sephadex G-75 in a buffer containing 6.0 M guanidine-hydrochloride and 10 mM
tris (pH 7.5). A single na~row cut was made ~rom the protein lS peak and desalted on a column of a dextran gel (Sephadex G-10) in sterile water. The purified sample so obtained was identifled as thymosin al The yield of thymosin al from fraction 5 was about 0.6%. The preparation was free of carbohydrate and nucleotide. ~`
The structure and complete amino acid sequence of thymosin al was determined using conventional methods such as enzymatic digestion by trypsin, chymotrypsin, thermolysin or subtilisin, separation of the digests by paper electrophoresis and/or chroma-tography and Edman degradation of the separated peptide fragments.
i ~' The following table demonstrates that thlymosin al is from 10 to 1,000 times more active than thymosin fraction 5 in an ` *Trade Mark , .
.
:
,: . : ~ . . , -4;~: - 4 -in vivo mouse mitogen assay, an in vitro ]ymphokine assay measuring MIF production, and an in vitro human E-rosette assay.
Thymosin Activity (~g) in Various Bioassays _ _ _ _ , __ _ MI~ E-~osette Mitogen*
. ~ _ , . ~
Thymosin Fraction 5 1-5 1-10 1-10 Thymosin 1 0.01-0.1 0.001-0.01 0.01-0.1 * In vivo 14 daily injections Thymosin al may be administered to warm blooded mammals by parenteral application either intravenously, subcutaneously ~ `
or intramuscularly. The compound is a potent immunopotentiating agent with a daily dosage in the range of about 1 to 100 ~g/kg of body weight per day for intravenous administration. Obviously -~the required dosage will vary with the particular condition being treated, the severity o~ the condition and the duration of the treatment. A suitable dosage form for pharmaceutical use is 1 mg. of lyophilized thymosin cl per vial to be reconstituted prior to use by the addition of sterile water or saline.
Also included within the scope of the present inventlon are the pharmaceutically acceptable acid addition and base salts of thymosin a1 such as the sodium or potasslum salt or salts with strong organic bases such as guanidine. Examples o~ acid addition salts are the hydrochloride, hydrobromide, sulfate, phosphate, maleate, acetate, citrate, benzoate,isuccinate, malate and ascorbate.
.:
,
Thymosin has been found to induce T cell differentiation and to ;~
enhance immunological functions in genetically athymic mice, in adult thymectomized mice, in NZB mice with severe autoimmune reactions, in tumor bearing mice and in mice with casein-induced amyloidosis. ~ -It is known that thymosin fraction 5 is a potent immuno-potentiating preparation and can act in lieu of the thymus gland to xeconstitute immune functions in thymic deprived and/or immunodeprived individuals. Ongoing clinical trials with fraction 5 suggest that thymosin is effecti~e in increasing T cell num-bers and normalizing immune function in children with thymic dependent primary immunodeficiency diseases and can increase T
cell numbers in immunodepressed cancer patients.
Mez/5.9.1977 ~
.
' ~ '~ , . ' ,'' : ' ~
84~
. :
Analytical polyacrylamide yel electrophoresis and iso-electric focusing have demonstrated that fraction 5 consists of 10-15 m~jor components and 20 or more minor components with molecular weights ranging form 1,000 to 15,000.
The present invention rela~es to the isolation and first complete structural determination of an acidic polypeptide isolated form thymosin fraction 5. Thi~; peptide has been termed :
thymosin 1 Thymosin ~1 has been ound to be 10 to 1,000 times more active than fraction S in several in vltro and in vivo assay systems designed to measure T cell differentiation and :~
function.
Thus the present invention provides a method of pre~
paring thymosin ~1 from thymosin fraction 5. The inventive method comprises the :Eollowing steps in combination: - ;~
(a) chromatographing lyophilized thymosin fraction 5 on a ~:
column of carboxymsthyl-cellulose in a sodium acetatej2-mer-captoethanol buffer of p~l 5.0, washing the column first with , :~
this buffer and then eluting with a linear gradient of said buffer and said buffer plus 1.0 M NaCl;
~b) fractionating the first protein peak of step (a) on a .
column of a dextran gel (Sephadex G-25); :
(c) Chromatographing the second protein peak from step (b) on a 2-diethylaminoethyl-cellulose column equilibrated with tris/2-mercaptoethanol buffer of pH 8.0, eluating with said buffer alone followed by a l.inear gradient of said buffer and :~
said buffer plus 0.8 M NaCl;
* Trade Mark.
, :
:
` - 2(a) - ~ `
(d) passing the first sixth of the eluted protein peak from step (c) through a column o a dextran gel (Sephadex G-7S) in quanidine-hydrochlor.ide/tris bufer of pH 7.5 and (e) taking a single narrow cut from the protein peak of step (d) and desalting on a column of a dextran gel (Sephadex G-10).
Thymosin al has a molecular weight of 3,108 and a pI in the range of 4.0-4.3 as determined by gel isoelectric focusing at a pH range o~ 3-5. The compound has the following amino acid sequence~
~N-acetyl)-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-. Thr-Thr-Lys~Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH.
Thymosin al was isolated from fraction 5 by a combination ~.
of ion-exchange chromatography and gel filtration in the follo~
wing way:
, ,.' ~:
Lyophylized thymosin fraction 5 was chromatographed on ~ .
.~ a column of carboxymethyl-cellulose in 10 mM sodium acetate buffer, pH 5.0, containing 1.0 mM 2 mercaptoethanol. The column * Trade Mark :~:"
~.......................................................................... .
~'.-. .
.
. - , .
~.~1318~
was washed first with the buffer followed by a linear gradient of 2 liters each of starting buffer ancl the same buffer con-taining 1.0 M NaCl. The first protein i-raction was filtered on a oolumn of a dextran gel ~Sephadex G-25) in sterile water.
The second protein peak from the dextran gel colu~n was applied on a 2-diethylaminoethyl-cellulose col~ (DE-3~) equilibrated with 50 mM tris-buffer containing 1.0 mM 2-mercaptoethanol ~pH 800). The colu~n was eluted with the starting buffer ~;
followed by a gradient of 1.3 liters each of starting buffer and the same buffer containing 0 8 M NaCl. The first sixth of the protein peak from the DE-32 calumn was further purified by being passed through a column of a dextran gel (Sephadex G-75 in a buffer containing 6.0 M guanidine-hydrochloride and 10 mM
tris (pH 7.5). A single na~row cut was made ~rom the protein lS peak and desalted on a column of a dextran gel (Sephadex G-10) in sterile water. The purified sample so obtained was identifled as thymosin al The yield of thymosin al from fraction 5 was about 0.6%. The preparation was free of carbohydrate and nucleotide. ~`
The structure and complete amino acid sequence of thymosin al was determined using conventional methods such as enzymatic digestion by trypsin, chymotrypsin, thermolysin or subtilisin, separation of the digests by paper electrophoresis and/or chroma-tography and Edman degradation of the separated peptide fragments.
i ~' The following table demonstrates that thlymosin al is from 10 to 1,000 times more active than thymosin fraction 5 in an ` *Trade Mark , .
.
:
,: . : ~ . . , -4;~: - 4 -in vivo mouse mitogen assay, an in vitro ]ymphokine assay measuring MIF production, and an in vitro human E-rosette assay.
Thymosin Activity (~g) in Various Bioassays _ _ _ _ , __ _ MI~ E-~osette Mitogen*
. ~ _ , . ~
Thymosin Fraction 5 1-5 1-10 1-10 Thymosin 1 0.01-0.1 0.001-0.01 0.01-0.1 * In vivo 14 daily injections Thymosin al may be administered to warm blooded mammals by parenteral application either intravenously, subcutaneously ~ `
or intramuscularly. The compound is a potent immunopotentiating agent with a daily dosage in the range of about 1 to 100 ~g/kg of body weight per day for intravenous administration. Obviously -~the required dosage will vary with the particular condition being treated, the severity o~ the condition and the duration of the treatment. A suitable dosage form for pharmaceutical use is 1 mg. of lyophilized thymosin cl per vial to be reconstituted prior to use by the addition of sterile water or saline.
Also included within the scope of the present inventlon are the pharmaceutically acceptable acid addition and base salts of thymosin a1 such as the sodium or potasslum salt or salts with strong organic bases such as guanidine. Examples o~ acid addition salts are the hydrochloride, hydrobromide, sulfate, phosphate, maleate, acetate, citrate, benzoate,isuccinate, malate and ascorbate.
.:
,
Claims (6)
1. A method of preparing thymosin .alpha.1 from thymosin fraction 5, said method comprising the following steps in combination:
(a) chromatographing lyophilized thymosin fraction 5 on a column of carboxymethyl-cellulose in a sodium acetate/2-mercapto-ethanol buffer of pH 5.0, washing the column first with this buffer and then eluting with a linear gradient of said buffer and said buffer plus 1.0 M NaCl;
(b) fractionating the first protein peak of step (a) on a *
column of a dextran gel (Sephadex G-25);
(c) chromatographing the second protein peak from step (b) on a 2-diethylaminoethyl-cellulose column equilibrated with tris/2-mercaptoethanol buffer of pH 8.0, eluting with said buffer alone followed by a linear gradient of said buffer and said buffer plus 0.8 M NaCl;
(d) passing the first sixth of the eluted protein peak from *
step (c) through a column of a dextran gel (Sephadex G-75) in guanidine-hydrochloride/tris buffer of pH 7.5 and (e) taking a single narrow cut from the protein peak of step *
(d) and desalting on a column of a dextran gel (Sephadex G-10).
(a) chromatographing lyophilized thymosin fraction 5 on a column of carboxymethyl-cellulose in a sodium acetate/2-mercapto-ethanol buffer of pH 5.0, washing the column first with this buffer and then eluting with a linear gradient of said buffer and said buffer plus 1.0 M NaCl;
(b) fractionating the first protein peak of step (a) on a *
column of a dextran gel (Sephadex G-25);
(c) chromatographing the second protein peak from step (b) on a 2-diethylaminoethyl-cellulose column equilibrated with tris/2-mercaptoethanol buffer of pH 8.0, eluting with said buffer alone followed by a linear gradient of said buffer and said buffer plus 0.8 M NaCl;
(d) passing the first sixth of the eluted protein peak from *
step (c) through a column of a dextran gel (Sephadex G-75) in guanidine-hydrochloride/tris buffer of pH 7.5 and (e) taking a single narrow cut from the protein peak of step *
(d) and desalting on a column of a dextran gel (Sephadex G-10).
2, The method of claim 1 wherein the buffer in step (a) is 10 mM sodium acetate and 1.0 mM 2-mercaptoethanol.
3. The method of claim 1 wherein the buffer in step (c) is 50 mM tris-HCl and 1.0 mM 2-mercaptoethanol.
*Trade Mark
*Trade Mark
4. The method of claim 1 wherein the buffer in step (d) is 6.0 M guanidine-hydrochloride and 10 mM tris.
5. The method of claim 1 wherein steps (b) and (e) are carried out using sterile water.
6. Thymosin a1 whenever prepared according to a method claimed in claim 1, 2 or 3 or by an obvious equivalent method thereof.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US73663876A | 1976-10-28 | 1976-10-28 | |
| US736,638 | 1976-10-28 | ||
| US766,638 | 1977-02-08 | ||
| US05/766,638 US4079127A (en) | 1976-10-28 | 1977-02-08 | Thymosin alpha 1 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1101842A true CA1101842A (en) | 1981-05-26 |
Family
ID=27113070
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA289,510A Expired CA1101842A (en) | 1976-10-28 | 1977-10-26 | THYMOSIN .alpha.1 |
Country Status (22)
| Country | Link |
|---|---|
| JP (1) | JPS5411220A (en) |
| AR (1) | AR214903A1 (en) |
| AT (1) | AT362493B (en) |
| AU (1) | AU514996B2 (en) |
| CA (1) | CA1101842A (en) |
| CH (1) | CH633258A5 (en) |
| DE (1) | DE2748213A1 (en) |
| DK (1) | DK149094C (en) |
| ES (1) | ES463588A1 (en) |
| FI (1) | FI56317C (en) |
| FR (1) | FR2369248A1 (en) |
| GB (1) | GB1590457A (en) |
| IL (1) | IL53218A (en) |
| IT (1) | IT1195254B (en) |
| LU (1) | LU78395A1 (en) |
| MC (1) | MC1167A1 (en) |
| NL (1) | NL188699C (en) |
| NO (1) | NO143346C (en) |
| NZ (1) | NZ185519A (en) |
| PT (1) | PT67204B (en) |
| SE (1) | SE442479B (en) |
| YU (1) | YU40314B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2919592A1 (en) * | 1979-05-15 | 1981-01-15 | Max Planck Gesellschaft | METHOD FOR PRODUCING THYMOSINE ALPHA 1 AND DERIVATIVES THEREOF |
| US4339427A (en) * | 1980-04-14 | 1982-07-13 | Hoffmann-La Roche Inc. | Radioimmunoassay of thymosinα |
| FR2492663B1 (en) * | 1980-10-24 | 1985-11-08 | Vtoroi Mo G | MEDICINAL PRODUCT REGULATING THE IMMUNITY T-SYSTEM AND ITS PREPARATION METHOD |
| DE3137231A1 (en) * | 1981-09-18 | 1983-04-14 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., 3400 Göttingen | BIS-THYMOSINE (ALPHA) (DOWN ARROW) 1 (DOWN ARROW) CONNECTIONS |
| JPH01250018A (en) * | 1988-03-30 | 1989-10-05 | Noble Sangyo Kk | Displacement detector electronic measuring instrument |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1195980A (en) * | 1966-08-24 | 1970-06-24 | Univ Yeshiva | Hormone-Like Preparations Derived from Thymus Gland and Methods of Producing the Same. |
-
1977
- 1977-10-05 CH CH1216177A patent/CH633258A5/en not_active IP Right Cessation
- 1977-10-14 AU AU29699/77A patent/AU514996B2/en not_active Expired
- 1977-10-25 IL IL53218A patent/IL53218A/en unknown
- 1977-10-26 NZ NZ185519A patent/NZ185519A/en unknown
- 1977-10-26 CA CA289,510A patent/CA1101842A/en not_active Expired
- 1977-10-26 JP JP12766677A patent/JPS5411220A/en active Granted
- 1977-10-26 SE SE7712071A patent/SE442479B/en not_active IP Right Cessation
- 1977-10-26 MC MC771268A patent/MC1167A1/en unknown
- 1977-10-26 IT IT29020/77A patent/IT1195254B/en active
- 1977-10-26 FR FR7732259A patent/FR2369248A1/en active Granted
- 1977-10-27 NO NO773681A patent/NO143346C/en unknown
- 1977-10-27 AR AR269752A patent/AR214903A1/en active
- 1977-10-27 NL NLAANVRAGE7711814,A patent/NL188699C/en not_active IP Right Cessation
- 1977-10-27 ES ES463588A patent/ES463588A1/en not_active Expired
- 1977-10-27 DK DK478277A patent/DK149094C/en not_active IP Right Cessation
- 1977-10-27 LU LU7778395A patent/LU78395A1/xx unknown
- 1977-10-27 DE DE19772748213 patent/DE2748213A1/en active Granted
- 1977-10-27 AT AT765877A patent/AT362493B/en not_active IP Right Cessation
- 1977-10-27 YU YU2578/77A patent/YU40314B/en unknown
- 1977-10-27 PT PT67204A patent/PT67204B/en unknown
- 1977-10-28 GB GB45001/77A patent/GB1590457A/en not_active Expired
- 1977-10-28 FI FI773221A patent/FI56317C/en not_active IP Right Cessation
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |