Purified thymosin and process

Claim 2

In a process for recovering a biologically active protein component from thymus tissue, said process comprising i. homogenizing and extracting thymic tissue in aqueous saline solution, then centrifuging to remove undissolved material; ii. heat treating the centrifuge supernatant at about 80° C., which treatment precipitates undesired fraction then centrifuging to remove the precipitated portions; iii. admixing the supernatant with a large excess of cold acetone, thereby precipitating the active fraction, and collecting said precipitate; iv. dissolving the acetone precipitate in phosphate buffer, then adding ammonium sulfate to about 25% of saturation and removing non-soluble impurity; and v. increasing the ammonium sulfate content to about 50% of saturation, thereby precipitating the active fraction; the improvement which comprises in combination A. dissolving the collected precipitate from step (v) in phosphate buffer and ultrafiltering the solution using a membrane having a molecular weight cut-off of 15,000; B. concentrating the ultrafiltration filtrate and treating the residue by column chromatography to thereby separate salt and nucleotides; C. isolating the protein peak from (B) and then additionally purifying the protein material by column chromatography followed by gel filtration and then gel electrophoresis.

Claim 3

The process of claim 1 wherein said ultrafiltration is conducted in a hollow fiber system.

Claim 4

The process of claim 1 wherein the column chromatography of step (B) is conducted on Sephadex G-25.

Claim 5

The process of claim 1 wherein the gel filtration of step (C) is conducted on Sephadex G-50.

Claim 6

The process of claim 1 wherein the gel electrophoresis is conducted on 15% polyacrylamide gel using pH 8.3 buffer.

Claim 7

The process of claim 1 wherein bovine thymus tissue is employed and the protein component isolated is essentially free of carbohydrate, lipid, nucleotide and cyclic AMP, is homogeneous on 15% polyacrylamide gel electrophoresis at pH 8.3 and 2.9, has a molecular weight of about 12,200 and an amino acid composition as follows: ______________________________________ Amino acid Residues/molecules ______________________________________ Lysine 4 Histidine 1 Arginine 2 Aspartic acid 22 Threonine 4 Serine 5 Glutamic acid 32 Proline 5 Glycine 6 Alanine 7 Cysteine 1 Valine 5 Methionine 2 Isoleucine 3 Leucine 6 Tyrosine 1 Phenylalanine 2 Total 108 ______________________________________

Claim 8

An improved process for preparing thymosin fraction 5 which process comprises ultrafiltering a solution of thymosin fraction 4 in phosphate buffer using a membrane having a molecular weight cut-off of 15,000 concentrating the ultrafiltration filtrate and treating the residue by column chromatography to thereby separate salt and nucleotide.

Claim 9

An improved process for preparing thymosin fraction 5 which process comprises ultrafiltering a solution of thymosin fraction 4 in phosphate buffer using a membrane having a molecular weight cut-off of 15,000 concentrating the ultrafiltration filtrate and treating the residue by column chromatography to thereby separate salt and nucleotide.

Claim 10

The process of claim 7 wherein said ultrafiltration is conducted in a hollow fiber system.

Claim 11

The process of claim 7 wherein said column chromatography is conducted on Sephdex G-25.