Cetus Corporation

Leland Stanford Junior University

Leo S Lin

David F Mark

Shi-da Yu Lu

Alice M Wang

Glenn J Dorin

Abla A Creasey

Martha B Ladner

Janelle N Van Arsdell

Wolfgang H Hanisch

James W Larrick

Arie Ben-Bassat

James W Thomson

Shi-Da Y Lu

Gordon M Ringold

Keith A Bauer

Sidney N Wolfe

Michael W Konrad

Frank M Torti

Ralph Yamamoto

Muteins of biologically active proteins such as IFN-.beta. and IL-2 in which cysteine residues that are not essential to biological activity have been deleted or replaced with other amino acids to eliminate sites for intermolecular crosslinking or incorrect intramolecular disulfide bridge formation. These muteins are made via bacterial expression of mutant genes that encode the muteins that have been synthesized from the genes for the parent proteins by oligonucleotide-directed mutagenesis.

Cysteine-depleted muteins of biologically active proteins

A process is disclosed for the purification of recombinantly produced biologically active proteins in which a solution containing a mixture of materials, including the biologically active protein, is passed through a continuous porous hydrophobic membrane, and the fraction enriched in the biologically active protein is recovered. Hydrophobic proteins such as TNF and recombinant ricin toxin A chain may be purified according to the process. Conditions for enhanced recovery of purified TNF using the process are disclosed. A highly purified TNF comprising 95% or greater TNF as determined by SDS-PAGE analysis, with an endotoxin content of less than 0.1 ng/mg TNF which is substantially free of pyrogens by the USP rabbit pyrogen test at a dosage range of 1.0 to 2.4.times.10.sup.5 U/kg, is obtained.

N-terminal derivatives of tumor necrosis factor purified by microporous

Structural genes, plasmids and transformed cells for producing cysteine

A refractile material containing a heterologous protein is recovered from a host microorganism cell culture transformed to produce the protein. One recovery process involves disrupting the cell wall and membrane of the host cell, removing greater than 99% by weight of the salts from the disruptate, redisrupting the desalted disruptate, adding a material to the disruptate to create a density or viscosity gradient in the liquid within the disruptate, and separating the refractile material from the cellular debris by high-speed centrifugation. Another version of such a recovery process comprises the further steps of solubilizing the refractile material under reducing conditions, organically extracting the solubilized refractile material, and isolating said refractile material from the extractant. Preferably the protein is recombinant IL-2 or IFN-.beta. and the salt removal step is carried out by diafiltration.

Process for recovering refractile bodies containing heterologous

A modified IFN-.beta. is provided wherein the cysteine residue at position 17 is deleted and serine is substituted therefor. DNA sequences coding for the modified protein, nucleotide primers used for the mutagenesis, appropriate cloning vectors, host organisms transformed with the vectors, methods for the production and use of the modified IFN-.beta. (IFN-.beta..sub.ser17) are also provided. The specific activity of IFN-.beta..sub.ser17 is found to be substantially the same as that of native IFN-.beta..

A method for controlling weight by suppressing the normal metabolism of adipose tissue is disclosed. Administration of tumor necrosis factor (TNF) or a pharmaceutical composition containing it results in suppression of anabolism of adipose cells.

Use of tumor necrosis factor as a weight regulator

Human tumor necrosis factor (TNF) has been prepared using recombinant methods. A human promyelocytic leukemia cell line has been induced using an improved induction procedure, and the TNF purified to homogeneity. Methods, vectors, and cells useful in obtaining human TNF in practical amounts are disclosed. Muteins having N-terminal deletions, which muteins have superior biological activity, are also disclosed.

Human tumor necrosis factor

Human Tumor necrosis factor (TNF) has been prepared using recombinant methods. A human promyelocytic leukemia cell line has been induced using an improved induction procedure, and the TNF purified to homogeneity. Methods, vectors, and cells useful in obtaining human TNF in practical amounts are disclosed. Muteins having N-terminal deletions, which muteins have superior biological activity, are also disclosed.

An improved method for purifying TNF, especially recombinantly produced TNF in bacteria, is disclosed. The purification employs a hydrophobic support in a chromatographic column which is then developed, preferably by HPLC. Preliminary purifications using anion exchange resins are also helpful.

Purification method for tumor necrosis factor

A method for improving the yield of heterologous protein such as IFN-.alpha., IFN-.beta., and IL-2, produced by recombinant bacteria by supplementing the nutrient medium in which the bacteria are grown with a water soluble alcohol and/or amino acid mixture during the terminal phase of the cultivation.

Method of improving the yield of heterologous protein produced by

Human recombinant cysteine depleted interferon-.beta. muteins

Human recombinant interleukin-2 muteins

A process for recovering IFN-.beta. from transformed bacteria comprising: disrupting the cell membranes of the bacteria; solubilizing the IFN-.beta. from the disruptate into an aqueous medium with a solubilizing agent such as sodium dodecyl sulfate; extracting the IFN-.beta. from the aqueous medium with 2-butanol, 2-methyl-butanol, or mixtures thereof under conditions that maintain phase separation between the aqueous meduim and the extractant; and isolating the IFN-.beta. from the extractant such as by precipitating the IFN-.beta. from an aqeous buffer mixture of the extractant by lowering the pH thereof.

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Fremont, CA, United States of America

Leo S Lin