Abstract: A method for refolding insoluble, improperly folded IGF-I is provided, wherein the IGF-I, precipitated from prokaryotic host cells, is concurrently solubilized, unfolded, and refolded into a biologically active conformation in a single buffer. The buffer contains reducing agent and chaotropic agent to solubilize the IGF-I at concentrations sufficiently low to allow solubilization and refolding to occur. Also provided is a triple-protease deficient E. coli host suitable for use in the process.

Abstract: Disclosed is an improved method for hemoglobin purification utilizing a novel two-step dual-aqueous-phase extraction technique to separate hemoglobin from red cell membrane stroma and other protein contaminants. In the first step, a first dual-aqueous-phase liquid system is prepared which comprises an upper aqueous phase containing polyethylene glycol in water and a lower aqueous phase containing a phosphate buffer at a pH of about 10. In the second step, a second dual-aqueous-phase liquid system is prepared which comprises an upper aqueous phase, which contains the polyethylene glycol phase containing the hemoglobin extracted in the first step, and a lower aqueous phase containing a new phosphate buffer at a pH of less than 7.5. After the second extraction step, purified hemoglobin solution can then be obtained by removing the phosphate salt and the minute amounts of polyethylene glycol contained in the lower phase.

Abstract: A method is described for isolating an exogenous polypeptide in a non-native conformation from cells, such as an aqueous fermentation broth, in which it is prepared comprising contacting the polypeptide with a chaotropic agent and preferably a reducing agent and with phase-forming species to form multiple aqueous phases, with one of the phases being enriched in the polypeptide and depleted in the biomass solids and nucleic acids originating from the cells. Preferably, the method results in two aqueous phases, with the upper phase being enriched in the polypeptide.

Abstract: The present invention relates to the development of bone growth factors as therapeutics for the prevention and treatment of pathological conditions involving bone tissue. The present invention provides biologically active proteinaceous factors comprising polypeptides containing the amino acid sequence M-A-G-L-G-D-E-F-G-D, [SEQ ID NO: 1], (X) F E T L F G A/V E D V I/D A L Q F V C G D, [SEQ ID NO: 2], or A-Y-I-P-I-E-T-L-E-G-I/G-E-L-V-D/Q-T-G/L-Q-F, [SEQ ID NO: 3], or biologically active fragments or sequence analogues thereof. Among the biological properties of the proteinaceous materials of the present invention is the capability to promote the growth and/or differentiation of osteoblastic cells.

Abstract: Primary-toxic chemical compounds with a molecular weight of less than 12000 Daltons, which may be isolated from plant material and which are potential IgE-binding allergens causing immediate-type allergy in predisposed individuals; methods for their isolation and their use for clinical purposes.

Abstract: The invention involves the supercritical or near-critical fluid disruption of microbial cells and extraction of intracellular components. First, a solvent that is a gas at ambient conditions and that has a critical temperature of between 0.degree. and 100.degree. C. is selected. This solvent is brought to near-critical pressure or higher and to near-critical temperature. The solvent then is combined with a slurry of cells to saturate the cells with the solvent under the prescribed conditions. Next, the pressure is released to cause a pressure drop which results in partial disruption of the cell membrane and release of solvent and other materials from the cell. Novel apparatus and associated methods are provided for carrying out the foregoing process continuously.

Abstract: A method of obtaining a novel polypeptide from a crude extraction product of polysaccharide peptide Coriolus versicolor comprising: a) boiling a water soluble powder of polysaccharide peptide Coriolus versicolor; b) centrifuging a boiled product from step a); c) filtering a centrifuged product from step b); d) purifying a solution from step c) by gel filtration chromatography; e) subjecting the purified material from step d) to HPLC using a reversed-phase at ambient temperature, wherein a solvent composition is at an acidic Ph and further includes KCl solvent, and wherein an elution system consists of a linear gradient of about 80% methanol applied at a rate of from between 0-40 minutes to obtain chromatographic peaks; f) analyzing the chromatographic peaks by monitoring for absorbance at about 230 nm, 1.0 AUFS for protein analysis and about 620 nm, 0.

Abstract: A process for the microbial production of a protein susceptible to inactivation in a fluid production medium by continuously and reversibly protecting said protein against said inactivation during the production stage, separating the protein from the production medium, deprotecting the protein, and recovering the protein product. The process is especially useful for obtaining substantially increased yields of the protein in question by continuously precipitating said protein.

Abstract: Processes for treating a zein containing material to decrease inherent color and smell as well as for obtaining zein component therefrom, which is decreased in color tone and smell. The removal of color and smell is carried out by contacting the material with an aqueous acetone solution having acetone concentration which causes almost no dissolution of the zein component. An extraction of zein component from the raw material is carried out in a conventional manner, for instance, with use of an aqueous ethyl alcohol.

Abstract: In a process for the production of a soluble native protein, such as immunoglobulin or methionine-prochymosin, in which an insoluble form of the protein is produced by a host organism transformed with a vector including a gene coding for the protein, the insoluble form of the protein is reversibly denatured in an alkaline aqueous solution at a pH selected to promote dissociation of a group or groups of the protein involved in maintaining the conformation of the protein, and the protein is subsequently allowed to renature by reducing the pH of the solution below a pH effective to denature the protein to produce the soluble native form of the protein. The pH of the alkaline aqueous is suitably in the range 9.0 to 11.5.

Abstract: A protein which inhibits collagen-stimulated platelet aggregation. The protein has a molecular weight of approximately 17,000. A method of isolating the protein from Ornithodoros moubata and using the protein to prevent or delay blood coagulation by blocking the stimulation of platelet aggregation by collagen is also described. The protein is useful in the prevention, prophylaxis, therapy and treatment of thrombotic diseases.

Abstract: A process for the in vitro production of chemically modified polyphenolic polymer (PPP). First, stable, highly active extracellular tyrosinase is produced from genetically transformed microorganism such as Streptomyces antibioticus. The tyrosinase is then incubated with a reaction substrate such as l-tyrosine, hydrolyzed protein, or an oligopeptide in combination with l-tyrosine. The ratio of the oligopeptide/tyrosine combination as well as variation in the concentration of tyrosinase can be used to modify the color, the molecular size, and the spectral absorbance properties of the PPP produced. Alternatively, or additionally, oxidants such as hydrogen peroxide or hypochlorite can be used to modify the color of the PPP, regardless of the method used to produce the PPP, and the PPP can subsequently be fractionated using molecular weight cut-off ultrafiltration. Organic solvents can also be used in the method of making PPP to produce PPPs having variable but reproducible physical properties.

Abstract: The production of recombinant chymosin is disclosed in which an insoluble form of chymosin precursor is produced by a bacterial host cell transformed by a vector including a coding sequence for said precursor. Solubilization of said insoluble form of chymosin precursor is accomplished using urea at a concentration of at least 7M or guanidine hydrochloride at a concentration of at least 6M prior to cleaving said precursor to form chymosin. Said solubilization preferably additionally involves the denaturation of said precursor in an alkaline aqueous solution, e.g., at a pH between a 9 and 11.

Abstract: A method of isolating cystine rich acid-stable, biologically active proteins comprising the steps of co-precipitation by acidification of said proteins together with at least one acid-sensitive protein; isolation of said proteins from the co-precipitate by its resuspension in aqueous solution and the subsequent recovery of the proteins from the supernatant.

Abstract: A method for refolding insoluble, improperly folded IGF-I is provided, wherein the IGF-I, precipitated from prokaryotic host cells, it concurrently solubilized, unfolded, and refolded into a biologically active conformation in a single buffer. The buffer contains reducing agent and chaotropic agent to solubilize the IGF-I at concentrations sufficiently low to allow solubilization and folding to occur. Also provided is a triple-protease deficient E. coli host suitable for use in the process.

Abstract: A process and apparatus are disclosed for the demineralization of ground bone particles or pieces of cancellous or cortical bone. The apparatus includes multiple solution reservoirs which supply solutions to be pumped into one or more columns filled with bone samples to be demineralized. Solvent outflowing from the column(s) can be monitored for pH, calcium ion concentration or conductivity as a basis for determining when demineralization is complete. During the delipidization phase of processing, lipid solute can be monitored by photometry, or a small assay of the eluent can be dropped into deionized water to see whether a precipitate develops. Flow through the apparatus, including the types and amounts of solutions to be flowed through the apparatus, can be manually or computer controlled.

Abstract: A recombinant spider silk protein can be obtained in a commercially useful form by cloning and the expression in a host cell of a polynucleotide encoding an endogenous spider silk protein or variant thereof. The sequencing of a spider silk protein is made possible by a method for solubilizing a spider silk protein.

Abstract: This invention describes processes for producing mature human members of the NGF/BDNF family of neurotrophic proteins that are fully biologically active. In addition, the gene encoding human BDNF and processes for obtaining the same are disclosed.A previously-unreported member of the NGF/BDNF family of neurotrophic proteins, NGF-3, has been identified and a portion of the gene encoding for the NGF-3 has been described. Processes for identifying additional previously unreported members of the NGF/BDNF family are also described.

Abstract: Recombinant structural and functional polymers are purified by lysing of the cellular host, separation of the solid materials, washing and extraction of contaminants using a detergent solution at elevated temperatures.

Abstract: The present invention relates to a naturally occurring, minimally modified LDL antigen which is present in human atherosclerotic lesions as well as in the serum of a high percentage of patients with coronary artery disease. The invention also comprises antibodies reactive with the antigen, hybridoma cell lines that produce the antibodies of the inveniton, and methods for using the antibodies in the diagnosis and treatment of atherosclerotic disease.

Abstract: A process is disclosed in which high purity protamine-DNA complexes are prepared by collecting nucleoprotamines specific developmental stages of a life form, specifically, amphibian, egg by low temperature processing. The process also includes the steps of sequential homogenization in a high concentration aqueous salt solution at a buffered low pH, followed by ultracentrifugation to remove insoluble matter. Either a crude mixture or pure isolate of the complexes may be produced. Pure isolates require aqueous chloroform extraction to isolate protein and to remove lipids. Lyophilization then removes chloroform and excess water. The isolate is then fractionated by single pass alumina chromatography. Dialysis against pure water removes salts. Repeated lyophilization removes excess water and concentrates single protamines and protamine-like proteins. The mixture may then be reconstituted with 5% weight/volume heterologous or homologous DNA, in order to shield from charge toxicity.

Abstract: Methods are provided for the purification of interleukin-2 (IL-2) from a wide variety of sources, including synthetic mixtures, culture medium conditioned by natural IL-2 producing cells, and mammalian and bacterial recombinant IL-2 expression systems. The methods of the invention employ IL-2 receptor-affinity adsorbents in which soluble IL-2 receptors have been immobilized on solid supports. Through the use of these affinity adsorbents, highly purified IL-2 can be produced in a single step from bacterial extracts or conditioned medium. The IL-2 thus purified is largely free of aggregated forms, which are often present when other purification methods are used.

Abstract: Methods are disclosed for increasing the solubility of antibodies and their radioisotope, toxin, or drug immunoconjugates and for reducing the non-specific uptake of antibody, either conjugated or unconjugated, into the RES organs such as via Fc receptor-mediated mechanisms. The methods involve incubation of the reactive component with amphipathic molecules, such as an anionic detergent, to achieve the desired result. A preferred anionic detergent in this regard is sodium dodecylsulfate.

Abstract: Methods are provided for the purification of interleukin-2 (IL-2) and chimeric proteins containing an IL-2 moiety from a wide variety of sources, including synthetic mixtures, cell culture conditioned medium and mammalian and bacterial recombinant expression systems. The methods of the invention employ IL-2 receptor-affinity adsorbents in which soluble IL-2 receptors have been immobilized on solid supports. Through the use of these affinity adsorbents, highly purified IL-2 and chimeric proteins containing an IL-2 moiety can be produced in a single step from bacterial extracts or conditioned medium. The proteins thus purified are largely free of aggregated forms, which are often present when other purification methods are used.

Abstract: A method of extracting granulocyte/macrophage colony stimulating factor (GM-CSF) from GM-SCF-expressing bacterial cells comprising treating a suspension of GM-CSF-containing bacterial cells with an acid and an enhancing agent, or with an acid that is itself an enhancing agent, removing substantially all of the suspension liquid from the cells, preparing a second suspension of the acidified cells, neutralizing said second suspension, and separating the GM-CSF-containing liquid from the suspended cells.

Abstract: A method for purifying from a mixture of hybrid compound which contains a portion of IL-2, which portion includes at least a region of the IL-2R binding domain of IL-2, which region is effective to cause the hybrid compound to bind to cells containing an IL-2R. The method includes passing the mixture containing the hybrid compound over a column having attached thereto molecules consisting of or containing a complex sugar moiety with affinity for the hybrid compound, and eluting the hybrid compound with a suitable eluant. The invention also features related complexes of the hybrid compound and assays for the hybrid compound.

Abstract: This invention provides a crystalline form of recombinant human granulocyte-macrophage colony-stimulating factor (r-h-GM-CSF) and methods for making such crystals.

Abstract: An aqueous two-phase protein partitioning system is disclosed which employs polyvinylpyrrolidone as the upper phase and maltodextrin as the lower phase and provides a low-cost system for protein partitioning. The system can also be employed with the amion derivatives of chlorotriazine dyes, which bind in a noncovalent manner with the PVP and serve as a ligand for the proteins to be separated.

Abstract: The present invention relates to a novel glycoprotein, extracted from the seeds of Trichosanthes kirilowii, called: trichokirin, as well as to its modified derivatives containing a free or blocked SH group.It relates to a process for its preparation, to its use and to pharmaceutical compositions in which it is present.

Abstract: Hepatitis B PreS2+S antigen is isolated from plasma by adsorption on an affinity chromatography column, elution with a chaotropic agent and treatment with concentrated urea at an elevated temperature. The process retains all or substantially all of the preS2+S antigen.

Abstract: Recombinant hepatitis B antigen bound to yeast membranes in yeast expression systems is rapidly purified by subjecting the membrane bound protein to agents that release undesired proteins, followed by agents that release the recombinant hepatitis B antigen.

Abstract: A method and therapeutic composition for the treatment of bleeding disorders, for example those characterized by a tendency toward hemorrhage or a hypercoagulative state, by the administration of tissue factor protein or antagonists thereof.

Abstract: Methods of purifying recombinant surface antigen of hepatitis B virus are disclosed. In one protocol, purification is achieved by selective extraction of the antigen from yeast membranes, followed by solubilization with urea and dithiothreitol.

Abstract: The present invention encompasses a method for removing proteins from a solution containing polynucleotides and proteins comprising filtering the solution through a nitrocellulose membrane at neutral or basic pH.

Abstract: A process is disclosed for continuously treating a solubilized protein solution containing a protein unfolded to some degree, in a small volume continuous flow reactor, to obtain the protein in a conformation exhibiting the protein's characteristic biological activity by continuously diluting out the solubilizing agent, while continuously withdrawing the refolded protein. The continuous process may be carried out using deionized water as a diluent, rather than buffer solutions.

Abstract: A method is provided for recovering a purified animal growth hormone or a polypeptide analog thereof having substantially the same amino acid sequence as, and the biological activity of, the corresponding naturally-occurring animal growth hormone from a bacterial cell in which the animal growth hormone or polypeptide analog has been produced by means of expression of a plasmid encoding the hormone or polypeptide analog which comprises: (a) disrupting the cell wall of the bacterial cell in a buffered neutral pH solution so as to produce a lysate containing precipitated hormone or polypeptide analog; (b) recovering the resulting precipitated hormone or polypeptide analog; (c) suspending the precipitated hormone or polypeptide analog so recovered in distilled water; (d) treating the resulting precipitate-containing suspension with a sodium hydroxide solution having an alkaline pH of about 11.

Abstract: A method for the recovery of proteins in a solubilized form from host cells including providing a source of host cells incorporating a synthesized or expressed protein; providing a source of at least one cationic surfactant; and treating the host cells with at least one cationic surfactant, in an amount sufficient to effect solubilization of the proteins.

Abstract: Purified glomerular proteoglycans are used as a basis for a diagnostic test for glomerulonephritis in humans involving an immunological reaction between the purified proteoglycans and patient area. A new method for purification of glomeruli proteoglycan antigens is described using guanidine extraction.

Abstract: A method for the preparation of proteins in biologically active form including providing a source of protein solubilized from inclusion bodies with a cationic surfactant; providing a weak denaturing agent; and contacting the solubilized protein with the weak denaturant in water in an amount sufficient to allow the protein to remain in a biologically active form.

Abstract: A process for the preparation of a spatial form, which has biological activity, of a protein from a biologically inactive spatial form is described and comprises the protein being dissolved with the addition of a denaturing agent and thus converted into the random coil form, and the solution being allowed to pass through a material which has molecular sieve properties and contains a liquid medium in which the protein can assume a spatial form which has biological activity, and this material having molecular sieve properties being selected so that the molecules of the denaturing agent can penetrate, but the protein molecules canot. It is possible by centrifugation, blowing or sucking out to remove the medium in the "external volume" of the molecular sieve and to increase the rate of passage of the solution through the molecular sieve.

Abstract: A method is provided for extracting human interleukin-4 (IL-4) from IL-4 expressing bacterial cells. The method comprises treating the bacterial cells with a deactivating agent, disrupting the cell membrane and recovering the IL-4.

Abstract: The present invention provides a process for the renaturation of denatured proteins in solution in a renaturation buffer, wherein a solution is prepared of the protein to be renatured in the critical concentration in a selected buffer and, after formation of the folding intermediate, further protein to be renatured is added in the amount necessary for the achievement of the critical concentration.

Abstract: A process for recovering substantially pure rIL-2 from transformed microorganisms in which the cells are disrupted, impure insoluble rIL-2 is separated from the bulk of the cellular components, the separated impure rIL-2 is solubilized and partially purified in a reduced form, the solubilized rIL-2 is oxidized, the oxidized rIL-2 is purified to clinically acceptable levels, and the oxidized, purified IL-2 is denatured by placing it into a solution of a chaotropic agent, solids are removed from the solution and rIL-2 is renatured from the solution.

Abstract: A process for recovering dimeric, biologically active CSF-1 from bacterially expressed recombinant CSF1 genes is described. The process comprises recovery of the solubilized monomeric form, followed by dimerization under refolding conditions and further purification of the dimer. Heterodimers may also be produced by this process.

Abstract: Biologically active lymphotoxin polypeptide species, and derivatives, fragments, aggregates and pharmaceutically acceptable salts are provided. The lymphotoxins are substantially homogenous, and are formulated into pharmaceutical compositions. The lymphotoxins are purified to a specific activity of at least 10.sup.6 units/mg protein by using hydrophobic substances and/or immobilized lentil lectin, or with the use of other chromatographic processes such as ion exchange chromatography, HPLC or gel filtration.

Abstract: A method of extracting granulocyte macrophage colony stimulating factor (GM-CSF) from GM-CSF-expressing bacterial cells comprising treating a suspension of GM-CSF-containing bacterial cells with an acid and an enhancing agent, removing substantially all of the suspension liquid from the cells, preparing a second suspension of the acidified cells, neutralizing said second suspension, and separating the GM-CSF containing liquid from the suspended cells.

Abstract: Basic Fibroblast Growth Factor (FGF) is substantially purified by the employment of affinity chromatography using heparin-linked support material. Described is a simplified three step procedure for extracting basic FGF from either mammalian brain or mammalian pituitary tissue. Salt precipitation, e.g., with ammonium sulfate is used to provide a partially purified precipitate that is then subjected to ion-exchange chromatography, e.g., using a Carboxymethyl-Sephadex column. Substantially pure basic FGF fractions are then obtained by fractionating the further partially purified fractions using affinity chromatography on a heparin-linked support e.g., Heparin-Sepharose.

Abstract: A bidifobacteria growth promoter contained in soybeans is extracted from defatted soybeans with an aqueous solution of alcohol.

Abstract: Immunologically intact protein or peptide immunogens are recovered from vaccines consisting of immunogen-aluminum hydroxide (alum) complexes. Recovery consists of dissolution of the complexes with an alkali metal salt of a carboxylic acid at a basic pH, reduction of the pH to physiological levels, removal of excess dissolvent and isolation of the protein or peptide immunogen.

Abstract: A method of purifying hydrolyzed protein compositions by contact with a substantially phase-incompatible fluid extractant is provided. A reduction in the concentration of chlorohydrins, measured as 3-monochloro-1,2-propanediol, in hydrolyzed protein compositions can be obrtained by contacting the hydrolyzed protein composition with such a phase-incompatible fluid extractant, e.g., ethyl acetate. The method allows removal of chlorohydrins without substantially affecting the organoleptic qualities of the hydrolyzed protein.