Chemistry: analytical and immunological testing – Peptide – protein or amino acid – Glycoproteins
Patent
1993-08-26
1997-02-04
Czaja, Donald E.
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
Glycoproteins
436 17, 436513, 436539, 436503, G01N 33487
Patent
active
055997198
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
Protein Isolation
Protein isolation is an important tool in biological research, clinical diagnostics and the production of pharmaceuticals, especially production by recombinant techniques. The scientific researcher must obtain a protein quickly while retaining high specific activity; the clinician must identify proteins in biological samples in order to make an accurate diagnosis; and the molecular biologist must recover and purify large quantities of proteins produced by recombinant organisms.
Scientists have traditionally isolated proteins by precipitating them from biological samples with salts, such as ammonium sulfate, or organic solvents, such as ethanol. Exposure to the chemicals used in such methods often causes protein denaturation. In addition, separation of the proteins from the precipitating chemical is difficult and may cause further denaturation.
The ammonium sulfate precipitation technique, also known as "salting out," is based on the fact that the solubility of most proteins decreases at high electrolyte concentration. Sulfate is used because multivalent ions are more effective than monovalent ions. This procedure is usually carried out in the cold (0.degree.-4.degree. C.) with control of pH close to neutrality. Different classes of proteins precipitate depending on the concentration of salt added. The disadvantage to this method is the difficulty of removing residual salt from the precipitate or supernatant. Often dialysis is used, but is very time consuming.
Organic solvents are often used for fractional precipitation of proteins. However, there is a risk that the solvent will denature the protein unless kept at a temperature near the freezing point. In addition, the solvent must be removed from the protein. A solvent such as ethanol is generally removed by lyophilizing the precipitated proteins.
Recent advances in protein purification have centered around the development of high performance ion exchange, affinity chromatography, hydrophobic interactions and gel filtration chromatography. The biological sample is loaded onto a chromatography column and is eluted with the appropriate solvent into fractions that are analyzed for protein activity. This method is expensive, time consuming, and poorly suited for large scale protein purification.
Many scientist continue to use traditional methods alone or in combination with the recently developed chromatography procedures. For example, after precipitation of a protein from a biological sample with ammonium sulfate, protein is separated from the ammonium sulfate salt by chromatography. Often the protein loses activity or becomes denatured during one or more steps of the procedure, resulting in a low yield or inaccurate identification.
Polymer Studies
In the mid 1960's, Polson et al., Biochim. Biophys. Acta 82:463-475 (1964), analyzed a variety of high molecular weight polymers for purifying proteins including polyethylene glycol (PEG), dextran, nonylphenol-ethoxylates (NPEs), polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP). Polson et al. concluded that PEG is the most suitable of the tested polymers for protein precipitation. The disadvantage to PEG is that it must be removed from the protein either by passage through a DEAE or cellulose column under conditions that adsorb the proteins and wash out the PEG with the effluent or by adding ethanol to precipitate the proteins from the supernatant, leaving the PEG in the supernatant. Polson et al. explicitly caution against the use of PVE, PVP and NPEs because of their high intrinsic viscosities and because, according to their observations, the polymers cause significant protein denaturation. The results of Polson et al. were confirmed and extended by Zeppezauer and Brishammar, Biochim. Biophys. Acta 94:581-583 (1965) who precipitated kidney proteins with three high molecular weight preparations of Polyox.TM., a PEG resin obtained from Union Carbide Corp. (Danbury, Conn).
Polyethylene glycol (PEG), also known as poly(oxyethylene) glycol, is a condensation p
REFERENCES:
patent: Re31268 (1983-06-01), Hansen
patent: 3631018 (1971-12-01), Shanbroom et al.
patent: 3652530 (1972-03-01), Johnson et al.
patent: 3790552 (1974-02-01), Johnson et al.
patent: 3869436 (1975-03-01), Falksveden et al.
patent: 3897414 (1975-07-01), Albertsson
patent: 4016039 (1977-04-01), Schreiber
patent: 4093606 (1978-06-01), Coval
patent: 4115375 (1978-09-01), Pederson
patent: 4124576 (1978-11-01), Coval
patent: 4164495 (1979-08-01), Hansen
patent: 4165370 (1979-08-01), Coval
patent: 4543210 (1985-09-01), Mitra et al.
patent: 4578218 (1986-03-01), Saundry et al.
patent: 4683294 (1987-07-01), Van Wijnendaele et al.
patent: 4692331 (1987-09-01), Uemura et al.
patent: 4740304 (1988-04-01), Tjerneld et al.
patent: 4822535 (1989-04-01), Ekman et al.
patent: 4874708 (1989-10-01), Makula et al.
patent: 4910182 (1990-03-01), Hums et al.
patent: 5135875 (1992-08-01), Meucci et al.
patent: 5177194 (1993-01-01), Sarno et al.
patent: 5288853 (1994-02-01), Bhattacharva et al.
Albertsson et al., "Seperation of Membrane Components by Partition in Detergent-Containing Polymer Phase System", J. of Chromatography, 215, 1981, pp. 131-141.
Hammar, L. et al., "The Use of Aqueous Two-Phase Systems to Concentrate and Purify Bovine Leukemia Virius Outer Envolope Protein g p51," Biotech and Appl. Biochem, Nov., 1986, pp. 296-306.
Green et al., "Protein Fractionation on the Basis of Solubility in Aqueous Solutions of Solk and Organic Solvents", Methods in Enzymology, Ed. C. Kaplan, 1, 99-121, 1955.
Ingham, "Precipitation of Proteins with Polyethylene Glycel" Methods in Enzymology, vol. 182, 301-306, 1990.
Polson et al., "The Fractionation of Protein Mixtures by Linear Polymers of High Molecular Weight", Biochim. ET Biophys. Acta, 82, 463-475, 1964.
Hasko et al., "Fractionation of Plasma Proteins with PEG" Hoematoligia, 14(2), pp. 199-206, 1981.
Farrugia et al., "Studies on the Prowrement of Coagulation Factor VIII: Selective Precipitation of Factor VII with Hydrophilic Polymers, " Thromb Horm., 51(3)338-342, 1984.
Spence et al., "Use of Water-Soluble Polymers in the Precipitation of Ikod Group Diagnostic Reagents", Med. Lab. Sciences, 42, 115-117, 1985.
Wiesen et al., "Gel Difusion Reactions and Biological Properties of Paeonia Tammin," Phytopath. 2, 93, 56-68, 1978.
Virella et al., "Isolation of Soluble Immune Compleros from Human Serum: Combined Use of PEG Preciptation, Gel Filtration . . . ", Methods in Enzymology, 74, 644-663, 1981.
Clamagirand, C., et al., "Partial Purification and Functional Properties of an Endoprotease from Bovine Neurosecretory Granules Cleaving Proocytocin/Neurophysin Peptides at the Basic Amino Acid Doublet," Biochemistry, 26(19): 6018-6023 (1987).
Feldman, J. A., et al., "Semiinterpenetrating Networks Based on Triazine Thermoset and N-Alkylamide Thermoplastics," Am. Chem. Soc. Symposia Series, 367: 244-268 (1988).
Georgiou, M., et al., "Functional and Physical Characteristics of Rat Leydig Cell Populations Isolated by Metrizamide and Percoll Gradient Centrifugation," Biology of Reproduction, 37: 335-341 (1987).
He, D., et al., "Mast-Cell Heterogeneity; Functional Comparison of Purified Mouse Cutaneous and Peritoneal Mast Cells," The Society for Investigative Dermatology, Inc., 95(2) (1990).
Heuck, C. C., et al., "Rapid Development of Immunoprecipitins in Agarose Gel," Clinica Chimica Acta, 98:195-199 (1979).
Ralston, G. B., "Effects of `Crowding` in Protein Solutions, " J. Chemical Education, 67(10) Oct. 1990, pp. 857-860.
Sanbar, et al., "Hypolipidemic Effect of Polyvinylpyrrolidone in Man," Circulation, 38: 771-776 (1968).
Schultze, H. E., et al., "Molecular Biology of Human Proteins--With Special Reference to Plasma Proteins," 1: 240-317 (1966).
Sperling, L. H., "Interpenetrating Polymer Networks and Related Materials," Plenum Press, New York (1981).
Strong, M. J., et al., "Isolation of Fetal Mouse Motor Neurons on Discontinuous Percoll Density Gradients," In Vitro Cellular & Developmental Biology, 25(10): 939-945 (1989).
van Suylic
Rothstein Fred
Woiszwillo James E.
Cano Milton I.
Czaja Donald E.
Middlesex Sciences, Inc.
LandOfFree
Method for isolating biomolecules from a biological sample with does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for isolating biomolecules from a biological sample with , we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for isolating biomolecules from a biological sample with will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-677886