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  Methods for removing suspended particles from soluble protein solutionsRelated Patent Categories: Chemistry: Natural Resins Or Derivatives; Peptides Or Proteins; Lignins Or Reaction Products Thereof, Proteins, I.e., More Than 100 Amino Acid Residues, Separation Or PurificationThe Patent Description & Claims data below is from USPTO Patent Application 20060142551. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application claims the benefit under 35 U.S.C. .sctn. 119 (e) of U.S. Provisional Application No. 60/241,967, filed Oct. 19, 2000, which is incorporated herein by reference in its entirety. 1. FIELD OF THE INVENTION [0002] The present invention relates to methods for removing suspended particles from soluble protein solutions. In particular, the methods of the invention are useful for removing suspended particles from secreted protein solutions and lysates, including bacterial lysates containing a heterologous protein. 2. BACKGROUND OF THE INVENTION [0003] Proteins play critical roles in functions such as metabolism, gene expression, signal transduction, cellular and extracellular structures, which are essential to the survival and/or reproduction of any living organism. Many proteins may be used in therapeutic and/or diagnostic applications, particularly when available in pure form. Contaminants often prevent realization of therapeutic and/or diagnostic goals and may endanger the health of a patient. [0004] Protein purification is often a significant challenge, especially when large amounts of protein are required for therapeutic or diagnostic purposes. Procedures that simply and rapidly provide the protein of interest in pure form and high yield are very desirable, regardless of scale. [0005] Removing suspended particles from soluble protein solutions is often an important practical problem in purifying proteins of therapeutic or diagnostic significance, particularly when heterologous proteins are expressed in either eukaryotic or procaryotic cells. Currently, several methods are used for removing suspended particles from soluble protein solutions. [0006] Centrifugation is a common method for removing suspended particles from soluble protein solutions. In some situations, suspended particles may be removed from soluble protein solutions by centrifugation alone. In other instances, prior to centrifugation, soluble protein solutions, particularly bacterial lysates, may be treated with a flocculating agent (e.g., polyethyleneimine ("PEI")) which typically removes macromolecules (e.g., DNA and endotoxins) and cell debris. However, large scale centrifugation equipment is very expensive capital equipment and is often a limiting factor in removing suspended particles from soluble protein solutions on a process scale. Major problems with centrifugation include low yields and air entrapment in the supernatant that can lead to substantial protein denaturation. Typical yields of protein after centrifugation are about 80%-85%. [0007] Aqueous two-phase partitioning is another method that has been used for removing cellular debris and suspended particles from soluble protein solutions. Liquid-liquid extraction relies on the incompatibility between two polymers in aqueous solution or one polymer and a salt present at high concentration. This incompatibility typically results in the formation of two separate phases of very different compositions. The protein molecules partition preferentially into one phase or the other, depending on their characteristics (Hayenga et al., U.S. patent application Ser. No. 09/307,549; Diamond et al., Advances in Biochem. Eng. Biotechn. 1992, 47:89-135). [0008] However, aqueous two-phase extraction is time consuming, expensive and requires large amounts of chemicals, which must be properly disposed in compliance with environmental regulations. Further, the chemicals used in extraction must be removed from the protein of interest and the two-phase distribution of protein may limit product yield. Finally, two-phase extraction lacks generality since only a limited number of proteins can be purified by this method. [0009] Microfiltration is another popular method for removing suspended particles from soluble protein solutions. Microfiltration uses membranes that either entrap particles on the membrane surface or within a bed of fibers found within the membrane. However, microfiltration on a process scale is a complicated operation that requires precise optimization of a number of variables such as transmembrane pressure, shear force, flow rate, concentration, pH, ionic strength, etc. Thus, process scale microfiltration frequently requires considerable development time. [0010] Accordingly, what is needed is a rapid and inexpensive process that removes suspended particles from soluble protein solutions in high yield, particularly on a process scale. Further, such a process should not require the use of expensive capital equipment or large amounts of chemicals that require costly disposal. 3. SUMMARY OF THE INVENTION [0011] The present invention addresses this need by providing rapid, efficient and inexpensive methods for removing suspended particles from soluble protein solutions. The present invention provides soluble protein solutions, free of suspended particles in high yield, while avoiding the use of expensive capital equipment or chemicals that require expensive disposal. [0012] The current invention provides a method for removing suspended particles from soluble protein solutions by filtering the soluble protein solution through highly purified diatomaceous earth. Preferably, the highly purified diatomaceous earth is Celpure.TM. P-1000. [0013] In one embodiment, the soluble protein solution is a secreted protein solution. In another embodiment, the soluble protein solution is a lysate. In a preferred embodiment, the lysate is a bacterial lysate. [0014] Preferably, the amount of DNA and endotoxins in a bacterial lysate is reduced. Then, the lysate is filtered through highly purified diatomaceous earth to remove suspended particles, which dramatically reduces lysate turbidity. In one embodiment, the highly. purified diatomaceous earth is packed in a filter press. [0015] In a preferred embodiment, flocculation with polyethyleneimine at between about pH 7.3 and about pH 7.7 reduces the amount of DNA and endotoxins in the lysate. Preferably, the amount of DNA in the lysate is reduced by between about 100-fold and about 150-fold. In one embodiment, the amount of endotoxins in the lysate is reduced by between about 1,000-fold and about 10,000-fold. In another embodiment, the turbidity of the lysate is reduced by between about 200-fold and about 300-fold. [0016] In another preferred embodiment, the lysate is filtered through highly purified diatomaceous earth with a filter press. In a more specific embodiment, the lysate is stirred with highly purified diatomaceous earth before filtering through the filter press. Preferably, the yield of the soluble protein solution is between about 95% and about 100% after filtration through highly purified diatomaceous earth. [0017] In yet another preferred embodiment, the lysate is a bacterial lysate containing a heterologous protein that was obtained by expression in bacteria. Preferably, the heterologous protein is SY161, which has the amino acid sequence shown in SEQ. ID. NO. 1. In a more specific embodiment, refractile bodies in the lysate are resolubilized. Preferably, the bacteria is E. coli. [0018] In one embodiment, the cysteine residues of the heterologous protein are blocked. Preferably, the cysteine residues are blocked with an oxidizing agent. More preferably, the oxidizing agent is a mixture of sodium sulfite and sodium tetrathionate. Even more preferably, about a 2:1 ratio of sodium sulfite and sodium tetrathionate are added to the heterologous protein at a pH of between about 7.8 and about 8.2. [0019] In another embodiment, the blocked cysteine residues of the heterologous protein are deblocked. Preferably, a reducing agent is used to deblock the heterologous protein. More preferably, the reducing agent is dithiothreitol. 4. BRIEF DESCRIPTION OF THE DRAWINGS [0020] FIG. 1 provides the amino acid sequence (SEQ ID NO 1) of SY161. Continue reading... Full patent description for Methods for removing suspended particles from soluble protein solutions Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Methods for removing suspended particles from soluble protein solutions patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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