Methods for removing suspended particles from soluble protein solutions

a technology of suspended particles and soluble protein, which is applied in the field of methods for removing suspended particles from soluble protein solutions, can solve the problems of high cost affecting the efficiency of large-scale centrifugation equipment, and affecting the quality of protein purification, so as to avoid the use of expensive capital equipment or chemicals, and improve the efficiency of large-scale centrifugation equipmen

Inactive Publication Date: 2006-01-26
NV ORGANON
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0011] The present invention addresses this need by providing rapid, efficient and inexpensive methods for removing suspended particles from soluble protein solutions. The present inve

Problems solved by technology

Contaminants often prevent realization of therapeutic and/or diagnostic goals and may endanger the health of a patient.
Protein purification is often a significant challenge, especially when large amounts of protein are required for therapeutic or diagnostic purposes.
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.
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.
This i

Method used

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  • Methods for removing suspended particles from soluble protein solutions

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example 1

6. EXAMPLE 1

Removing Suspended Particles from E. coli Lysate Containing SY161

[0055] 6.1. Lysis of E. coli Cells Expressing SY161

[0056] SY161 may be produced in E. coli strain TG1 transformed with plasmid pMc5-SY161-S3C. This clone represents 13 mutations from the original Staphylokinase gene subcloned from Staphylococcus aureus.

[0057] The E. coli cells expressing SY161 were harvested by centrifugation and stored at −70° C. prior to use. The frozen cell paste was broken into pieces and suspended in about 7.0 volumes (weight / volume) of lysate buffer (50 mM sodium phosphate, pH 9.5 containing 5 mM EDTA) using an overhead mixer set at between about 500 RPM to about 1000 RPM. Mixing was continued until the cell paste was completely suspended in the lysate buffer. A microfluidizer unit was assembled by connecting the required air pressure lines, coolant lines and hoses. The microfluidizer was then purged with lysate buffer and the pressure was adjusted to between about 13,000 psi to ab...

example 2

7. EXAMPLE 2

Removing Suspended Particles from E. coli Lysate

[0065] 7.1 Lysis of E. coli cells that do not Express a Heterologous Protein

[0066]E. coli null cells (E. coli TGI, pMc5-8 (Δ clone)) for expression of SY161 were harvested by centrifugation and stored at −70° C. prior to use. The frozen cell paste was suspended in about 7.0 volumes (weight / volume) of lysate buffer (50 mM sodium phosphate buffer, pH 9.5, containing 5 mM EDTA). The frozen cell paste was stirred for about 0.5 hour with a Silverson Lab Mixer Emulsifier (Model L4R) at about 3,000 rpm to resuspend the cells. A microfluidizer (Model 110Y) was connected to compressed air and the cooling chamber was filled with ice. The homogenizer was purged with lysate buffer and the pressure was adjusted to between about 13,000 psi to about 14,000 psi. The suspended cells were fed into a homogenizer and lysed under the operational pressure of between about 13,000 psi to about 14,000 psi. The once-lysed cell suspension was colle...

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PUM

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Abstract

The present invention provides soluble protein solutions, free of suspended particles in high yield. More particularly, 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.

Description

[0001] This application claims the benefit under 35 U.S.C. § 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...

Claims

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Application Information

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IPC IPC(8): C07K1/34
CPCC07K1/34B01J20/14
Inventor WAN, MINRABIDEAU, SUSAN
Owner NV ORGANON
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