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Extraction of integral membrane proteins

A protein and diafiltration technology, applied in the preparation methods of peptides, microorganism-based methods, peptides, etc., can solve the problems that centrifugation is not suitable for large-scale protein extraction and troublesome.

Inactive Publication Date: 2002-07-10
WYETH HOLDINGS CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, centrifugation is not suitable for large-scale extraction of these proteins because centrifugation is a cumbersome process

Method used

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  • Extraction of integral membrane proteins
  • Extraction of integral membrane proteins
  • Extraction of integral membrane proteins

Examples

Experimental program
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Effect test

Embodiment 1

[0129] Membrane extraction of lipidated rP4 with different detergents

[0130] The overall process of extraction of lipidated rP4 from bacterial cells (such as E. coli cells) involves microfluidization or cell lysis and membrane extraction with different detergents. The fermentation broth was collected and 5mM EDTA was added to inhibit protein degradation caused by possible metalloproteases. The culture solution was then diluted to below 5% (w / v) cell wet weight concentration and lysed with a high-pressure microfluidizer (Microfluidics, Newton, MA). Use includes a surface area of ​​0.002m 2 1000KD regenerated cellulose Millipore membrane tangential flow system per g wet weight of cells, adding buffers in a specific order to diafilter lysed cells. The order of buffer addition was chosen to solubilize inner membrane proteins first, followed by outer membrane proteins including rP4. During diafiltration, appropriately sized soluble proteins that are smaller than th...

Embodiment 2

[0144] Another Extraction Method of Lipidated rP4

[0145] This example presents the data obtained by other four extraction methods of lipidated rP4. In each operation, 5 mM EDTA was first added to the recombinant E. coli fermentation broth to inhibit possible protein degradation by metalloproteases. Then the wet cell concentration of the fermentation medium was adjusted to 10%, and lysed by Microfluidics microfluidizer. This cell lysate was then divided into aliquots containing 500 g of cells and frozen at -70°C.

[0146] Remove a 500-gram aliquot of the lysed E. coli fermentation broth at -70°C and thaw it in a water bath whose temperature does not exceed 40°C. The cell lysate was then diluted to 5% wet weight of cells. The 5% cell lysate was then extracted with different detergents as described in Example 1 using tangential flow diafiltration. There is only a slight difference in step (4), where diafiltration was done 2 times instead of 3 times.

[01...

Embodiment 3

[0156] Membrane extraction of lipidated rP6 with different detergents

[0157] The method for extracting lipidated rP6 is similar to the method for extracting lipidated rP4. However, the diafiltration process requires more steps because lipidated rP6 is tightly bound to peptidoglycan. The fermentation broth of E. coli cells expressing lipidated rP6 was treated with 10 mM EDTA and then diluted to below 10% cell wet weight / volume before homogenization. Cells were then lysed using a high-pressure microfluidizer and sequentially diafiltered with buffers at room temperature using a flow-through membrane filtration device. It was determined that the minimum membrane area that allows the transport of effective mass of dissolved protein across the membrane is approximately 0.002m 2 / g cell wet weight. Soluble proteins with a size smaller than the 1000KD molecular weight cut-off of the membrane pass through the membrane with the permeate, while larger molecules and insolubl...

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Abstract

A process is described for extracting gram-negative integral membrane proteins from bacteria or bacterial host cells containing a recombinant vector by differential detergent tangential flow diafiltration. This process has several advantages over alternate processes. First, it combines the clarification and extraction processes into one unit operation. The product is extracted from the cells and it is separated from cell debris with only one continuous diafiltration process. Second, the membrane proteins are extracted in a semi-purified state, which simplifies the downstream processing steps. Third, this process is very scalable because the only requirement is that the surface area of the membranes be increased proportionally with the amount of cells.

Description

technical field [0001] The invention relates to a method for extracting internal proteins of gram-negative bacterial membranes from bacteria or bacterial host cells containing recombinant vectors by using different detergents to flow through the diafiltration method. Background technique [0002] Gram-negative bacteria have an inner membrane and an outer membrane. The proteins contained in these membranes are collectively referred to as membrane-intrinsic proteins. Relatively small amounts of native membrane-intrinsic proteins can be extracted from Gram-negative bacteria. Recombinant expression technology enables bacteria to increase the expression of these proteins. [0003] Small-scale batch purification of these native or recombinant membrane-intrinsic proteins involves an extraction step using centrifugation to extract the protein from bacterial cell lysates, followed by downstream purification using conventional techniques. [0004] However,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C07K1/34C07K14/245C07K14/285C12P21/00C12R1/19
CPCC07K14/285
Inventor S·拉科蒂亚M·R·比尔
Owner WYETH HOLDINGS CORP
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