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On-column refolding and purifying of lipoproteins

A lipoprotein, refolding technology, applied in apolipoprotein, animal/human protein, organic chemistry, etc., can solve problems such as immunogenicity

Inactive Publication Date: 2015-08-19
MERCK & CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Histidine tags bound to proteins enable alternative purification schemes but can lead to immunogenicity

Method used

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  • On-column refolding and purifying of lipoproteins
  • On-column refolding and purifying of lipoproteins
  • On-column refolding and purifying of lipoproteins

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Experimental program
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Embodiment Construction

[0060] I. General approach.

[0061] Standard methods in molecular biology are described in Sambrook, Fritsch and Maniatis (2nd edition 1982 & 1989, 3rd edition 2001) Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, version 3 , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA , Vol. 217, Academic Press, San Diego, CA). Standard methods also appear in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols.1-4 , John Wiley and Sons, Inc. New York, NY, which describes cloning and DNA mutagenesis in bacterial cells (Volume 1), cloning in mammalian cells and yeast (Volume 2), glycoconjugates and Protein Expression (Volume 3) and Bioinformatics (Volume 4).

[0062] Methods for protein purification are described, including immunoprecipitation, chromatography, electrophoresis, centrifugation and crystallization (Coligan, et al....

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Abstract

Provided are methods of refolding and purifying lipoproteins on a chromatography column. In particular methods of refolding ApoL1 polypeptides using a hydrophobic interaction column (HIC). The present invention provides methods for the purification of an active form of a human lipoprotein, such as ApoL1. More particularly, the present invention relates to a method for renaturing an inclusion body of proteins expressed in a large quantity in E. coli into an active form using a hydrophobic interaction column and removal of impurities, e.g., endotoxins.

Description

field of invention [0001] The present invention provides methods for purifying the active form of human lipoproteins, such as ApoL1. More specifically, the present invention relates to a method for annealing inclusion bodies of proteins expressed in large quantities in E. coli into an active form and removing impurities (eg, endotoxin) using a hydrophobic interaction column. Background of the invention [0002] The manufacture of recombinant therapeutic proteins presents many purification challenges, including the removal of process and product-related impurities such as endotoxins, host cell proteins, and protein fragments. These challenges are amplified with the production of apolipoproteins, as these hydrophobic proteins tend to have high binding affinity for impurities and will co-purify, making isolation difficult (see, e.g., Caparon, et al. (2010) Biotechnol. Bioeng. 105:239-249). Interaction of apolipoproteins with impurities adds complexity to the purification pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/775
CPCC07K14/775C07K1/20
Inventor R.A.齐米洛夫斯基C.卡特勒H.李T.O.林登
Owner MERCK & CO INC