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Integrated solution to purification challenges in the manufacture of a soluble recombinant protein in E. coli
Author(s) -
Caparon Maire H.,
Rust Kevin J.,
Hunter Alan K.,
McLaughlin Joseph K.,
Thomas Kristen E.,
Herberg John T.,
Shell Robert E.,
Lanter Paul B.,
Bishop Bruce F.,
Dufield Robert L.,
Wang Xing,
Ho Sa V.
Publication year - 2009
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.22542
Subject(s) - protease , recombinant dna , fermentation , biochemistry , escherichia coli , chemistry , protein purification , biology , enzyme , gene
Apolipoprotein A 1 Milano (ApoA‐1M), the protein component of a high‐density lipoprotein (HDL) mimic with promising potential for reduction of atherosclerotic plaque, is produced at large scale by expression in E. coli . Significant difficulty with clearance of host cell proteins (HCPs) was experienced in the original manufacturing process despite a lengthy downstream purification train. Analysis of purified protein solutions and intermediate process samples led to identification of several major HCPs co‐purifying with the product and a bacterial protease potentially causing a specific truncation of ApoA‐1M found in the final product. Deletion of these genes from the original host strain succeeded in substantially reducing the levels of HCPs and the truncated species without adversely affecting the overall fermentation productivity, contributing to a much more efficient and robust new manufacturing process. Biotechnol. Bioeng. 2010; 105: 239–249. © 2009 Wiley Periodicals, Inc.