Studies on two major contaminating proteins of the cytoplasmic inclusion bodies in Escherichia coli

Veeraragavan, Kannappan

doi:10.1016/0378-1097(89)90187-0

Cited by 4 publications

(3 citation statements)

References 15 publications

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“…This association takes place, at least partly, during the isolation of the protein (Frankel et ai. 1991;Marston, 1986;Veeraragavan, 1989) and is presumed to be caused by the non-native conformation of the protein in the inclusion body. We suggest that the Bac-OM proteins also associate with membranous material in a similar manner.…”

Section: Discussionmentioning

confidence: 99%

Secretion of the Escherichia coli outer membrane proteins OmpA and OmpF in Bacillus subtilis is blocked at an early intracellular step

Puohiniemi

Simonen

Muttilainen

et al. 1992

Molecular Microbiology

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When the genes coding for the outer membrane (OM) proteins OmpA and OmpF of Escherichia coli are fused to a signal sequence of a bacillar exoenzyme and expressed in Bacillus subtilis they remain cell-bound and the signal sequence is not cleaved. To identify the step of arrest in the export of these proteins we studied their accessibility to protease applied to intact protoplasts; they remained resistant indicating fully intracellular localization. Both proteins appeared associated with the cell membranes in sedimentation and flotation centrifugation experiments. However, OmpA and OmpF proteins synthesized in B. subtilis without a signal sequence were similarly associated with membranes in centrifugation experiments whereas electron microscopy showed the presence of intracytoplasmic inclusion bodies not obviously attached to the cytoplasmic membrane. We conclude that OmpA and OmpF proteins even when provided with a functional signal sequence do not enter the export pathway in B. subtilis, probably owing to lack of a specific export component in B. subtilis.

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Section: Discussionmentioning

confidence: 99%

Secretion of the Escherichia coli outer membrane proteins OmpA and OmpF in Bacillus subtilis is blocked at an early intracellular step

Puohiniemi

Simonen

Muttilainen

et al. 1992

Molecular Microbiology

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“…The HCP composition is impacted by the proteome complexity of the utilized host expression system [7–9], the manner in which the therapeutic protein is expressed [10–13], and the purification process itself [10,12]. Moreover, all methods for analytical HCP characterization face challenges due to the dynamic range of HCP abundance at proteome and final drug level.…”

Section: Introductionmentioning

confidence: 99%

Identification and Monitoring of Host Cell Proteins by Mass Spectrometry Combined with High Performance Immunochemistry Testing

et al. 2013

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Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS). However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP) was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day) manner.

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“…Alternatively, in E. coli, the protein of interest may be engineered to be deposited as inclusion bodies in the cytoplasm or periplasm. Each of the different expression modalities and techniques can produce different populations of HCPs (Hart et al, 1990;Rinas and Bailey, 1992;Rinas et al, 1993;Veeraragavan, 1989). Thirdly, the HCP composition at various steps of the manufacturing processes is related to the purification process itself (Hart et al, 1990;Rinas et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

Host cell proteins in biologics development: Identification, quantitation and risk assessment

Wang

Hunter

Mozier

2009

Biotech & Bioengineering

202

184

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Host cell proteins (HCPs) are those produced or encoded by the organisms and unrelated to the intended recombinant product. Some are necessary for growth, survival, and normal cellular processing whereas others may be non-essential, simply carried along as baggage. Like the recombinant product, HCPs may also be modified by the host with a number of post-translational modifications. Regardless of the utility, or lack thereof, HCPs are undesirable in the final drug substance. Though commonly present in small quantities (parts per million expressed as nanograms per milligrams of the intended recombinant protein) much effort and cost is expended by industry to remove them. The purpose of this review is to summarize what is of relevance in regards to the biology, the impact of genomics and proteomics on HCP evaluation, the regulatory expectations, analytical approaches, and various methodologies to remove HCPs with bioprocessing. Historical data, bioinformatics approaches and industrial case study examples are provided. Finally, a proposal for a risk assessment tool is provided which brings these facets together and proposes a means for manufacturers to classify and organize a control strategy leading to meaningful product specifications.

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Studies on two major contaminating proteins of the cytoplasmic inclusion bodies in Escherichia coli

Cited by 4 publications

References 15 publications

Secretion of the Escherichia coli outer membrane proteins OmpA and OmpF in Bacillus subtilis is blocked at an early intracellular step

Secretion of the Escherichia coli outer membrane proteins OmpA and OmpF in Bacillus subtilis is blocked at an early intracellular step

Identification and Monitoring of Host Cell Proteins by Mass Spectrometry Combined with High Performance Immunochemistry Testing

Host cell proteins in biologics development: Identification, quantitation and risk assessment

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