Details

Autor(en) / Beteiligte

• Alete, Daniel E.
• Racher, Andrew J.
• Birch, John R.
• Stansfield, Scott H.
• James, David C.
• Smales, C. Mark

Titel

Proteomic analysis of enriched microsomal fractions from GS-NS0 murine myeloma cells with varying secreted recombinant monoclonal antibody productivities

Ist Teil von

• Proteomics (Weinheim), 2005-12, Vol.5 (18), p.4689-4704

Ort / Verlag

Weinheim: WILEY-VCH Verlag

Erscheinungsjahr

2005

Quelle

Wiley-Blackwell Journals

Beschreibungen/Notizen

• The folding, transport and modification of recombinant proteins in the constitutive secretory pathway of eukaryotic cell expression systems are reported to be a bottleneck in their production. We have utilised a proteomic approach to investigate the processes catalysed by proteins constituting the secretory pathway to further our understanding of those processes involved in high‐level antibody secretion. We used GS‐NS0 cell populations differing in qmAb to prepare enriched microsome fractions from each cell population at mid‐exponential growth phase. These were analysed by 2‐D PAGE to characterise the microsome protein component and test the hypothesis that bottlenecks in recombinant protein synthesis exist in these compartments, which are alleviated in high producers by the up‐regulation of key secretory pathway proteins. Proteins whose abundance changed in a statistically significant manner with increasing qmAb were involved in a range of cellular functions: energy metabolism, mAb folding/assembly, cytoskeletal organisation and protein turnover. Amongst these were BiP and PDI, chaperones resident in the ER that interact with nascent immunoglobulins during their folding/assembly. However, our results suggest that there are diverse mechanisms by which these cells achieve qmAb. The results imply that cell‐engineering strategies for improving qmAb should target proteins associated with altered functional phenotype identified in this study.