Proteomic analysis of enriched microsomal fractions from GS-NS0 murine myeloma cells with varying secreted recombinant monoclonal antibody productivities.
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.
Alete
Daniel E
DE
Protein Science Group, Research School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK.
Racher
Andrew J
AJ
Birch
John R
JR
Stansfield
Scott H
SH
James
David C
DC
Smales
C Mark
CM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics; D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D005974:Glutamate-Ammonia Ligase / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D009101:Multiple Myeloma / Q000378:metabolism; D020543:Proteome / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
D000911:Antibodies, Monoclonal; D020543:Proteome; D011994:Recombinant Proteins; D005974:Glutamate-Ammonia Ligase