title: Proteomic analysis of enriched microsomal fractions from GS-NS0 murine myeloma cells with varying secreted recombinant monoclonal antibody productivities.
abstract: 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.
authors.affiliation: Protein Science Group, Research School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK.
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Alete
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Racher
authors.forename: John R
authors.initials: JR
authors.lastname: Birch
authors.forename: Scott H
authors.initials: SH
authors.lastname: Stansfield
authors.forename: David C
authors.initials: DC
authors.lastname: James
authors.forename: C Mark
authors.initials: CM
authors.lastname: Smales
mesh_terms: 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
chemical_list: D000911:Antibodies, Monoclonal; D020543:Proteome; D011994:Recombinant Proteins; D005974:Glutamate-Ammonia Ligase