Abstract:
:Many recombinant proteins that are produced in Escherichia coli have to be targeted to the periplasm to be functional. N-terminal signal peptides can be used to direct recombinant proteins to the membrane-embedded Sec translocon, a multiprotein complex that translocates proteins across the membrane into the periplasm. We have recently shown that the cotranslational targeting of the single-chain variable antibody fragment BL1 saturates the capacity of the Sec translocon leading to impaired translocation of secretory proteins and protein misfolding/aggregation in the cytoplasm. In turn, protein production yields and biomass formation were low. Here, we study the consequences of targeting BL1 posttranslationally to the Sec translocon. Notably, the posttranslational targeting of BL1 does not saturate the Sec translocon capacity, and both biomass formation and protein production yields are increased. Analyzing the proteome of cells producing the posttranslationally targeted BL1 indicates that the decreased synthesis of endogenous secretory and membrane proteins prevents a saturation of the Sec translocon capacity. Furthermore, in these cells, highly abundant chaperones and proteases can clear misfolded/aggregated proteins from the cytoplasm, thereby improving the fitness of these cells. Thus, the posttranslational targeting of BL1 enables its efficient production in the periplasm due to a favorable adaptation of the E. coli proteome. We envisage that our observations can be used to engineer E. coli for the improved production of recombinant secretory proteins.IMPORTANCE The bacterium Escherichia coli is widely used to produce recombinant proteins. To fold properly, many recombinant proteins have to be targeted to the E. coli periplasm, but so far the impact of the targeting pathway of a recombinant protein to the periplasm has not been extensively investigated. Here, we show that the targeting pathway of a recombinant antibody fragment has a tremendous impact on cell physiology, ultimately affecting protein production yields in the periplasm and biomass formation. This indicates that studying the targeting and secretion of proteins into the periplasm could be used to design strategies to improve recombinant protein production yields.
journal_name
Appl Environ Microbioljournal_title
Applied and environmental microbiologyauthors
Ytterberg AJ,Zubarev RA,Baumgarten Tdoi
10.1128/AEM.00671-19subject
Has Abstractpub_date
2019-06-17 00:00:00issue
13eissn
0099-2240issn
1098-5336pii
AEM.00671-19journal_volume
85pub_type
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