Depression of protein synthetic capacity due to cloned‐gene expression in E. coli

Thomas K. Wood, Steven W. Peretti

Research output: Contribution to journalArticle

36 Scopus citations

Abstract

Recombinant bacterial systems exhibit limited capacities for heterologous protein production. As seen with this and other systems, cloned‐gene protein production reaches an upper limit despite further increases in gene dosage. A series of closely related plasmids which contain mutations affecting their copy number has been used to investigate the macromolecular impediments to enhanced recombinant protein production. Within a common host, HB101, the level of the ampicillin resistance gene, bla, was varied using five plasmids which differ solely in their replication machinery. Separate fermentations were conducted in which the plasmid copy number was varied from 0 to over 400 while the specific growth rate was fixed at 0.6 h−1 for each chemostat cultivation. The effects of constitutive expression of the bla gene as copy number was elevated were then determined using pulse‐labelling and RNA–DNA hybridizations. Specifically, the steady‐state level, synthesis rate, and stability of β‐lactamase messenger RNA and ribosomal RNA were determined as a function of copy number. The results indicate that as copy number rises, both β‐lactamase mRNA synthesis rates and steady‐state mRNA levels increase. Therefore, β‐lactamase production in these strains does not appear to be limited by the level of β‐lactamase mRNA. However, as the copy number was amplified, the stability of rRNA decreased to the point that steady‐state levels of rRNA decreased. These data indicate that a limitation develops within the translational capacity of the cell at high levels of cloned‐gene expression. The results suggest that strategies designed to enhance recombinant protein expression should include manipulation of translation as well as transcription.

Original languageEnglish (US)
Pages (from-to)865-878
Number of pages14
JournalBiotechnology and bioengineering
Volume36
Issue number9
DOIs
StatePublished - Nov 1990

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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