ISSN:
0006-3592
Keywords:
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
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.
Additional Material:
8 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/bit.260360902
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