Stringent Regulation and High-Level Expression of Heterologous Genes in Escherichia Coli Using T7 System Controllable by the araBAD Promoter

Biotechnol Prog. Mar-Apr 2002;18(2):394-400. doi: 10.1021/bp0101785.

Abstract

The recombinant Eschreichia coli strain BL21 (BAD) was constructed to carry a chromosomal copy of T7 gene 1 fused to the araBAD promoter. To further characterize this expression system, strain BL21 (BAD) was transformed with the plasmid containing the carbamoylase gene from Agrobacterium radiobacter driven by the T7 promoter. Upon induction with L-arabinose, recombinant cells produced 100-fold increase in carbamoylase activity in comparison with uninduced cells on M9 semidefined medium plus glycerol. This protein yield accounts for 30% of total cell protein content. In addition, it was found that after 100 generations the plasmid harboring the carbamoylase gene remained firmly stable in strain BL21 (BAD), but its stability dropped to only 20-30% in strain BL21 (DE3), a commercial strain bearing T7 gene 1 regulated by the lacUV5 promoter in its chromosome. In an attempt to enhance the total protein yield, fed-batch fermentation process was carried out using a two-stage feeding strategy to compartmentalize cell growth and protein synthesis. In the batch fermentation stage, the culture was grown on glucose to reach the stationary growth phase. Subsequently, glycerol was fed to the culture broth and L-arabinose was augmented to induce protein production when cells entered the late log growth phase. As a result, a carbamoylase yield corresponding to 5525 units was obtained, which amounts to a 337-fold increase over that achieved on a shake-flask scale. Taken together, these results illustrate the practical usefulness of T7 system under control of the araBAD promoter for heterologous protein production.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amidohydrolases / analysis
  • Amidohydrolases / biosynthesis*
  • Arabinose / genetics
  • Arabinose / pharmacology
  • Bacteriophage T7 / drug effects
  • Bacteriophage T7 / genetics*
  • Carrier Proteins
  • Cell Count
  • Chromosomes, Bacterial / genetics
  • DNA-Directed RNA Polymerases / drug effects
  • DNA-Directed RNA Polymerases / genetics
  • Escherichia coli / classification
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics*
  • Escherichia coli / growth & development
  • Escherichia coli Proteins
  • Feasibility Studies
  • Fermentation
  • Gene Expression Regulation*
  • Models, Molecular
  • Promoter Regions, Genetic / genetics*
  • Rhizobium / enzymology
  • Rhizobium / genetics
  • Sensitivity and Specificity
  • Viral Proteins

Substances

  • AraF protein, E coli
  • Carrier Proteins
  • Escherichia coli Proteins
  • Viral Proteins
  • Arabinose
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases
  • Amidohydrolases