What are some secretory pathways that can be used to secrete a recombinant bacterial enzyme from E. coli?

I have a recombinant protein (29kDa) that I will express in E. coli BL21 cells. For now I have been sonicating E. coli to extract the enzyme, but in the future I would like E. coli to secrete the enzyme.

  • $\begingroup$ I think E.coli doesnt have secretory system. You might have to use Bacillus spp. which have a secretory system. Also, you need to add a secretory signal sequence at the N-terminal of the protein. $\endgroup$
    Commented Sep 5, 2013 at 11:53

1 Answer 1


According to the 2012 iGEM Kyoto team:

The Twin Arginate Translocation pathway(TAT) is [an endogenous] secretion system in E.coli. This system can carry proteins that have torA signal amino acid sequences at N terminal. TatA, TatB, TatC and TatD compose Tat complex on inner membrane. Tat complex recognizes torA signal peptide and then it transports protein (with torA) from cytoplasm to periplasm. In addition, protein that has passed through TAT pathway cut off the torA signal. Proteins which are secreted by this system have no tag that obstruct the activation of the protein.

The team created a cassette with the TAT genes to increase the expression of the pathway and also included the Kil gene, which perforates the outer membrane, possibly aiding the external diffusion of the secreted protein. They noted, rather tongue-in-cheek, that

Needless to say, function of outer membrane as membrane is essential for E.coli to survive. In other words, overexpression of Kil causes cell death. In this reason, we must find suitable amount of expression.

Note: according to their lab notebook, the team never observed extracellular GFP via confocal microscope (search that page for 'confocal'). So. Beware :)

Their project page also includes some references to the following literature:

  • Tracy Palmer and Ben C. Berks "The twin-arginine translocation (Tat) protein export pathway"
  • J. H. Choi. S. Y. Lee "Secretory and extracellular production of recombinant proteins using Escherichia coli"
  • G. Miksch · E. Fiedler · P. Dobrowolski · K. Friehs "The kil gene of the ColE1 plasmid of Escherichia coli controlled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase"
  • Brad A. Seibel* and Patrick J. Walsh "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage"

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