Protein secretion in E.coli

[Duke]

Hi everybody.

I hope this is the right place to ask the following questions.
I am looking for a possibility to express proteins in E.coli which should end up as secreted proteins in the medium.

I know that E.coli usually do not secret proteins. I have found a publication (Golemi et al. J Am Chem Soc, 2000, 122, 6132) in which they use a OmpA leader sequence and 400 mM sorbitol and 2,5 mM betaine in the medium and end up with a sectered protein in the medium. How does it work?
Usually leader sequences like ompA or pelB direct proteins into the periplasma but don´t lead to secretion into the medium. Sorbitol to my knowledge stabilizes the membrane which would lead to the opposite effect (it was also reported, that sorbitol in the medium increases solubility of proteins and therby reducing the amount of inclusion bodies, but this should play any role here).

Does anybody have an explanation for this effect of secretion into the medium and are there other methods to acheive this?

For example, has anybody tried adding lysozyme in small amounts to the medium. My hope would be, that it digests parts of the wall and thereby releasing the periplasmatic proteins into the medium. The "protoplasts" could be stabilized by sorbitol addition. Would E.coli grow and express proteins under theses conditions?

Thank you for your suggestions.

[admin]

Hey Duke,
Very interesting questions. I am not sure exactly how it works although this would help immensely in E.coli protein expression methods.

I will do some reading and get back to you on this.
Cheers

[danfive]

Here is a great review titled "Recombinant Protein Secretion in E. coli"

Abstract
The secretory production of recombinant proteins by the Gram-negative bacterium Escherichia coli has several advantages over intracellular production as inclusion bodies. In most cases, targeting protein to the periplasmic space or to the culture medium facilitates downstream processing, folding, and in vivo stability, enabling the production of soluble and biologically active proteins at a reduced process cost.

This review presents several strategies that can be used for recombinant protein secretion in E. coli and discusses their advantages and limitations depending on the characteristics of the target protein to be produced.

Link ---> http://www.sciencedirect.com/science...9f923a425f2b3#

[danfive]

Article Outline::Recombinant Protein Secretion in E.coli

1. Introduction
2. Recombinant protein secretion
2.1. Type I secretion mechanism
2.2. Type II secretion mechanism
2.2.1. Cytoplasmic membrane translocation
2.2.1.1. SecB-dependent pathway
2.2.1.2. SRP pathway
2.2.1.3. TAT pathway
2.2.2. Extracellular secretion
2.2.3. How are recombinant proteins really transported?
3. Conclusions
Acknowledgements
References

Keywords: Recombinant proteins; Escherichia coli; Secretion; Periplasm; Type II
---> ScienceDirect - Biotechnology Advances : Recombinant protein secretion in Escherichia coli