After expression of a recombinant protein in pET system, does E.coli secrete it out of cell or it stays inside cytoplasm or any other compartment of cell? If secretion is dependent to any factor like, E.coli strain, Vector type, protein, or medium please provide me related literature.
Information about Protein: S100 proteins; small Ca-ion binding poteins, only express in vertebrates.
That depends on your protein. pET does not contain any secretion signals, so a cytoplasmic protein will stay in the cytoplasm. But even if yor protein is secreted in vertebrates, it will likely lack the signals needed for secretion in prokaryotes, i.e. either a Sec or a Tat signal sequence.
If you want to get secretion, you will have to include one of those in your protein sequence (they are removed by the cell upon secretion). Sec results in "secretion during synthesis", i.e. secretion starts while the protein is still synthesized. In contrast, Tat-tagged proteins are secreted after folding in the cytoplasm.
http://www.athenaes.com/tech_brief_ACESyebf.php
That depends on your protein. pET does not contain any secretion signals, so a cytoplasmic protein will stay in the cytoplasm. But even if yor protein is secreted in vertebrates, it will likely lack the signals needed for secretion in prokaryotes, i.e. either a Sec or a Tat signal sequence.
If you want to get secretion, you will have to include one of those in your protein sequence (they are removed by the cell upon secretion). Sec results in "secretion during synthesis", i.e. secretion starts while the protein is still synthesized. In contrast, Tat-tagged proteins are secreted after folding in the cytoplasm.
http://www.athenaes.com/tech_brief_ACESyebf.php
Thanks a lot.
This article is very informative, but frankly it will make my protocol very difficult.
I found that pET vectors are also available fused with some leaders like pelB or ompT that could help my proteins till periplasmic space. Now please comment, is it helping for complete secretion?
That article has 4 possible methods to deal with problem. First one is very easy and practical for me, “use chemical, biochemical, enzymatic or physical treatment to induce outer membrane leakage resulting in the release of the target protein to the medium.” Can you please name any method that is not harmful for my proteins?
I usually work with Gram-positive bacteria, so I do not know how efficient these signals are. But according to SignalP, they should do their job.
Concerning the question of complete secretion: That I cannot tell for sure, as I usually do not use E.coli for secretion. As your proteins seem to be rather small (~10-20 kDa) they might simply diffuse through natural outer membrane porins. If that does not happen or ist not efficient enough, you might try to co-express OprF from Pseudomonas fluorescens. That is an outer membrane porin that allows the passage of large molecules.
In addition to what Christian answered, protein expression in soluble form can be achieved altering protein induction conditions to 16-20 hours at 20°C instead of normally 37°C.
Treatments examples you've asked:
Chemical: Detergens such Triton-X100, NP-40, Tween 20
Enzimatic: Lysozyme
Physical: Frech Press, Sonication
For extraction: Try using detergent such Triton-X100 (0,5% to 2%) and lysozyme (1 mg/mL) in your extraction buffer followed by mild sonication to release proteins from bacteria.
Here are some references you may be interested in:
- Kim et al 2013 - PlosOne (doi:10.1371/journal.pone.0056168)
- Johnson et al 2013 - MaterMethods (http://dx.doi.org/10.13070/mm.en.3.163)
- Harper 2011 - Methods in Molecular Biol (doi: 10.1007/978-1-60761-913-0_14)
- AMERSHAM handbook - http://sevierlab.vet.cornell.edu/resources/GE_Recombinant_protein.pdf
Good luck!
If you want to isolate the perispalic proteins gently, usually a hypoosmotic shock is used. Again, I am a Gram-positive person, but our last years iGEM team used the following protocol:
http://2013.igem.org/Team:Bielefeld-Germany/Labjournal/Molecular#Cold_osmotic_shock
Depends on protein localization, intrcellular or extracellular.
That iGEM team assay is realy simple, and i think it will work without harming my proteins. Thanks guys for help.
could you guys give me an example protein that is not secreted in E.coli, or has problems to be secreted, but that works well in gram positive bacteria? Thank you
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