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Team:Imperial College/SecretionHelp
From 2013.igem.org
Secretion Guide
Alternative strategies for protein secretion in E.coli:
N terminal secretion-signal peptides are recognised by the sec pathway. The pathway transports proteins to the periplasm and additional mechanisms are usually required to further export the protein to the extracellular space. An approach for using this pathway is the addition of an N terminal signal peptide to the target protein which can obtain as high as 90% efficiency in secretion to the extracellular space. Such signals are pelB and phoA tags which are available as biobricks. PelB can be cleaved off by pelB peptidase in the periplasm whereas phoA will anchor your protein to this localisation.
Fusion partners are endogenous proteins that are naturally secreted in E.coli and can be fused to the target protein. Some such proteins were demonstrated to be a powerful carriers of medically relevant human proteins in E.coli. The yebF and ompF proteins exit the cell via the sec pathway and use additional mechanisms for leaving the periplasm, where they interact with outer-membrane porins for extracellular secretion. The osmY is available as a Biobrick and we have submitted ompF this year.
The porin proteins such as ompF and ompA, can be used as fusion partners too and anchor proteins to the outer surface of E.coli.
ABC transporters use ATP for transport of specific proteins across the bacterial membrane. The ABC transporter of Erwinia chrysanthemi can be expressed in E.coli and export proteins that contain the LARD1 domain as a C terminal fusion. The system is biobricked in two separate plasmids (transporter, LARD1) with different antibiotic resistance and it is important to use both at the same time.
You can find a list of localisation tags in the registry [http://parts.igem.org/Protein_domains/Localization here]. It contains both eukaryotic and prokaryotic localisation signals. We have put together the tables below to help you choose an E.coli secretion strategy that is suitable for your project.
Extracellular Secretion in E.coli:
| Description | Biobrick | Team | |
|---|---|---|---|
| pelB | type II secretion signal peptide, cleaved off in periplasm | BBa_K208004 BBa_K1149022 BBa_K208004 BBa_J32015 | Imperial 2013 |
| yebF | large fusion-protein, Sec-pathway and additional mechanism | BBa_K1149001 | Imperial 2013 |
| osmY | large fusion-protein, Sec-pathway and additional mechanism | BBa_K892008 | Washington 2012 |
| LARD 1 | Lipase ABC transporter recognition domain | BBa_K258001 | METU-GEne 2009 |
Outer Membrane Anchors and Periplasmic Expression in E.coli:
| Description | Biobrick | Team | |
|---|---|---|---|
| ompF | porin protein that can be fused to a protein, anchors to outer membrane | BBa_K864204 | Uppsala 2012 |
| ompA | porin protein that can be fused to a protein, anchors to outer membrane | BBa_K103006 | Wasraw 2008 |
| INP | short domain,anchors in periplasm | BBa_K523013 (INP-YFP) BBa_K632002 (INP-Silicatein) | Minnesota 2011 Edinburgh 2011 |
| torA | anchors in periplasm | Dundee 2013 | |
| phoA | tag for sec pathway, anchors to OM | BBa_K808028 | Darmstadt 2011 |
Assembly methods and toolkit:
A problem with standard biobrick assembly is that the scar site contains a STOP codon, therefore [http://parts.igem.org/Protein_domains alternative strategies are needed]. One strategy is to use Infusion/Gibson assembly. We have constructed a biobrick where LARD1 is after a constitutive promoter and RBS in order to facilitate quicker cloning with less assembly steps, [http://parts.igem.org/Part:BBa_K1149021 BBa_K1149021].
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