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 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 tag for tat pathway, anchors in periplasm BBa_K1012002 Dundee 2013
phoA tag for sec pathway, anchors to OM BBa_K808028 Darmstadt 2011
malE tag for sec pathway, exports to periplasm BBa_K1012004 Dundee 2013

Assembly methods and toolkit:

A problem with standard biobrick assembly is that the scar site contains a STOP codon, therefore 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, BBa_K1149021.

References

  1. Economou A. Following the leader: bacterial protein export through the Sec pathway. Trends in microbiology 1999;7(8) 315-320.
  2. Thanassi DG, Hultgren SJ. Multiple pathways allow protein secretion across the bacterial outer membrane. Current opinion in cell biology 2000;12(4) 420-430.
  3. Sletta H, Tondervik A, Hakvag S, Aune TEV, Nedal A, Aune R, et al. The presence of N-terminal secretion signal sequences leads to strong stimulation of the total expression levels of three tested medically important proteins during high-cell-density cultivations of E.coli. Applied and Environmental Microbiology 2007;73(3) 906-912.
  4. Qian Z, Xia X, Choi JH, Lee SY. Proteome-based identification of fusion partner for high-level extracellular production of recombinant proteins in E.coli. Biotechnology and bioengineering 2008;101(3) 587-601.
  5. Prehna G, Zhang G, Gong X, Duszyk M, Okon M, McIntosh LP, et al. A Protein Export Pathway Involving Escherichia coil Porins. Structure 2012;20(7) 1154-1166.
  6. Binet R, Letoffe S, Ghigo JM, Delepelaire P, Wandersman C. Protein secretion by Gram-negative bacterial ABC exporters - A review. Gene 1997;192(1) 7-11.
  7. Chung CW, You J, Kim K, Moon Y, Kim H, Ahn JH. Export of recombinant proteins in E.coli using ABC transporter with an attached lipase ABC transporter recognition domain (LARD). Microbial Cell Factories 2009;8 11.

Our Sponsors

TueSponsorsEppendorf.png Logo-bioline.gif Invitrogen.jpg Geneart.jpg CSynBI.JPG