Oct 27, 2020

Public workspaceProtein expression on the surface of Escherichia coli

DOI
dx.doi.org/10.17504/protocols.io.bfh4jj8w
  • 1Technical University of Denmark
Maja Rennig
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DOI: dx.doi.org/10.17504/protocols.io.bfh4jj8w
Protocol Citation: Maja Rennig, Kristoffer Bach Falkenberg, Cristina Hernandez Rollan, Andreas Birk Bertelsen, Morten Norholm 2020. Protein expression on the surface of Escherichia coli. protocols.io https://dx.doi.org/10.17504/protocols.io.bfh4jj8w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 24, 2020
Last Modified: October 28, 2020
Protocol Integer ID: 36124
Abstract
Display of proteins on the bacterial cell surface has always been an attractive technique for the production of functional cell-anchored proteins, thereby reducing the cost, time and effort related to enzyme purification. Thus, surface display can enable fast and easy screening of protein libraries, e.g. activity variants, or screening of different enzyme substrates, while maintaining a connection between the phenotype and the genotype. Additionally, cells displaying functional enzymes can potentially be used as whole-cell catalysts.

This protocol describes how to express proteins on the surface of E. coli BL21 (DE3) using a L-rhamnose inducible expression system.


Guidelines
Display of proteins on the bacterial cell surface is very attractive for fast and easy screening of protein variants, because it maintains a connection between the phenotype and the genotype. For some enzymes to be active, it is crucial whether they are cell-anchored via the C- or N-terminus. The choice of display construct is therefore of great importance. In our lab, we use two different constructs:
pBAD42-LppSP-OmpA-TEV-ccdB and pBAD42-ccdB-TEV-C-IgAP

The constructs are adapted from Wendel et al. (2016) A nanobody:GFP bacterial platform that enables functional enzyme display and easy quantification of display capacity. Microb Cell Fact 15:71, DOI 10.1186/s12934-016-0474-y
Materials
MATERIALS
ReagentBL21(DE3) Competent E.coli - 6x0.2 mlNew England BiolabsCatalog #C2527I
ReagentTrizma® baseMerck MilliporeSigma (Sigma-Aldrich)Catalog #93350
ReagentKanamycinResearch Products International Corp (RPI)Catalog #K22000-25.0
ReagentLBResearch Products International Corp (RPI)Catalog #L24400-2000.0
ReagentL-rhamnoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #W373011-100G-K
Safety warnings
This protocol describes the handling of GMO classified organisms. Make sure that the local GMO and safety legislations are respected.
Before start
Prepare a fresh transformation of your expression vector in E. coli BL21 (DE3) cells.
Pre culture - Day 1
Pre culture - Day 1
Pick a fresh colony of your BL21 (DE3) strain with your expression vector, and inoculate it in LB supplemented with relevant antibiotics. Grow the culture at Temperature37 °C at 250 RPM shaking DurationOvernight . The volume of the overnight culture depends on the volume of the expression culture and should be at least 1/100 of the expression culture
Inoculation, Induction and expression - Day 2
Inoculation, Induction and expression - Day 2
Dilute the overnight culture 1:100 in fresh LB supplemented with relevant antibiotics
Grow the culture at Temperature37 °C with 250 RPM shaking until an OD600 = 0.5 - 0.6

Induce expression by adding L-rhamnose to a final concentration of Concentration5 millimolar (mM)

Let the culture grow at Temperature30 °C with 250 RPM shaking for up to Duration20:00:00

Assaying surface displayed proteins - Day 3
Assaying surface displayed proteins - Day 3
Spin the culture down at Centrifigation4000 x g, 4°C, 00:05:00 and discard the supernatant
Resuspend pellet in the desired assay buffer (e.g. Concentration10 millimolar (mM) - Concentration50 millimolar (mM) Tris-HCl)

Run activity assay in liquid or on agar plates.
Alternatively: Shaving-off of surface displayed proteins - Day 3
Alternatively: Shaving-off of surface displayed proteins - Day 3
Spin the culture down at Centrifigation4000 x g, 4°C, 00:05:00 and discard the supernatant
Resuspend pellet in Concentration10 millimolar (mM) Tris-HCl or Amount100 µL /ODU TEV cleavage buffer (Concentration10 millimolar (mM) Tris-HCl Ph7.5 , Concentration150 millimolar (mM) NaCl2, Concentration0.5 millimolar (mM) EDTA)



Add purified and activated TEV protease
Incubate at TemperatureRoom temperature DurationOvernight (~Duration16:00:00 )
Note
It is also possible to shorten the incubation time, so that the protein harvest can still be performed on the same day.




Protein harvest - Day 4
Protein harvest - Day 4
Spin the cells at Centrifigation5000 x g, 4°C, 00:10:00 and collect the supernatant
Keep the extractions TemperatureOn ice when working with it and at Temperature4 °C for storage