Jun 19, 2020
Version 5
Heterologous protein expression in E. coli V.5
- Diep R Ganguly1,
- Timothy Rhodes2,
- Nay Chi Khin2,
- Estee E Tee2,
- Kai Xun Chan2
- 1University of Pennsylvania;
- 2The Australian National University
- Pogson Group
Protocol Citation: Diep R Ganguly, Timothy Rhodes, Nay Chi Khin, Estee E Tee, Kai Xun Chan 2020. Heterologous protein expression in E. coli. protocols.io https://dx.doi.org/10.17504/protocols.io.bdjti4nn
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: March 12, 2020
Last Modified: June 19, 2020
Protocol Integer ID: 34131
Keywords: heterologous protein expression, E. coli
Abstract
Protocol for recombinant protein expression in E. coli for protein purification and subsequent enzyme assays, protein crystallography etc.
Guidelines
This protocol will take a few days so be sure to have all buffers, cell strains, and plasmids on hand. Different sections do not need to be performed immediately after each other - there are various safe stopping steps where cells can be stored at -20/-80 ºC until you are ready to continue. However, for convenience, the entire protocol is described here.
Adjust volumes, taking care to ensure appropriate vessels are used to allow proper aeration (e.g. grow 800 mL culture in 2 L flasks or 2 L culture in 5 L flasks), depending on the desired downstream application and expected protein yield. We commonly use BL21 (DE3) strains for T7 expression (i.e. IPTG induction).
Materials
MATERIALS
Potassium chlorideP212121
Petri DishP212121Catalog #LI-PD01100
Lysozyme from chicken egg whiteMerck MilliporeSigma (Sigma-Aldrich)Catalog #L6876
Luria-Bertani (LB) broth, makes 1LAmrescoCatalog #K488
EDTA
cOmplete™, EDTA-free Protease Inhibitor CocktailMerck MilliporeSigma (Sigma-Aldrich)Catalog #05056489001
1.5 mL Eppendorf tubes
Electroporation System Gene Pulser XCellBio-Rad Laboratories
37˚C Incubator
DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632
14ml Polystyrene Cell Culture TubesAlkali ScientificCatalog #CT5250
4X Bolt LDS Sample BufferInvitrogen - Thermo FisherCatalog #B0007
NaClMerck MilliporeSigma (Sigma-Aldrich)Catalog #53014
IPTGBio Basic Inc.Catalog #IB0168.SIZE.100g
BL21(DE3) or BL21-Star(DE3) or Rosetta2(DE3) or etc for protein purification
Magnesium chloride hexahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M2670
Electroporation Cuvette 1mm Bio-Rad LaboratoriesCatalog #1652089
Falcon® Conical Tubes, 50 mL 500 Tubes
STEMCELL Technologies Inc.Catalog #38010
Tris-HClLife TechnologiesCatalog #AM9855
28°C incubator without CO2Thermo Fisher Scientific
Disodium phosphateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S7907
Monopotassium phosphateMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9791
42°C water bath
ImidazoleMerck MilliporeSigma (Sigma-Aldrich)Catalog #I5513
UV/Vis spectrophotometer
GelCode™ Blue Stain ReagentThermo Fisher ScientificCatalog #24590
Q125 SonicatorCatalog #Part #Q125
Safety warnings
Ensure use of appropriate asceptic technique. Use caution if using a bunsen burner and ethanol.
Before start
Make sure you have your verified plasmid transformed into your desired E. coli strain for protein expression e.g. BL21 Star (DE3). These should be plated on selective LB media to produce positive colonies for starter cultures. Prepare all the buffers described in Step 1, except make fresh IPTG stocks. Add DTT to buffers prior to use.
Prepare buffers
Prepare buffers
Buffer recipes
Auto-induction TB medium (alternatively, liquid LB media for IPTG induction)
- 5 g/L yeast extract
- 20 g/L tryptone
- 85.5 mM NaCl (5 g/L)
- 22 mM KH2PO4 (2.99 g/L)
- 42 mM Na2HPO4 (5.96 g/L)
- Supplement fresh: 0.6% glycerol, 0.05% glucose, 0.2% lactose
10X PBS
Dissolve the following in 800 mL H2O:
- 80 g of NaCl (1.37 M)
- 2.0 g of KCl (27 mM)
- 14.4 g of Na2HPO4 (100 mM)
- 2.4 g of KH2PO4 (18 mM)
Adjust pH to 7.4.
- Add H2O to 1L.
- Autoclave
Store 10X stock at 4 °C from which you can dilute 1:10 to make 1X working stock to keep at room temp. Make PBS-T by supplementing with 0.05 - 0.1% Tween20.
Can replace with TBS-T http://cshprotocols.cshlp.org/content/2015/3/pdb.rec085670.full?text_only=true.
Re-suspension buffer
- 50 mM Tris-HCl pH 8
- 2 mM EDTA
Cell lysis buffer
- 50 mM NaH2PO4 pH 8 (or ~1-2 pH units away from pI of expressed protein)
- 500 mM sodium chloride
- 10 mM imidazole
- 0.5% Triton X-100
- 10% glycerol
- 2 mM DTT (add fresh before use)
- 1x Protease-inhibitor cocktail (add fresh before use)
Denaturing buffer
- 8 M Urea
- 4% CHAPS
- 35 mM Tris-HCl pH 8
- Adjust pH to 7.5 if necessary for dissolving urea (or warm to 40 °C).
Transformation
Transformation
12h
12h
Transform desired E. coli cell strain with plasmid to be expressed using desired method (e.g. heat shock or electroporation depending on type of competency).
For electrocompetent cells:
- Add 0.5 - 1 µL purified plasmid to 50 µL cells (thawing on ice, 15 minutes)
- Gently flick with finger to mix
- Transfer mixer to chilled electroporation cuvette ensuring there are no bubbles. Keep on ice until ready to electroporate
- Set machine to 1.8 kV, 25 µF, 200-400 Ω
- Dry the outside of the cuvette and place into electroporation chamber.
- Prepare p1000 and p200 pipettes to be ready
- Close chamber and electroporate
- Immediately remove cuvette and add 1 mL LB. Transfer contents to microfuge tube using both p1000 and p200.
- Let cells recover at 37 ºC with ~200 rpm shaking for > 1 hour.
For chemically competent cells:
- Add 0.5 - 1 µL purified plasmid to 50 µL cells (thawing on ice, 15 minutes)
- Gently flick with finger to mix
- Sit on ice for 30 minutes, set water bath to heat to 42 ºC
- Depending on cells, incubate in water bath for 30 - 90 seconds.
- Return to ice for 5 minutes
- Add 1 mL LB and let cells recover at 37 ºC with ~200 rpm shaking for > 1 hour.
1h
Plate recovered transformed cells (~100 µL of transformed cells) onto selective LB media and grow O/N @ 37 ºC. Adjust volume as needed in order to obtain single colonies that can be picked for subsequent innoculation.
12h
Pick single bacterial colony and inoculate culture ( 3-5 mL LB or TB medium) supplmented with the appropriate antibiotic. Grow O/N @ 37 ºC with ~200-250 rpm shaking.
12h
Protein expression
Protein expression
Setup desired culture volume for protein expression with auto-induction TB medium (or just LB medium if using standard IPTG induction). An additional control culture can be setup as a control that lacks lactose (or will have not have IPTG added). Inoculate larger culture using the starter culture generated from the previous step at 1:50 dilution (e.g. 1 mL in 49 mL culture). Ensure culture is in an appropriate vessel that facilitates effective aeration.
1m
Induce protein expression, e.g. using IPTG to induce T7 promoter:
- If using LB culture with IPTG induction, grow larger culture at 37 ºC until OD600 = 0.8. Then supplement the culture with 0.2 - 0.4 mM IPTG (keep an aliquot of culture without IPTG). Grow cultures overnight at desired temperature (e.g. lower temperature can imrpove protein folding and solubility e.g. 18-25 ºC ). If desired, Measure OD600 for difference between induced vs non-induced. Non-induced should be higher by at least 0.1 (minimum difference).
- If using auto-induction TB medium, grow culture overnight (>12 hrs) or until OD600 = 2. Repeated sampling over time can be performed to ensure induction of expected protein fragment.
12h
Pellet cells by centrifugation at 4 ºC (7,000 rcf for 10 minutes). If you want to run a quick test for protein expression, remove a 1-3 mL aliquot of culture into a clean microfuge tube and store at -20 ºC.
5m
Remove supernatant and wash cells in re-suspension buffer. Pellet as above and discard supernatant. Snap-freeze pellet in LN2 and store at -80 ºC.
QC protein expression
QC protein expression
It may be worthwhile testing for successful recombinant protein expression on an aliquot of induced culture (Step 7).
Pellet culture by centrifugation at max speed for 3 minutes then remove supernatant.
4m
Resuspend cells in 100 μL 1X PBS (per 1 mL culture). Store the resuspended crude lysate at -20 °C when not in use.
10m
Add the appropriate amount of:
- 4X LDS (or appropriate SDS-PAGE sample buffer)
- [DTT]final = 50 mM, and
- [MgCl2]final = 100 mM.
These should be calculated first, and a mastermix of LDS, DTT and MgCl2 can be prepared then added to each sample accordingly.
30m
Calculate how much crude lysate to load, based on OD600 and concentration factor (CF):
CF = volume of culture / volume of resuspension
For example, CF for resuspending 1 mL culture in 100 μL 1X PBS = 1,000 / 100 = 10x)
μL to load = [180/CF]/OD
20m
Heat sample @ 72 ºC for 10 minutes in water bath.
10m
Place samples on ice for 5 minutes then spin for 15 minutes at max speed.
15m
Transfer supernatant to new tubes, taking care to avoid any "sticky" DNA coating the tube.
10m
Run supernatant on SDS-PAGE gel and perform Coomassie staining. Alternatively, transfer to PVDF membrane and perform Western blot analysis (ideally, recombinant protein is epitope-tagged).
3h
Solubilisation
Solubilisation
To isolate soluble protein, re-suspend pelleted cells in lysis buffer.
Note: ensure pH of lysis buffer is >1 pH units away from the isoelectric point of your expressed protein (calculate: http://isoelectric.org/index.html).
Keep samples on ice at all times.
20m
Transfer cell re-suspension to clean microfuge tube (or appropriate vessel for probe sonication). Retain any remaining cell suspension at -20 °C.
10m
Sonicate samples with the Q125 ultrasonicator (125 watt, 20 kHz, 60% amp, 3x30 seconds).
Keep samples on ice during and between rounds of sonication.
30m
Spin at 16,000g for 30 minutes at 4 °C.
30m
Recover supernatant in a clean tube. Retain the pellet at -20 °C to for insoluble protein fraction.
10m
To isolate the insoluble fraction; resuspend the remaining pellet in denaturing buffer .
Repeat the sonication and centrifugation steps as per the soluble fraction. Recover supernatant in a clean tube.
1h
Perform a Bradford assay to determine protein concentration in the soluble and insoluble fractions. Use these concentrations to normalise input volumes for each sample to a desired loading amount (e.g. 5 µg).
2h
Run samples on SDS-PAGE and stain gel with Coomassie or perform Western blot to verify expression.
3h