TITLE: Heterologous protein expression in E. coli
AUTHORS: Diep Ganguly, Timothy Rhodes, Nay Chi Khin, Estee E Tee, Kai Xun Chan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for recombinant protein expression in E. coli for protein purification and subsequent enzyme assays, protein crystallography etc.</div></div>

[STEPS]
?. [Prepare buffers]
Buffer recipesAuto-induction TB medium (alternatively, liquid LB media for IPTG induction)5 g/L yeast extract20 g/L tryptone85.5 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% lactose10X PBSDissolve 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.AutoclaveStore 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 buffer50 mM Tris-HCl pH 82 mM EDTACell lysis buffer50 mM NaH2PO4 pH 8 (or ~1-2 pH units away from pI of expressed protein)500 mM sodium chloride10 mM imidazole0.5% Triton X-10010% glycerol2 mM DTT (add fresh before use)1x Protease-inhibitor cocktail (add fresh before use)Denaturing buffer8 M Urea4% CHAPS35 mM Tris-HCl pH 8Adjust pH to 7.5 if necessary for dissolving urea (or warm to 40 °C).10X PBSDissolve 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.AutoclaveStore 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 buffer50 mM Tris-HCl pH 82 mM EDTACell lysis buffer50 mM NaH2PO4 pH 8 (or ~1-2 pH units away from pI of expressed protein)500 mM sodium chloride10 mM imidazole0.5% Triton X-10010% glycerol2 mM DTT (add fresh before use)1x Protease-inhibitor cocktail (add fresh before use)Denaturing buffer8 M Urea4% CHAPS35 mM Tris-HCl pH 8Adjust pH to 7.5 if necessary for dissolving urea (or warm to 40 °C).Adjust pH to 7.4.Add H2O to 1L.AutoclaveStore 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 buffer50 mM Tris-HCl pH 82 mM EDTACell lysis buffer50 mM NaH2PO4 pH 8 (or ~1-2 pH units away from pI of expressed protein)500 mM sodium chloride10 mM imidazole0.5% Triton X-10010% glycerol2 mM DTT (add fresh before use)1x Protease-inhibitor cocktail (add fresh before use)Denaturing buffer8 M Urea4% CHAPS35 mM Tris-HCl pH 8Adjust pH to 7.5 if necessary for dissolving urea (or warm to 40 °C).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 buffer50 mM Tris-HCl pH 82 mM EDTACell lysis buffer50 mM NaH2PO4 pH 8 (or ~1-2 pH units away from pI of expressed protein)500 mM sodium chloride10 mM imidazole0.5% Triton X-10010% glycerol2 mM DTT (add fresh before use)1x Protease-inhibitor cocktail (add fresh before use)Denaturing buffer8 M Urea4% CHAPS35 mM Tris-HCl pH 8Adjust pH to 7.5 if necessary for dissolving urea (or warm to 40 °C).Cell lysis buffer50 mM NaH2PO4 pH 8 (or ~1-2 pH units away from pI of expressed protein)500 mM sodium chloride10 mM imidazole0.5% Triton X-10010% glycerol2 mM DTT (add fresh before use)1x Protease-inhibitor cocktail (add fresh before use)Denaturing buffer8 M Urea4% CHAPS35 mM Tris-HCl pH 8Adjust pH to 7.5 if necessary for dissolving urea (or warm to 40 °C).Denaturing buffer8 M Urea4% CHAPS35 mM Tris-HCl pH 8Adjust pH to 7.5 if necessary for dissolving urea (or warm to 40 °C).
?. [Transformation]
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.
?. [Transformation]
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.
?. [Transformation]
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.
?. [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.
?. [Protein expression]
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.
?. [Protein expression]
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.
?. [Protein expression]
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]
It may be worthwhile testing for successful recombinant protein expression on an aliquot of induced culture (Step 7).
?. [QC protein expression]
Pellet culture  by centrifugation at max speed for 3 minutes then remove supernatant.
?. [QC protein expression]
Resuspend cells in 100 μL 1X PBS (per 1 mL culture). Store the resuspended crude lysate at -20 °C when not in use.
?. [QC protein expression]
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.
?. [QC protein expression]
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
?. [QC protein expression]
Heat sample @ 72 ºC for 10 minutes in water bath.
?. [QC protein expression]
Place samples on ice for 5 minutes then spin for 15 minutes at max speed.
?. [QC protein expression]
Transfer supernatant to new tubes, taking care to avoid any "sticky" DNA coating the tube.
?. [QC protein expression]
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).
?. [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.
?. [Solubilisation]
Transfer cell re-suspension to clean microfuge tube (or appropriate vessel for probe sonication). Retain any remaining cell suspension at -20 °C.
?. [Solubilisation]
Sonicate samples with the Q125 ultrasonicator (125 watt, 20 kHz, 60% amp, 3x30 seconds). Keep samples on ice during and between rounds of sonication.
?. [Solubilisation]
Spin at 16,000g for 30 minutes at 4 °C.
?. [Solubilisation]
Recover supernatant in a clean tube. Retain the pellet at -20 °C to for insoluble protein fraction.
?. [Solubilisation]
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.
?. [Solubilisation]
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).
?. [Solubilisation]
Run samples on SDS-PAGE and stain gel with Coomassie or perform Western blot to verify expression.