TITLE: E. coli protein expression and purification
AUTHORS: Diep R. 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 enzyme assays, protein crystallography etc.</div></div>

[STEPS]
?. [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).
?. [Protein expression]
Innoculate bacterial colony from selective media into LB + antibiotic of choice. Use p100-200 pipette tip to scrape colony and drop into 3-5 mL broth in 10 mL culture tube. Grow O/N @ 37 ºC with ~200 rpm shaking.
?. [Protein expression]
Innoculate larger culture using the starter culture generated from Step 3 at 1:100 dilution (e.g. 0.5 mL in 50 mL LB + antibiotic). Larger culture volume will depend on what you plan to do with the protein. For example, for large-scale protein production you may use 1 - 2 L cultures - ensure you use an appropriate vessel to allow appropriate aeration (see suggestions in guidelines). For smaller scale tests for protein induction and solubility, you may prefer 10 - 15 mL cultures in 50 mL falcon tubes.
?. [Protein expression]
Grow larger culture at 37 ºC and check OD600 after 2.5 - 3 hours (times may vary depending on total culture volume and quality of cells).
?. [Protein expression]
When OD600 is between 0.6 - 0.8, take an aliquot of culture (up to 15 mL) as non-induced control. To the remaining culture, add induction media. We typically use BL21 (DE3) derived strains and, thus, add 100 mM IPTG to achieve [IPTG]final = 1 mM (i.e. 1:100 dilution, 100 μL IPTG to 10 mL culture).
?. [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.
?. [Protein expression]
Grow cultures for approx. 5 hours, then check OD for difference between induced vs non-induced. Non-induced should be higher by at least 0.1.
?. [Store cells for purification]
If you are confident with your protein induction (see QC) and want to preserve the cell culture for protein purification, spin down cells at 4 ºC (~7000  rcf for 5 minutes with gentle/no brake). Remove supernatant.
?. [QC protein induction]
If you are running small scale test induction (e.g. testing constructs, induction, protein solubility), we can run SDS-page gels on the crude lysate (~1-2 mL culture) before trying larger cultures. With larger cultures, you may want to take an aliquot of the culture to check induction before proceeding with purification. If you are confident, proceed to step 21 for washing and storage.
?. [Prepare buffers]
10X PBS:    - Dissolve the following in 800ml distilled H2O.        - 80 g of NaCl        - 2.0 g of KCl        - 14.4 g of Na2HPO4        - 2.4 g of KH2PO4- 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.Resuspension buffer:50 mM Tris-HCl pH 82 mM EDTA1X Binding Buffer:50 mM NaH2P04, pH 8300 mM NaCl10 mM Imidazole1X Wash Buffer:50 mM NaH2P04, pH 8300 mM NaCl20 mM Imidazole1X Elute Buffer:50 mM NaH2P04, pH 8300 mM NaCl250 mM ImidazoleDigestion / Storage buffer:50 mM Tris-HCl pH 8150 mM NaCl20 mM KCl
?. [Transformation]
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]
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.
?. [QC protein induction]
Spin down culture (~1 - 3 mL)  at max speed for 3 minutes and remove supernatant. This pellet can be stored O/N @ -20 ºC until you are ready to proceed with checking induction of your expressed recombinant protein.
?. [QC protein induction]
Resuspend cells in 100 μL 1X PBS (per 1 mL culture).
?. [QC protein induction]
Calculate how much crude lysate to load onto gel based on OD (μL = 180/OD).
?. [QC protein induction]
To these volumes, add the appropriate amount of 4X LDS (or required SDS-PAGE sample buffer), DTT (final concentration = 50 mM) and MgCl2 (final concentration = 100 mM). These should be calculated first, and a mastermix of LDS, DTT and MgCl2 can be prepared and added to samples accordingly.
?. [QC protein induction]
Heat sample @ 72 ºC for 10 minutes on heat transfer block.
?. [QC protein induction]
Place samples on ice for 5 minutes, then spin for 15 minutes max speed.
?. [QC protein induction]
Transfer supernatant to new tubes, taking care to avoid any "sticky" DNA coating the tube.
?. [QC protein induction]
Run supernatant on SDS-PAGE gel (160V, 40-45 minutes).
?. [QC protein induction]
Stain gel with Coomasie or Gel Code Blue (or other stain of choice). Alternatively, perform Western blot if antibodies are available or recombinant proteins are epitope tagged (e.g. MYC).
?. [Protein purification]
Take stored cells out of -80 ºC and let thaw on ice.
?. [Store cells for purification]
Wash cells in resuspension buffer, spin down and remove supernatant.
?. [Store cells for purification]
Snap-freeze pellet in LN2 and store at -80 ºC. Likely, this will be done pre-emptively until results from the QC are obtained, then cells can be discarded or retained accordingly.This is a safe stopping point where you can continue with protein purification as needed.