Studier Method for Autoinduction of Protein Expression in the T7 System

This protocol describes the production of recombinant proteins in bacteria using the T7 polymerase expression system originally developed by F. W. Studier and colleagues (often known as the pET system, sold by Novagen (PDF), but available from other suppliers such as Promega, Stratagene, and Invitrogen). This protocol uses a method developed by Studier that provides for “autoinduction” of protein expression without the need to add inducers such as IPTG during mid-log phase of the cultures. The method is based upon a buffered medium that contains a mixture of carbon sources, including lactose. The bacteria initially use glucose; when the glucose is exhausted, lactose can enter the cell and induce expression of the T7 polymerase from the DE3 lambda lysogen. The medium provides high culture densities and high yields of proteins, usually in inclusion bodies. It is used extensively by the Tetramer Core Facility for the production of MHC subunits used during the preparation of MHC tetramers, but may also be used for the expression of other recombinant proteins that are ultimately driven by the lac promoter.

Innoculum cultures are grown in P-0.5G, a defined minimal medium for growth to saturation at high densities with little or no induction of expression of the target protein. According to Studier:

Cultures grown (in P-0.5G) at 37 °C typically saturate overnight at A600 ~ 4-7 and pH ~6.5, are quite stable for a week or more in the refrigerator, and grow subcultures with little lag. Fresh overnight cultures in P-0.5G make freezer stocks that remain viable indefinitely and generate cultures that produce high levels of target protein.

Autoinduction cultures are grown overnight (16-20 hours) in ZYP-5052 media.

Additional details, protocols, and recipes for minimal defined media (suitable for metabolic labeling of recombinant proteins with seleno-methionine) are available. We do not require these for our purposes.

Table of Contents

Reagents and stock solutions

Reagent List

ZY

20x NPS

Component 100 ml 1 liter mol/liter
dd H2O 90 ml 900 ml -
(NH4)2SO4 6.6 g 66 g 0.5 M
KH2PO4 13.6 g 136 g 1 M
Na2HPO4 14.2 g 142 g 1 M

50x5052:  (5052 = 0.5 % glycerol, 0.05% glucose, 0.2% α-lactose)

Component 100 ml 1 liter
Glycerol (weigh in beaker) 25 g 250 g
H2O 73 ml 730 ml
Glucose (Fisher FLBP350-1) 2.5 g 25 g
a-lactose (Sigma cat# L3625) 10 g 100 g

1 M MgSO4

40% glucose (w/v)

Component 100 ml 300 ml
Glucose (Fisher FLBP350-1) 40 g 120 g
H2O 74 ml 222 ml

80% glycerol (v/v) (= 100% w/v)

20% α-lactose (w/v)

Component 100 ml 600 ml
a-lactose (Sigma cat# L3625) 20 g 120 g
H2O 87.5 ml 525 ml

1000x trace metals mixture (100 ml in ~50 mM HCl)

Prepare all metal stock solutions in ddH2O, except for FeCl3, which is dissolved in ~0.1M HCl, as noted in the table below. Combine the metal solutions as in the table below.

Component Vol MW 1x conc
H2O 36 ml - -
0.1 M FeCL3•6H2O
(dissolved in ~0.1 M HCl = 100-fold dil of conc HCl)		 50 ml 270.30 50 µM Fe
1M CaCl2 2 ml 110.99 20 µM Ca
1M MnCl2•4H2O 1 ml 197.91 10 µM
1 M ZnSO4•7H2O 1 ml 287.56 10 µM Zn
0.2 M CoCl2•6H2O 1 ml 237.95 2 µM Co
0.1 M CuCl2•2H2O 2 ml 170.486 2 µM Cu
0.2 M NiCl2•6H2O 1 ml 237.72 2 µM Ni
0.1 M Na2MoO4•2H2O 2 ml 241.98 2 µM Mo
0.1 M Na2SeO3•5H2O 2 ml 263.03 2 µM Se
0.1 M H3BO3 2 ml 61.83 2 µM H3BO3

Autoclave the stock solutions of the individual metals, except 0.1 M FeCl3 in 1/100 volume conc HCl.

A brief precipitate appeared upon addition of Na2SeO3, which redissolved rapidly

Store at room temperature

When making growth media, add the metals mix before NPS.  If NPS is already present when 1000x metals mix is added, a precipitate forms which disperses but retains a light turbidity.  If the metals are diluted to near their final concentration before NPS is added, the medium remains clear.  The metals also precipitate and disperse or redissolve when added to ZY, a precipitate caused by yeast extract.  Although apparently not a problem, the precipitate could be avoided by diluting the metals in the water before dissolving the yeast extract in making ZY.

Antibiotics

P-0.5G defined minimal medium for growth to saturation with little or no induction

Component 50 ml 100 ml 200 ml Final concentration
ddH2O (sterile) ~46.8 ml ~93.6 ml ~187.3 ml -
1 M MgSO4 50 µl 100 µl 200 µl 1 mM
10000x metals mix 5 µl 10 µl 20 µl 1
40% glucose 0.625 ml 1.25 ml 2.5 ml 0.5%
20x NPS 2.5 ml 5 ml 10 ml 1x
antibiotics Choose only 1 from the list below
kanamycin (25 mg/ml) 0.2 ml 0.4 ml 0.8 ml 100 µg/ml
chloramphenicol (25 mg/ml) 50 µl 100 µl 200 µl 25 µg/ml
ampicillin (50 mg/ml) 50 µl 100 µl 200 µl 50 µg/ml

ZYP-0.8G

Component 50 ml 100 ml 200 ml 400 ml Concentration
ZY ~46.5 ml ~93 ml ~186 ml ~372 ml -
1 M MgSO4 50 µl 100 µl 0.2 ml 0.4 ml 1 mM
40% glucose 1 ml 2 ml 4 ml 8 ml 0.8%
20x NPS 2.5 ml 5 ml 10 ml 20 ml 1x
Antibiotics, as needed
Choose only 1 from the list below
kanamycin (25 mg/ml) 200 µl 0.4 ml 0.8 ml 1.6 ml 100 µg/ml
chloramphenicol (25 mg/ml) 50 µl 100 µl 0.2 ml 0.4 ml 25 µg/ml
ampicillin (50 mg/ml) 50 µl 100 µl 0.2 ml 0.4 ml 50 µg/ml

ZYP-5052 rich medium for auto-induction

Component 200 ml 400 ml 500 ml 1 liter Concentration
ZY ~186 ml ~372 ml ~464 ml ~928 ml -
1 M MgSO4 0.2 ml 0.4 ml 0.5 ml 1 ml 1 mM
50x 5052 4 ml 8 ml 10 ml 20 ml 1x
20x NPS 10 ml 20 ml 25 ml 50 ml 1x
Antibiotics Choose only 1 from the list below
kanamycin (25 mg/ml) 0.8 ml 1.6 ml 2 ml 4 ml 100 µg/ml
chloramphenicol (25 mg/ml) 0.2 ml 0.4 ml 0.5 ml 1 ml 25 µg/ml
ampicillin (50 mg/ml) 0.2 ml 0.2 ml 0.5 ml 1 ml 50 µg/ml

LB Agar plates + 1% glucose

Resuspension Buffer

Procedure

  1. At the end of day 1, streak plasmid-transformed BL21(DE3) bacteria  from a frozen glycerol stock onto an LB + 1% glucose agar plate with an appropriate antibiotic. Place in a 37°C incubator overnight.

    Alternate strains carrying carrying the T7 polymerase under control of the lacUV5 promoter in the DE3 lambda lysogen may be used. Examples include the BL21(DE3)RIL and BL21(DE3)RP strains from Stratagene which carry pACYC-based plasmids expressing tRNAs that are rare in E. coli.

    This procedure may also work for expression systems using the lac or tac promoters, such as the pHN1 plasmids (Garboczi et al.) which we use for expression of HLA-A2-BSP and human b2m.

  2. First thing in the morning on day 2, transfer a single colony from the fresh agar plate into 2 ml of ZYP-0.8G plus appropriate antibiotic in a 18 x 150 mm snap cap tube. Shake at 300 RPM at 37°C for 6-8 hours, until the culture is turbid but not saturated.

    Alternatively, you can use LB + 1% glucose, but you’re chances of failure increase because bacteria cultures in LB media are more likely to lose the expression plasmid when the cultures approach saturation.

  3. While the Inoculum Culture in step 2 is growing, prepare 6 2-liter baffled Erlenmayer flasks with 400 ml each of ZYP-5052 media plus appropriate antibiotic.

    Adequate aeration of the cultures is essential to achieving optimal protein expression using this method. It is essential to use baffled flasks, and to keep the volume of liquid in the 2-liter flask at or below 400 ml.

    The 5052 in ZYP-5052 stands for 0.5% glycerol, 0.05% glucose, and 0.2% a-lactose.

  4. Towards the end of day 2, transfer 200 µl of the Inoculum Culture into each of the 6 2-liter flasks containing 400 ml ZYP-5052 + antibiotic. Shake at 300 RPM at 37 °C overnight.
  5. First thing in the morning on day 3, cool the cultures by placing the Erlenmayer flasks containing the cultures ice buckets. Use flat, shallow ice buckets.
  6. While the cultures are cooling, take a 1.0 ml sample from each flask and place in a labeled 1.5 ml microcentrifuge tube. Prepare a 1:10 dilution of each sample by transferring 0.1 ml of each into each of a second set of microcentrifuge tubes containing 0.9 ml of fresh ZYP-5052. Prepare an additional microcentrifuge tube containing 1.0 ml of fresh ZYP-5052 to use as a blank.
  7. Measure the OD(600) of the 1:10 dilutions of each of the cultures and record the result in the Inclusion Body QC Database.

    This database is used in the Altman Lab to record data from Inclusion Body Preps. Others may simply record this data in their notebooks

  8. Pellet the bacteria in the remaining 0.9 ml by spinning the microcentrifuge at 1/2 of full speed for 2 minutes. Aspirate the supernatant.
  9. Resuspend the bacteria in the microcentrifuge tubes in 1X SDS-PAGE reducing sample buffer (SDS-rSB) in two stages: (1) add 50 µl of SDS-rSB (or water?) to each of the pellets and vortex vigorously to resuspend the pellet; (2) add 400 µl of SDS-rSB to each of these suspensions, to bring the total volume of SDS-rSB to 1/2 of the culture volume. These samples will be used for subsequent analysis of total bacterial protein by SDS-PAGE.

    The two step protocol is used to facilitate complete resuspension of the bacteria. If done in one stage by the addition of 450 µl of sample buffer, the pellets are difficult to resuspend.

    You won’t have to run samples from all 6 flasks, but you should run them from at least 1 or maybe two.

    Finally, it is a good idea to prepare an additional tube containing a 1:10 dilution of the SDS samples (in SDS-rSB) to run on the gel, in case the full-strength samples badly overload the gel.

  10. Check to see that the chromosomal DNA in the samples has been thoroughly sheared by vortexing. Tell-tale signs of unsheared DNA are a “stringy” consistency that is observed when the tubes are opened or when a small volume is drawn up into a pipette tip. If residual unsheared DNA remains, it can be sheared by further vigorous vortexing. As a last resort, the DNA can be sheared by sonication for 2-3 minutes using a microtip sonicator at 50% duty cycle, with the samples cooled on ice.

    Complete shearing of the DNA is essential to achieving a high quality SDS-PAGE gel.

  11. Combine the contents of two of the Erlenmayer flasks into a single 1-liter centrifuge bottle, resulting in 800 ml of bacterial culture in the flasks. Similarly combine the contents of remaining flasks into 2 additional centrifuge bottles, yielding a total of three centrifuge bottles each containing 800 ml of culture. Equalize the volume of bacteria in the cultures by eye, transferring culture liquid from one or more bottles to one a bottle that may contain slightly less liquid. Balance the bottles on a pan balance by moving liquid with a pipette from heavier to lighter bottles.
  12. Pellet the bacteria in the 1 liter bottles by spinning for 20 minutes at 5000 x g at 4°C.
  13. While the bacterial cultures are in the centrifuge, prepare an SDS-PAGE gel for analysis of total protein. You should be able to prepare at least the separating portion of the gel before the centrifuge run is completed.
  14. Decant the supernatants from the 1-liter centrifuge bottles into a large bucket or beaker, treat with a few hundred ml of 100% bleach, and then pour down the drain, rinsing with copious water.
  15. Add 20 ml of resuspension buffer to each of the bacterial pellets in the 1-liter bottles. Resuspend to homogeneity by vigorous shaking on the platform rocker in the cold room. This can take up to 20-30 minutes.

    Alternatively, you can resuspend the bacteria by vigorous pipetting, but this takes much more manual intervention and is a waste of your valuable time.

  16. While the bacteria is shaking in resuspension buffer, finish preparing your SDS-PAGE gel. In addition, set up a full 500-1000 ml beaker of water to boil for heating up your SDS-PAGE samples.
  17. Run your SDS-PAGE gel while you are processing the bacteria to isolate the inclusion bodies.

    Save the samples in SDS sample buffer for a few days, even if you've run the gel. You might want to run another gel which contains both the bacterial lysate and the washed inclusion bodies.

  18. Isolate and wash the inclusion bodies, following the “Inclusion Body Preparation” protocol. Do this on the same day that you harvest your bacteria, so that there is no need to freeze the bacteria.

    If absolutely necessary, you can snap freeze the bacteria in 50 ml screw cap plastic centrifuge tubes for processing at a later date, but you might was well get the whole thing over with.