Oct 27, 2020
Protein expression in Bacillus subtilis
- Kristoffer Bach Falkenberg1,
- Cristina Hernandez Rollan1,
- Maja Rennig1,
- Andreas Birk Bertelsen1,
- Morten Norholm1
- 1Technical University of Denmark
Protocol Citation: Kristoffer Bach Falkenberg, Cristina Hernandez Rollan, Maja Rennig, Andreas Birk Bertelsen, Morten Norholm 2020. Protein expression in Bacillus subtilis. protocols.io https://dx.doi.org/10.17504/protocols.io.bdmui46w
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 routinely and it works for us
Created: March 13, 2020
Last Modified: October 28, 2020
Protocol Integer ID: 34196
Keywords: B. subtilis, Bacillus, Bacillus subtilis, Protein expression
Abstract
B. subtilis is a gram-positive bacteria used by both academia and industry as a protein production workhorse. This is due to its' their excellent fermentation properties, high production titers, and capacity to secrete proteins into the extracellular medium.
This protocol describes how to express proteins in B. subtilis. The protocol is developed using KO7-S, although it might also work for other strains as well. The method is adapted from Rasmussen, M. D.; Bjoernvad, M. E.; Diers, I. Pectate Lyase Fusion for Expression and Secretion of Polypeptides. WO 00/75344, 2000 and Jensen, K.; Østergaard, P. R.; Wilting, R.; Lassen, S. F. Identification and Characterization of a Bacterial Glutamic Peptidase. BMC Biochem.2010, 11 (1), 47. https://doi.org/10.1186/1471-2091-11-47.
Materials
MATERIALS
Sodium molybdate dihydrate
manganese sulfate
Iron(III) chloride hexahydrateCatalog #236489
Zinc sulfate heptahydrateSigma AldrichCatalog #204986
Magnesium sulfate heptahydrate
Sodium Phosphate dibasicFisher ScientificCatalog #S373-500
Copper (II) sulfate pentahydrateSigma – AldrichCatalog #209198
Yeast extract
Nalgene™ Rapid-Flow™ Sterile Disposable Bottle Top Filters with PES Membrane,150mL, 0.45μm pore, 45mm neckThermo FisherCatalog #296-4545
Maltodextrin (DE 13.0-17.0)Sigma AldrichCatalog #419680
Pluronic L-61Sigma AldrichCatalog #435422
Safety warnings
Be sure to wear protective equipment when adjusting the pH of the media. Follow local safety regulations
Before start
Make sure you have your expression strain freshly streaked on an agar plate.
Cal18-2 media preparations
Cal18-2 media preparations
Prepare a stock solution of 2.0g/L Na2MoO4. Sterilize by filtration
Prepare a trace metal solution consisting of
- 4.48g/L MnSO4·H2O
- 3.33g/L FeCl3·6H2O
- 0.625g/L CuSO4·5H2O
- 7.12g/L ZnSO4·7H2O
Sterilize by filtration
Fill a blue cap bottle to ~80% of the desired final volume with MQ water.
Add a magnetic stirrer to the blue cap bottle and place the bottle on a stirring plate. Turn on the stirring, and make sure it's mixing well.
Add the following to the bluecap bottle:
- 40g/L yeast extract
- 1.3 g/L MgSO4·7H2O
- 50 g/L maltodextrin (DE ~ 12)
- 20 g/L NaH2PO4·2H2O
- 6.7mL/L 2.0g/L Na2MoO4 stock solution
- 6.7mL/L Trace metal solution
- 100μL/L Pluronic L-61
Make sure that all of the ingredients are dissolved
Adjust to pH 6 with 5M NaOH
Add MQ water to the desired final volume
Sterilize by filtration
Note
The media easily clogs filters, so choose a 0.45μM vacuum bottle top filter for this step and be prepared to use a few filters per liter
Store the media at 4 °C until needed
Overnight culture - Day 1
Overnight culture - Day 1
Inoculate between 3 mL to 50 mL LB media with a single colony of the expression strain. Depending on the expression volume and overnight OD. The culture can be grown in in a 24-deepwell plate, a falcon tube or a shake flask
Grow the strain at 37 °C Overnight
Note
Make sure to not incubate the overnight culture for longer than 16:00:00 . Using an overnight culture that has been incubating for longer than this, often results in non-reproducible results
Expression - Day 2 - 4/5
Expression - Day 2 - 4/5
Prepare the desired volume of expression media in the desired vessel
Note
Vessel and media volume has a massive influence on the final protein titers. In our experience, 24-deepwell plates in an incubator with a large shaking amplitude and CSTRs work well, while shake flasks give low protein yields. This is likely highly dependant on the equipment (e.g. incubators and the shape and baffle pattern of the shake flasks), and thus should be optimized for the individual labs
Inoculate the expression media to an OD600 of 0.1
Incubate the expression culture at 20 °C with 250 RPM shaking between 48:00:00 and 72:00:00
Note
The expression temperature and duration is dependant on the target protein, although the specified values seem to be a good starting point in our experience
Harvesting - Day 4/5
Harvesting - Day 4/5
Harvest the culture by centrifuging at 6000 x g, 4°C, 00:05:00
Note
Depending on what the samples are for, the supernatant from the first centrifugation can additionally be centrifuged at 16000 x g, 4°C, 00:30:00 to clear out cell debris and other smaller contaminants
Keep the sample On ice when working with it and at -20 °C for storage