Aug 27, 2025
Medium throughput protein expression and purification for protein design using heat lysis
- Ida K. Grene1,2,3,
- Nadja Joachim4,
- Francisca Pinheiro1,2,3,
- Vili Lampinen1,2,3,
- Kaare Teilum4,
- Magnus Kjaergaard1,2,3
- 1Molecular Biology and Genetics, Aarhus University, Denmark;
- 2The Danish National Research Foundation Center for Proteins in Memory (PROMEMO), Aarhus University;
- 3The Danish Research Institute for Translational Neuroscience (DANDRITE), Nordic EMBL Partnership for Molecular Medicine, Aarhus University;
- 4University of Copenhagen
Protocol Citation: Ida K. Grene, Nadja Joachim, Francisca Pinheiro, Vili Lampinen, Kaare Teilum, Magnus Kjaergaard 2025. Medium throughput protein expression and purification for protein design using heat lysis. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpq6b5lzp/v1
Manuscript citation:
Pinheiro F, Nowak JS, Zueva E, Pheasant EC, Grene IK, Lampinen V, et al. Screening de novo designed protein binders in unpurified lysate using flow induced dispersion analysis. Protein Science. 2025; 34(10):e70286. https://doi.org/10.1002/pro.70286
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: May 07, 2025
Last Modified: August 27, 2025
Protocol Integer ID: 217837
Keywords: de novo protein design, protein expression, heat lysis, Medium throughput, screening, bacterial expression, protein purification, small scale, multiwell plates, purification for protein design, medium throughput protein expression, pure lysate sample of the protein, protein design, heat lysis this protocol, using heat lysi, protein, purification step, purification, preparation of heat, using heat, pure lysate sample, stable de novo, lysi, heat
Funders Acknowledgements:
Danish National Research Foundation
Grant ID: DNRF133
Independent Research Fund Denmark
Grant ID: 026-00069B
Lundbeck Foundation
Grant ID: R449-2023-1396
Carlsberg Foundation
Grant ID: CF20-0610
Abstract
This protocols describes medium throughput protein expression in E. coli using heat-lysis as a built in purification step. It is intended for preparation of heat-stable de novo designed proteins for screening in vitro.1-3 It provides a small but relatively pure lysate sample of the protein within 2 working days.
Guidelines
Remember to always keep track of the samples when transferring between plate types.
Gene of interest should be in a pET system or another IPTG inducible expression vector.4
Materials
Equipment:
Suggested products will be in parantheses
- Flowbench, appropriate for working with bacteria
- Water bath
- Shaking incubator, preferably with sticky surface for securing plates
- Centrifuge with adaptor for multiwell plates
- Multichannel pipettes taking 1-1000 µL volumes
- Heatblock that can evenly heat 96 wells to 95°C (Eppendorf‱ ThermoMixer‱with deepwell plate smartblock)
- Platereader for absorption at 600nm
- SDS-page setup
Materials:
Suggested products will be in parantheses
- Plasmid DNA at ≈ 100 ng/µL . (We use synthetic genes cloned into the NdeI-XhoI sites of pET28a+ and stored in 96-well plates)
- Chemically competent E. coli BL21 (DE3) strain
- Lennox Broth medium (Pr. liter: 10.0 g Tryptone, 5.0 g Sodium Chloride, 5.0 g Yeast Extract)
- Appropriate antobiotic (e.g. kanamycin for pET28a+)
- ZYM-5052 autoinduction medium5
- Sterile reservoirs for multichannel pipettes.
- Sterile 96-well plates (UNIPLATE Collection and Analysis Microplate, 96-well, 2 ml, natural polypropylene, round well bottom)
- Sterile 24-well plates (UNIPLATE Collection and Analysis Microplate, 24-well, 10 ml, natural polypropylene, round well bottom)
- Breathable film (AeraSeal‱ film)
- 96-well plate able to withstand 95°C (Eppendorf‱ Deepwell Plate 96/1000μL)
- 96-well filter plate (AcroPrep‱ Advance filter plates for Lysate Clearance - 1 mL, 3.0 µm Glass Fiber/0.2 µm Supor membrane)
- 96-well clear plate for the plate reader
- Lysis buffer (pH 7.4 phosphate buffer + 300 mM NaCl or similar)
- Polyacrylamide gels, loading dye, appropriate size marker and running buffer
- BCA Protein Assay Kit
If going for chemical lysis:
- Bacterial protein extraction reagent (B-PER‱ Complete Bacterial Protein Extraction Reagent)
If going for purification:
- 96-well IMAC plate (Cytiva His MultiTrap FF)
- Imidazole stock for buffers
Troubleshooting
Before start
If you have not bought your 96- and 24-well plates, medium and reservoirs sterile, make sure to autoclave them or sterilize in another way before starting.
If keeping the medium in the fridge, bring it to room temperature before adding to the cells.
Prepare the lysis buffer before starting day 2.
Transformation (day 1):
1h 53m
Before starting:
- Thaw competent E. coli BL21 On ice
- Thaw plasmid DNA Room temperature
- Cool a sterile 96-well plate On ice
- Preheat the water bath 42 °C
15m
Add 10 µL E. coli BL21 per well in the 96-well plate.
10m
Add 1 µL plasmid DNA solution to the bacteria. Stir with pipette tip to connect droplets, do not pipette up and down.
Note
Alternatively, spin the plate after adding the DNA to make sure that cells and DNA come into contact.
10m
Incubate the plate 00:15:00 On ice .
15m
Heat-shock bacteria in water bath by holding the plate half submerged in the water 42 °C 00:00:30 . Put directly back on ice afterwards.
1m
Add 50 µL sterile LB medium to each well.
2m
Incubate transformation mixtures 400 rpm, 37°C, 01:00:00 .
1h
Expression (day 1)
12h 22m
Prepare autoinduction medium with appropriate antibiotic in a sterile reservoir.
2m
Add 2 mL autoinduction medium to each well in 4 sterile 24-well plates.
5m
Add 50 µL of the transformation mixture to each well in the 24-well plates. Resuspend any pelleted bacteria before transferring.
Note
To keep the layout compatible with a multichannel pipette, this template can be used (assuming samples 1-96)
96 well plate:
| 1 | 2 | 3 | 4 | 5 | |
| A | 1 | 2 | 3 | 4 | 5 |
| B | 13 | 14 | 15 | 16 | 17 |
| C | 25 | 26 | 27 | 28 | 29 |
| D | 37 | 38 | 39 | 40 | 41 |
| E | 49 | 50 | 51 | 52 | 53 |
| F | 61 | 62 | 63 | 64 | 65 |
| G | 73 | 74 | 75 | 76 | 77 |
| H | 85 | 86 | 87 | 88 | 89 |
| 6 | 7 | 8 | 9 | 10 | |
| A | 6 | 7 | 8 | 9 | 10 |
| B | 18 | 19 | 20 | 21 | 22 |
| C | 30 | 31 | 32 | 33 | 34 |
| D | 42 | 43 | 44 | 45 | 46 |
| E | 54 | 55 | 56 | 57 | 58 |
| F | 66 | 67 | 68 | 69 | 70 |
| G | 78 | 79 | 80 | 81 | 82 |
| H | 90 | 91 | 92 | 93 | 94 |
| 11 | 12 | |
| A | 11 | 12 |
| B | 23 | 24 |
| C | 35 | 36 |
| D | 47 | 48 |
| E | 59 | 60 |
| F | 71 | 72 |
| G | 83 | 84 |
| H | 95 | 96 |
24-well plate 1:
| 1 | 2 | 3 | 4 | 5 | |
| A | 1 | 2 | 3 | 4 | 5 |
| B | 13 | 14 | 15 | 16 | 17 |
| C | 25 | 26 | 27 | 28 | 29 |
| D | 37 | 38 | 39 | 40 | 41 |
| 6 | |
| A | 6 |
| B | 18 |
| C | 30 |
| D | 42 |
24-well plate 2:
| 1 | 2 | 3 | 4 | 5 | |
| A | 7 | 8 | 9 | 10 | 11 |
| B | 19 | 20 | 21 | 22 | 23 |
| C | 31 | 32 | 33 | 34 | 35 |
| D | 43 | 44 | 45 | 46 | 47 |
| 6 | |
| A | 12 |
| B | 24 |
| C | 36 |
| D | 48 |
24-well plate 3:
| 1 | 2 | 3 | 4 | 5 | |
| A | 49 | 50 | 51 | 52 | 53 |
| B | 61 | 62 | 63 | 64 | 65 |
| C | 73 | 74 | 75 | 76 | 77 |
| D | 85 | 86 | 87 | 88 | 89 |
| 6 | |
| A | 54 |
| B | 66 |
| C | 78 |
| D | 90 |
24-well plate 4:
| 1 | 2 | 3 | 4 | 5 | |
| A | 55 | 56 | 57 | 58 | 59 |
| B | 67 | 68 | 69 | 70 | 71 |
| C | 79 | 80 | 81 | 82 | 83 |
| D | 91 | 92 | 93 | 94 | 95 |
| 6 | |
| A | 60 |
| B | 72 |
| C | 84 |
| D | 96 |
15m
Cover plates with breathable film.
Incubate cultures 400 rpm, 37°C Overnight .
12h
Harvest (day 2)
47m
Add 90 µL LB per well in a 96-well plate.
Note
Should be plate reader compatible.
2m
Add 10 µL of culture per well.
Note
Make sure to resuspend if pelleted.
5m
Measure OD600 in the plate reader. Remember to include a couple of wells with just LB as blank controls.
Note
OD of the culture will then be 10x this number. This step is qualitative and meant as a quick check of the growth. The values vary between experiments.
10m
Take a 15 µl sample of each production from diluted 96-well plate samples and save for SDS-PAGE analysis.
10m
Pellet bacteria in the 24-well plate 2500 x g, Room temperature, 00:15:00
15m
Remove the supernatant. Freeze 24-well plate or proceed to lysis.
5m
Decide on your lysis protocol. This will depend on the stability of your proteins of interest.
If you have heat stable proteins16 steps
This protocol will remove many of the endogenous E. coli protein in your samples6.
Preheat Thermomixer 95 °C
15m
Resuspend the pellets in 500 µL lysis buffer and move to a heat-stable 96-well plate.
Note
If the lysate is very viscous you can increase the volume, but this will decrease the final protein concentration.
10m
Heat-lyse the bacteria 95 °C 00:15:00 .
15m
Optional to ease filtration: Centrifuge 3000 x g, Room temperature, 00:10:00 and take only the supernatant to the next step.
10m
Transfer the lysate to a 96-well filter plate stacked on a collection plate (96 deep-well plate).
5m
Centrifuge 1500 x g, Room temperature, 00:05:00 . If the liquid in some wells has not gone fully through, resuspend with pipette and centrifuge again.
5m
Take 15 µl samples from cleared lysates for SDS-PAGE.
15m
Estimate protein concentration in the lysates using the Bicinchoninic Acid (BCA) Method.
Affinity chromatography purification (day 2, optional)
1h 10m
Prepare 200 mL binding buffer (20 mM imidazole) and 60 mL elution buffer (500 mM imidazole). Open the plate according to instructions and place it on top of a collection plate (96 deep-well).
15m
Centrifuge the plate to remove the storage solution from the resin 500 x g, 4°C, 00:02:00
5m
Add 500 μL deionized water/well 500 x g, 4°C, 00:02:00 .
5m
Add 500 μL binding buffer/well and mix briefly to equilibrate the resin, centrifuge 500 rpm, 4°C, 00:02:00 . Repeat once.
10m
Apply filtered lysate to the wells, mix briefly, and incubate 00:03:00
5m
Remove the flow-through by centrifuging 100 x g, 4°C, 00:04:00 , or until the wells are empty
5m
Add 500 μL binding buffer/well and mix briefly to wash out unbound sample, centrifuge 500 x g, 4°C, 00:02:00
Repeat once.
10m
Add 200 μL of elution buffer/well and mix for 00:01:00 . Change collection plate and centrifuge the plates 500 x g, 4°C, 00:02:00 and collect the fractions.
Repeat twice.
Note
If required, change collection plate between each elution (to prevent unnecessary dilution of the
target protein).
15m
Protocol references
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Citations
Francisca Pinheiro, Jan S. Nowak, Elena Zueva, Emily C. Pheasant, Ida Kjærsgaard Grene, Vili Lampinen, Magnus Kjaergaard. Screening de. novo designed protein binders in unpurified lysate using flow induced dispersion analysis
https://doi.org/10.1101/2025.06.17.6601270Acknowledgements
We thank Nadja Joachim and Kaare Teilum for sharing their protein expression protocol.