Nov 22, 2025
Small-scale test expression of recombinant protein in E. coli
- Ainsley Lederer1,
- Prem Shrestha1
- 1University of Pittsburgh
Protocol Citation: Ainsley Lederer, Prem Shrestha 2025. Small-scale test expression of recombinant protein in E. coli. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkydkdg5r/v1
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: September 04, 2025
Last Modified: November 22, 2025
Protocol Integer ID: 226473
Keywords: protein expression, test expression, protein production, small scale production, scale test expression of recombinant protein, small scale test production of recombinant protein, recombinant protein, small scale test production, scale test expression, procedure
Abstract
This procedure is meant to be used for the small scale test production of recombinant protein in E. coli before scaling up to larger volumes.
Materials
Ni-binding buffer (for resuspension):
50 mM Tris-HCl pH 7.5
100 mM NaCl
10% glycerol
5 mM beta-mercaptoethanol
Troubleshooting
Day 1
3h
Take LB agar plates with transformed E. coli. Select a single colony and add to 4 mL LB media with antibiotic. Place tube in 37 °C shaker.
It is convenient to keep 1000x stocks of antibiotics in the freezer.
| A | B | C | D | |
| Antibiotic | 1000X Concentration | 1X Concentration | Solvent | |
| Ampicillin | 100 mg/mL | 100 ug/mL | water | |
| Kanamycin | 50 mg/mL | 50 ug/mL | water | |
| Chloramphenicol | 34 mg/mL | 34 ug/mL | ethanol |
Shake tubes until media becomes cloudy, typically 03:00:00 (2-3 hours) depending on cell strain.
3h
Take 500 µL of cells and add to500 µL of 50% glycerol solution (so final of 25% glycerol). Store this at -80ºC.
Note
The purpose of creating a glycerol stock at this step is to be able to use this exact colony of cells again in the future if the results are good! Each colony on a plate originated from a single E. coli. Yes, theoretically all colonies which grow on the antibiotic plate should act the same, but there are other factors at play that can affect the efficiency of protein production. So, if you plan to scale-up your protein production after the test expression, it is best to use the same stock of cells.
Take 1 mL of cells and put in a microcentrifuge tube. Centrifuge 14000 rpm, 4°C , 1 minutes . Aspirate the media completely and store the pellet at -20 °C until day 2. This is your control, uninduced sample.
Add 2.5 µL of 1000X IPTG to the tube. IPTG concentration may vary depending on protein you are trying to express, but 0.5 millimolar (mM) final (0.5M stock) is a good starting point.
Reduce temperature of shaker to 18 °C and put tubes back in. Allow tubes to shake in incubator overnight.
Day 2
Take tubes from shaker. Pipette 1 mL of cells into a microcentrifuge tube and centrifuge 14000 rpm, 4°C for 1 minute. Aspirate media completely. This is your induced sample.
Place both uninduced and induced pellet samples on ice. Add 400 µL resuspension buffer. We use Ni-binding buffer (recipe in materials). Pipette to resuspend each pellet.
Sonicate each cell suspension. For QSonica sonicator, use the smallest tip. The settings are: 1.5 minutes, 03 s on, 08 s off, 30% amplitude.
Each cycle takes about 5 minutes. Make sure that the sonicator tip is submerged in the cell suspension, but not pressed against the side of the tube.
After all tubes have been sonicated, take 15 µL of each sample and label as "uninduced total" and "induced total". This represents all soluble and insoluble protein.
Spin cells down at 14000 rpm, 4°C for 00:10:00 . Take 15 uL of the supernatant, and label as "uninduced soluble" and "induced soluble". This represents the fraction of protein that is soluble in your buffer.
Add 5 µL 4X Laemmli sample dye to each tube. Place tubes in 95 °C heat block for 2 minutes. Load samples onto SDS-PAGE gel and analyze by Coomassie staining.
Note
What would the ideal result be? Ideally, you will see non-specific proteins in the uninduced samples, both total and soluble. In your induced samples, you will hopefully see a thick band at the expected MW for your recombinant protein in both the total and soluble samples. If you do not see your target protein, or if you see protein only in the induced total fraction, it indicates a solubility/expression issue that should be addressed before you scale up your protein production!