7 Methods for Protein Expression

Autoinduction Media Preparation: Media should be prepared with the sterile solutions and sterile equipment obtained in Week 1.

ADVICE: All of these steps should be done using sterile technique with a flame on. Both the pipettes and the benchtop should be cleaned with water and ethanol before starting.  Make sure to only use sterile tips, flasks, pipettes, water, and media.

Preparation of Autoinduction Media for Expressions (100 mL) Scale to make amount you need for your experiment based on the following amounts per 100 mL.  Prepare fresh and do not plan on storing extra AIM.

Aspartate (5%, pH 7.5)                       5 mL

Glycerol (10%)                                     5 mL

25x Mineral Salts                                4 mL

Glucose (40%)                                     0.125 mL

MgSO4 (1 M)                                        0.2 mL

Arabinose (20%)                                 0.25 mL

Trace metals (5000x)                         20 microliter

18 AA mix (25x) stored @ 4°C         4 mL

Add appropriate antibiotics (ampicillin to final concentration of 100 microgram/mL and tetracycline to 25 microgram/mL).

Add sterile water to a final volume of 100 mL.

Recommended volumes and flasks for the expressions during weeks 2 and 3:

Wild-type protein                                                             75 mL expression in a 250 mL flask

ncAA protein 1                                                            75 mL expression in a 250 mL flask

ncAA protein 1 (negative control -ncAA)                5 mL expression in culture tube

ncAA protein 2                                                            75 mL expression in a 250 mL flask

ncAA protein 2 (negative control -ncAA)                5 mL expression in culture tube

sfGFP wild-type control (-ncAA)                               5 mL expression in culture tube

ADVICE (EXAMPLE FOR 250 mL—SCALE ACCORDING TO YOUR NEEDS): It is convenient to start with a 250 mL bottle of sterile water and remove about 50 mL of it by placing it into another sterile vessel (for later use). Then all of the media components can be added to the 200 mL of sterile water and topped off with sterile water to a final volume of 250 mL. Then add 250 microliter of 100 mg/mL ampicillin to the media, and then place 75 mL of it in a 250 mL baffled flask for expression of wild-type protein. 175 microliter of 25 mg/mL tetracycline can then be added to the remaining media (175 mL) and separated into expression flasks. ncAAs will be added later.

Each lab section should use the appropriate code of tape color to label flasks so it is easy to recover them.  Remember there are 4 sections, over 80 students and some weeks 80-100 flasks growing.

Addition of Cells from Starter Cultures:

To the prepared flasks (75 mL media/flask), saturated cell cultures in non-inducing media of the appropriate cell line must be added to start the expressions. Dilute cells of appropriate overnight starter culture 1:100-1:200 to each flask containing 75 mL. Incubate these cultures for 0.5 -1 hr before adding the ncAA (this incubation may be shortened for certain ncAAs).

ncAA Preparation and Addition:  (Week 3)

The concentration recommended for the ncAA is >1 mM final ncAA concentration in the expression media.  The ncAAs may need assistance dissolving before adding to the media, depending on the chemical characteristics of the ncAA. It is recommended to weigh out slightly more than the appropriate amount of ncAA directly into a microcentrifuge tube. Start by adding 0.5 mL of sterile water and mixing – if it dissolves, add it to the appropriate expression flask. If most of the ncAA dissolves, first try adding an additional 0.5 mL of sterile water. If very little is dissolved after the first 1 mL, try adding 1 molar equivalent of NaOH from the 8 M stock solution (add 5 µL 8 N NaOH at a time and do not exceed 20 µL total).  Adding an excess of NaOH can damage some of the ncAAs and cause problems with cell growth. Once ncAAs are added to their appropriate flasks, let them incubate while shaking at 250-300 rpm at 37°C for up to 48 hours (this long incubation is designed to meet the schedule of BB 494; typically cultures are grown for 24-40 hours before harvesting).

To obtain a zero time point of protein expression, remove 250 microliter of cell culture from the original starter cells in non-inducing medium and centrifuge at 3000 – 5000 rcf for 5-10 min. Discard the supernatant and store the small cell pellet at -20°C in your freezer box (label all samples well, so you and your partners can identify in the future).

Express protein for 24-48 hours. Before harvesting cells by centrifugation, remember to remove 250 microliter cells for final time points for a crude gel first. Also, determine the OD600 of the cultures. Divide each cell sample into 25-35 mL aliquots in pre-weighed conical tubes. Spin the cells in 50 mL conical tubes for 10 min at 5000-8000 rcf. in the bench top centrifuge (not the Sorvall).  Remove the supernatant by pouring it off, determine the mass of each cell pellet, and store the cell pellets at -80 °C. Again label tubes well with section, group number, date, and sample.  Store cell pellets in small Ziploc bags that are well labeled with pen directly on the bag with your lab section, group number, and date. Do not use tape to label tubes or bags—it falls off at -80 °C.