Editing Protein Expression using Vmax (section)

=== Seed culture protocol  ===

1. Pick the first/largest colonies to appear from the transformation plates. 

2. Inoculate a culture tube containing 2–15 mL growth media that is at room temperature, 30°C, or 37°C. 

Note: The volume of the seed culture medium should be at least 1/100th the volume of desired protein expression culture. 

3. Culture cells at 30°C or 37°C at 250 rpm for ~3 hours for rapid protein expression or for 6–16 hours if the culture will be used as a standard seed culture or used to create glycerol stocks for long-term storage at –80°C. 

Note: Refer to the following table for additional information.

Glycerol stocks 

For critical clones, we recommend preparing multiple glycerol culture aliquots to avoid freeze/ thaw cycles. 

1. To prepare a glycerol stock, choose from one of the following options: 

• Add 250 µL of sterile 100% glycerol to a 1-mL cryopreservation vial or other −80°C compatible sterile tube. Add 750 µL of bacterial seed culture (prepared as described above) and mix for a final concentration of 25%&nbsp;glycerol. 

• Alternatively, spin 1 mL of bacterial seed culture in a microcentrifuge tube for ~1&nbsp;minute at 10 × g. Aspirate the media and resuspend the pellet in 1 mL sterile media supplemented with 25%&nbsp;glycerol.

2. Place vials in –80°C freezer for storage. 

3. To recover culture from glycerol stock, use a sterile inoculation loop to scoop frozen culture and transfer to liquid or solid growth medium with appropriate antibiotics and incubate at room temperature, 30°C, or 37°C. 

Note: Always incubate kanamycin-resistant strains grown on solid agar medium at 30°C or room temperature.



Protein expression protocol 

The following protocol is a standard protocol for protein expression using Vmax™ Express cells. This protocol has been used to successfully express multiple proteins with high yields. For some proteins, optimization of expression temperature and/or expression times may improve yield, solubility, or activity. Vmax™ Express has been shown to grow and produce recombinant proteins at temperatures as low as 16°C. If expression at lower temperatures is required, we recommend evaluating expression at multiple temperatures. 

For rapid expression 

To perform a rapid expression assay, pick a single colony, inoculate a seed culture in selective media, and grow to an OD600 of 0.3 (~3 hours in 25 mL of media) before inoculating a growth flask (see "Preparing seed cultures" on page 14 for additional details). 

Before starting

• To allow sufficient aeration, ensure the expression culture will not exceed ¼ of flask volume (e.g, for a 500-mL Fernbach baffled flask, grow a maximum of 125&nbsp;mL of expression culture).

• Optimal expression media are Vmax™ Enriched Growth Medium or Enhanced 2xYT medium. Other liquid media listed in Table 2 on page 8 can also be used (see "Media recipes" on page 18). 

Protocol 

1. On the day of expression, inoculate a seed culture (OD600 of 2–7 for standard protocol, >0.3 for rapid protocol) into growth medium containing appropriate antibiotics. Use 1/100&nbsp;volume of seed culture relative to the total amount of medium to inoculate the growth flask (e.g. for 125 mL of growth medium, add 1.25 mL of seed culture). 

2. Grow culture at 30°C for ~1–2 hours on a rotating shaker incubator at 250 rpm until the OD600 reaches >0.5. Note: Expression can be performed at 37°C, but for controls and most proteins tested, expression is optimal when cells are grown at 30°C on a rotating shaker incubator at 250&nbsp;rpm. 

3. After reaching OD600 of ≥0.5, induce protein expression by adding IPTG to a final concentration of 1&nbsp;mM. Note: Although induction is typically performed 1–2 hours after inoculation, Vmax™ Express cells can be induced within a wide range of time points during growth phase. The optical density at induction can be between 0.5–1 OD600 with no apparent difference in expression. If auto-induction medium is used, expression will initiate during overnight culture. 

4. Incubate induced cells for 4–24 hours to express recombinant protein. 

Note: For most proteins, longer post-induction incubation times will allow for increased biomass, which typically results in higher protein yields. Although some proteins may express high levels sooner, we recommend overnight induction to maximize yield. Vmax™ Express has been shown to maintain soluble protein expression after overnight culture. 

5. Harvest cells by transferring to a centrifugation tube and spinning at 4000–5000 × g for 10 minutes at room temperature or 4°C. Carefully remove supernatant.

6. Store the cell pellet at −80°C or proceed to lysis and protein purification. Note: If you are using 96-well plates for expression, apply an adhesive plate seal prior to centrifugation or storage.&nbsp;




[[Category:Protein Expression]][[Category:Protocols]]