Protein Extraction: Effect of Solubilization Additives

The Effect Of Solubilization Additives

In some cases, the protein of interest could not be observed in the cell extracts, either with the commercially available lysis products or with Aperto. A large number of additives were tested with Aperto, at concentrations generally ranging from 12.5 – 50% final volume and 5 additives were found to have broad application. In the example below 4 of the 5 additives improve solubility when compared to Aperto alone (A) and the competing product (C).

Examples requiring more stringent conditions are shown below

GROWTH CONDITIONS

Growth conditions had a significant effect on expression and the ability to recover the difficult proteins tested. Cells were typically grown in defined media then transferred to a modified Studier Media for induction2. The following conditions were found to yield the best results.

FROM A FROZEN GLYCEROL STOCK

  • 2% inoculum in Teknova Azure Media (Part number 3H5000) w/ 1% glucose and antibiotic.
  • Grow for 16 hr at 20–23°C.
  • Transfer 25 µL of this culture to a fresh 96-well plate containing 500 µL of Azure Media 1% glucose + antibiotic.
  • Grow for 16 hr at 20‐23C then raise temp to 37°C and continue to grow for 3 hr only (37°C increases cell mass.....non-inducing conditions).
  • Transfer 25 µL of this culture to a fresh 96-well plate containing 500 µL Studier 2PInduction Media (Part Number 3S2000) + antibiotic and grow at 20–23°C for 16–27 hr.

FROM A SINGLE COLONY ON A PLATE

  • Prepare 500 µL of Teknova Azure Media (Part number 3H5000) w/ 1% glucose and antibiotic in a 2 mL deep-well 96-well plate.
  • Inoculate w/ a single colony from a fresh plate.
  • Grow for 16 hr at 20–23°C then raise temp to 37°C and continue to grow for 3 hr only (37°C increases cell mass.....non-inducing conditions).
  • Transfer 25 µL of this culture to a fresh 96-well plate containing 500 µL Studier 2P Induction Media (Part Number 3S2000) + antibiotic and grow at 20–23°C for 16–27 hr.

References:

  1. Cells were generously provided by The Seattle Structural Genomics Center for Infectious Disease F.W. Studier (2005) Protein production by induction in high‐density shaking cultures. Prot. Exp. Pur. 41, 207–234.