GST SpinTrap Purification Module From GE Healthcare

GST SpinTrap Purification Module provides Glutathione Sepharose 4B prepacked in SpinTrap columns. Each kit contains 50 columns with each column containing 50 µl of Glutathione Sepharose 4B. This is theoretically enough for purifying up to 400 µg of recombinant GST-tagged protein. The SpinTrap GST columns are ideal for screening, analysis of expression, and purification prior to scaling up. The columns are also ideal for use in a microcentrifuge. Each is pre-equilibrated in 1× phosphate buffered saline (PBS) and 0.05% Kathon (an antibacterial preservative). The kit contains the following components: 10× PBS, IPTG, reduced glutathione, dilution buffer, Glutathione Sepharose 4B in the spin columns and an instruction booklet. It is recommended that you supply a sonicator and protease inhibitors unless you plan to use another type of lysis system.

Yield of fusion protein is highly variable and is affected by the nature of the fusion protein, the host cell and the culture conditions used. Fusion protein yields can range from 1–10 μg/ml and must be determined by the end user. However, a safe assumption is to start with 5 μg/ml. Therefore, a basic protocol would require growing of bacteria in 80 ml culture to an OD₆₀₀ 0.6-1.0 (also should be user-determined for each protein) and induced with 1 mM IPTG (optimal concentration for induction should also be user-determined for each protein) for 3 hours at 37°C. The bacteria are then pelleted down by centrifugation at 5000 rpm for 10 min at 4ºC. Pelleted cells are suspended in 50 μl of ice-cold PBS for each ml of culture that was used (~4 mls) by pipetting up and down. Sonication takes place and this really should be optimized for your sonicator and settings so as to maximize cell lysis and minimize protein degradation. However, 10-30 second bursts at about 20-30% is a good starting point. Make sure that the extract does not overheat as this will denature your protein. At this point, protease inhibitors can be added. The bacterial cell lysate is separated from the insoluble material by centrifugation at 20,000 x g at 4ºC for 30 mins. Place a column into a 1.5-2.0 ml eppendorf tube (preequilibrate the column in PBS beforehand). The supernatant is then applied to the column and centrifuged for 1 minute at 735 x g. No more than 600 μl of culture sonicated lysate or buffer can be applied the SpinTrap column; this is the major hinderance of this protocol. Repeat until all supernatant is applied. Wash the colum with 2-3 600 ul washes of PBS (the stringency can be increased with various salts and detergents and must be user-determined). The protein is eluted with 100 ul x 3 elutions with glutathione elution buffer (prepared per protocol supplied). With each elution, the elution buffer should be applied to the column and allowed to stand at room temperature for 10 minutes. Collect the protein and analyze by SDS-PAGE or other procedure.

We use the modules as a primary screening to determine, after transformation and plating, which bacterial colonies produce the highest level of protein. We screen by picking several colonies, growing small cultures and then splitting each culture in half. One half we save; the second half we induce for protein expression. We then purify the protein using the columns and combine the elutions and analyze by SDS-PAGE gel using Imperial Protein Stain from Pierce Biotechnology (now part of Thermo Fisher Scientific). Once we determine which colony produces the largest amount of protein, we use the saved bacteria to grow up large culture volumes.