PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

This protocol to purify the enzyme tryptophan synthase from Salmonella typhimurium is fast, simple, and short. Ammonia sulfate fractionation and size exclusion chromatography make it possible to purify the wild type and mutant tryptophan synthase complex in a single day. This technique can be applied to purify other related proteins since ammonia sulfate fractionation and size exclusion chromatography are commonly used to purify non-tagged proteins.

To begin, disrupt the cell pellet by sonication on an ice water bath at 80%amplitude for 20 cycles using 10-second pulses and 20-second rests. Centrifuge the cell lysate at 30, 000 times g for 30 minutes at 25 degrees Celsius, and aspirate the supernatant without dislodging the pellet. Filter the clarified supernatant fraction with a 0.45-micrometer filter at room temperature.

Slowly add small amounts of ammonium sulfate to the filtrate until 20%saturation is reached. Carry out ammonium sulfate fractionation at 25 degrees Celsius, and gently stir the solution for 10 minutes, taking care to avoid bubbles. Centrifuge the solution again for 10 minutes, and transfer the supernatant into a clean flask for further use.

Gently resuspend the pellet in 20 milliliters of sample buffer, and prepare a sample to run SDS-PAGE. Add ammonium sulfate to the supernatant until 30%saturation is reached, and stir the solution, avoiding bubbles. Centrifuge the solution again for 10 minutes, and transfer the supernatant to a clean flask for further use.

Gently resuspend the obtained 40%pellet fraction in 10 milliliters of sample buffer two, and prepare a sample to run SDS-PAGE. Then microcentrifuge the 40%protein sample at 10, 000 times g for 20 minutes at four degrees Celsius, and load the obtained supernatant on a previously equilibrated HiPrep 16/60 Sephacryl S-200 HR column attached to a fast protein liquid chromatography instrument at a flow rate of 0.5 milliliters per minute. Assess the collected peak fractions and the ammonium sulfate fractions on a 12%SDS-PAGE gel stained with Coomassie brilliant blue.

Concentrate the wild type or mutant alpha-two beta-two Salmonella typhimurium tryptophan synthase complex with a 100-kilodalton centrifugal filter by spinning at 3, 000 times g, at four degrees Celsius. Transfer the concentrated protein to a fresh two-milliliter tube, and again centrifuge at 10, 000 times g for 10 minutes at four degrees to remove aggregates. After determining the protein concentration of the sample, prepare 0.5 milliliter aliquots at 20 to 25 milligrams per milliliter.

Flash freeze them in liquid nitrogen, and store them at minus 80 degrees. Prepare a five-milliliter stock solution of 200-millimolar spermine in water, and store it in 500 microliter aliquots at minus 20 degrees. Then prepare a 50-milliliter stock solution of 30%polyethylene glycol 8, 000 in water, and keep 25 milliliters in a 50-milliliter disposable centrifuge conical flask at 25 degrees Celsius.

Prepare 25-milliliter stock solutions of one-molar cesium chloride and sodium chloride, each in water, and keep them at 25 degrees Celsius. Prepare three sets of 50-milliliter stock solutions of one-molar bicine, and titrate it with cesium hydroxide or sodium hydroxide to obtain buffered solutions at pH 7.6, 7.8, and eight. Keep 25-milliliter aliquots of the solutions at four degrees Celsius.

Thaw a sample of alpha-two beta-two Salmonella typhimurium tryptophan synthase complex in an ice bath. Then microcentrifuge it at 10, 000 times g for 10 minutes at 25 degrees Celsius. Transfer the clear supernatant fraction into a clean centrifuge tube.

Estimate the protein concentration of the supernatant, and dilute the aliquots to 15 milligrams per milliliter with 50-millimolar bicine cesium hydroxide or bicine sodium hydroxide of pH 7.8, containing 50-millimolar cesium or sodium chloride. Prepare 500 microliters of reservoir solutions for three 24-well sitting drop plates in sterile 1.5-milliliter microcentrifuge tubes. Vary the concentration of PEG 8, 000 and spermine, and change buffer pH as described in the text manuscript for each set.

Cap the tubes, and vigorously vortex for 10 seconds, before centrifuging them at 10, 000 times g for 10 minutes at 25 degrees Celsius to remove air bubbles. Dispense 500 microliters of this solution in each reservoir. Dispense five microliters of protein solution at 15 micrograms per liter into each sitting drop well using a P-10 micropipette.

Then add five microliters of each corresponding reservoir solution to the protein drop. Avoid air bubbles during mixing, and do not pipette up and down. Finally, cover the plate with transparent adhesive tape, and store it at 25 degrees.

The crystals appear in two to five days and grow to their full dimensions within two weeks. This protocol was used to subclone the alpha subunit and the beta subunit of the Salmonella typhimurium tryptophan synthase into the modified pET SUMO vector. Representative SDS-PAGE results of two strong bands corresponding to the His6-SUMO-alpha and His6-SUMO-beta tryptophan synthase fusion protein are shown here.

Each protein was purified by nickel-nitrilotriacetic acid affinity chromatography and ammonium sulfate precipitation, followed by His-SUMO-tag cleavage, removal of His-SUMO-tag traces, and protein concentration measurement. The elution profile of the alpha subunit, beta subunit, and alpha-two beta-two Salmonella typhimurium tryptophan synthase complex on SEC, and the purity of their peak fractions are shown here. After generating mutant forms of the tryptophan synthase complex, ammonium sulfate fractionation was used to remove the contaminant proteins from the heterologous expression system.

A representative elution profile with a relative elution position of the alpha-two beta-two Salmonella typhimurium tryptophan synthase complex on an exclusion chromatography column is shown here. The purity of the excluded peak fractions was analyzed using SDS-PAGE before pooling. Large single crystals were obtained through a fine crystallization optimization by varying PEG 8, 000 and spermine concentrations and bicine buffer pH.

Crystals with different morphologies appeared in two to five days and grew to full size within two weeks. The validation of electron density maps for the crystal structure of the wild type alpha-two beta-two Salmonella typhimurium tryptophan synthase complex was elucidated after crystal structure refinement. When performing this protocol, complete the initial purification steps at room temperature as soon as possible to prevent tryptophan synthase activity loss.

The crystal structures of the mutants are anticipated to provide new insights into the mechanism and roles played by key residues in L-tryptophan synthesis.