PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase
September 26th, 2020
This article presents a series of consecutive methods for the expression and purification of Salmonella typhimurium tryptophan synthase comp this protocol a rapid system to purify the protein complex in a day. Covered methods are site-directed mutagenesis, protein expression in Escherichia coli, affinity chromatography, gel filtration chromatography, and crystallization.
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.
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Overview
This article presents a rapid and efficient protocol for the expression and purification of tryptophan synthase from Salmonella typhimurium. The methods described allow for the purification of both wild type and mutant protein complexes in a single day.
Key Study Components
Area of Science
- Biochemistry
- Protein Purification
- Enzyme Characterization
Background
- Tryptophan synthase is an essential enzyme in the biosynthesis of tryptophan.
- Salmonella typhimurium serves as a model organism for studying this enzyme.
- Efficient purification methods are crucial for biochemical studies.
- Common techniques include ammonium sulfate fractionation and size exclusion chromatography.
Purpose of Study
- To develop a streamlined protocol for purifying tryptophan synthase.
- To enable the study of both wild type and mutant forms of the enzyme.
- To provide a method applicable to other related proteins.
Methods Used
- Site-directed mutagenesis
- Protein expression in Escherichia coli
- Affinity chromatography
- Gel filtration chromatography
Main Results
- The protocol allows for purification within a single day.
- Both wild type and mutant tryptophan synthase can be effectively purified.
- Ammonium sulfate fractionation and size exclusion chromatography are key steps.
- The method is adaptable for purifying other non-tagged proteins.
Conclusions
- This protocol provides a rapid and efficient means of purifying tryptophan synthase.
- It can facilitate further studies on enzyme function and structure.
- The methods described are versatile for other protein purification tasks.
Frequently Asked Questions
Executive Industry Relevance
Rapid purification of wild type and mutant tryptophan synthase complexes enables efficient structural and functional studies of PLP-dependent enzymes, supporting target validation in early discovery. The streamlined workflow reduces timelines for protein production, accelerating hit-to-lead progression in enzyme-focused drug discovery programs. This approach enhances predictive confidence by providing high-purity, reproducible protein samples for mechanistic de-risking and assay development.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of catalytic mechanisms and allosteric behavior in tryptophan synthase to validate therapeutic hypotheses.
- Operational Value: Facilitates production of pure wild type and mutant complexes for structure-guided target de-risking.
Screening & Assay Development
- Scientific Value: Provides standardized, high-yield protein preparations suitable for developing biochemical and biophysical assays.
- Operational Value: Ensures batch-to-batch consistency in protein purity and concentration, critical for assay reproducibility.
Translational & Preclinical Research
- Scientific Value: Supports continuity from target validation to preclinical studies by enabling structural analysis of mutant forms linked to functional alterations.
- Operational Value: Allows generation of well-characterized protein variants to assess structure-function relationships in disease-relevant contexts.
Pipeline & Workflow Integration
The method integrates into early discovery workflows by delivering purified protein complexes for downstream applications in enzymology and structural biology.
- Discovery Biology: Supports hypothesis testing and pathway clarification through rapid access to wild type and mutant enzyme complexes.
- Screening: Enables assay readiness via reproducible purification of soluble, active protein suitable for ligand-binding and activity assays.
- Analytics: Generates quantitative outputs such as protein yield and purity metrics that inform go/no-go decisions in target validation.
- Translational Research: Connects discovery to preclinical work by providing protein samples for mechanistic studies that inform target selection.
- Enterprise Reuse: Establishes a reusable purification platform applicable to other non-tagged proteins across multiple projects.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target validation by reducing mechanistic ambiguity through structural insights.
- Operational Value: Improves standardization and scalability of protein production, reducing variability in experimental outcomes.
- Strategic Value: Enhances capital efficiency by shortening protein production timelines and enabling parallel testing of multiple mutants.
- Portfolio Impact: Supports risk-adjusted prioritization of targets by providing reliable structural and functional data early in the discovery pipeline.
Implementation Considerations
- Requires expertise in molecular biology, protein expression, and chromatographic purification techniques.
- Depends on access to sonication equipment, centrifuges, FPLC systems, and SDS-PAGE apparatus for analysis.
- Necessitates cross-team standardization of ammonium sulfate fractionation and SEC protocols to ensure reproducibility across laboratories.
- Involves adaptation considerations when applying the method to different protein systems, particularly regarding buffer conditions and salt concentrations.
- Includes practical limitations such as the need to perform initial steps promptly to prevent activity loss of tryptophan synthase during purification.
Biopharma FAQs
Chapters in this video
0:04
Introduction
0:44
Purification of Wild Type or Mutant Form of α2β2 StTS Complex
3:53
Optimized Crystallization for Wild Type and Mutant Form of the α2β2 StTS Comple
6:49
Results: Purification of the α- and β -Subunits of the Tryptophan Synthase and the Wild Type and Mutant α2β2 StTS Complexes
8:50
Conclusion
