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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.
Structural studies with tryptophan synthase (TS) bienzyme complex (α2β2 TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated α- and isolated β-subunit allowed the purification of high amounts of pure subunits and α2β2 StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC). To better understand the role of key residues at the enzyme β-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant α2β2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of α2β2 StTS complex in a single day. Both purification protocols described in this work have considerable advantages when compared with previous protocols to purify the same complex using PEG 8000 and spermine to crystalize the α2β2 StTS complex along the purification protocol. Crystallization of wild type and some mutant forms occurs under slightly different conditions, impairing the purification of some mutants using PEG 8000 and spermine. To prepare crystals suitable for x-ray crystallographic studies several efforts were made to optimize crystallization, crystal quality and cryoprotection. The methods presented here should be generally applicable for purification of tryptophan synthase subunits and wild type and mutant α2β2 StTS complexes.
The tryptophan synthase (TS) bienzyme complex (α2β2) is an allosteric enzyme, catalyzing the last two steps in the biosynthesis of the amino acid L-Tryptophan in bacteria, plants, and fungi1,2,3. Bacterium Salmonella enterica serovar typhimurium (St) causes a severe gastrointestinal infection in humans and other animals. Since humans and higher animals do not have TS (EC 4.2.1.20), the inhibition of S. typhimurium α2β2 TS complex (α2β2 StTS) has been explored as a potential drug target for the treatment of cryptosporidiosis and tuberculosis4, genital and ocular infections5, and for potential herbicide utilization in agriculture6. The α-subunit catalyzes the aldolytic cleavage of indole-3-glycerol-phosphate (IGP) to glyceraldehyde-3-phosphate (GAP) and indole, through the formation of an indolenine tautomer intermediate and subsequently carbon-carbon bond cleavage to produce GAP and indole3,6. The β-catalytic site contains a pyridoxal 5′-phosphate (PLP) cofactor molecule bound to β-Lys87 via a Schiff base, which functions as an electron sink in the course of the reactions at the enzyme β-subunit3,7. The β-site catalyzes the replacement of the L-Serine side-chain hydroxyl by indole to give L-Tryptophan and a water molecule in a PLP-dependent reaction. StTS serves as a longstanding model for the investigation of substrate channeling and allosteric communication within multi-enzyme complexes2,3. Bidirectional allosteric communication between the α- and β-subunits of TS is necessary to synchronize the catalytic steps and prevent indole release during L-Tryptophan synthesis3. To extend this effort, we have prepared several mutants (β-Gln114Ala, β-Lys167Thr, and β-Ser377Ala) by single point mutation to be used in further explorations of the relationship between enzyme structure, mechanism and function at the catalytic site of the StTS β-subunit.
Detailed research on the catalytic mechanism of α2β2StTS was initiated by the research group of Edith W. Miles. Early studies with native Escherichia coli α2β2 TS complex have focused on the purification and characterization of the isolated α-subunit8,9, isolated β-subunit10,11 and the reconstitution of the α2β2 TS complex from the isolated subunits12. Purification was carried out by ammonium sulfate precipitation, sample dialysis, DEAE-Sephadex chromatography, dialysis, and a second chromatographic round on a DEAE-Sephadex column12. In another protocol, the purification of the same complex was improved by loading the clarified cell lysate on a DEAE-Sephadex column followed by a chromatographic step on a Sepharose 4B column, ammonium sulfate precipitation and dialysis13. Both purification protocols last for 4-5 days. Escherichia coli α2β2 TS complex crystallized but crystals were not suitable for X-ray diffraction at that time.
In a novel study, recombinant and wild type forms of S. typhimurium α2β2 TS complex were purified and crystalized14,15. The recombinant α2β2 StTS complex was overexpressed in E. coli strain CB149 carrying the pEBA-10 expression vector. Initial crystallization and X-ray diffraction data collection and analysis of the α2β2 StTS complex were reported14. However, long and thin needle like α2β2 StTS crystals impaired structural studies. In an attempt to collect better X-ray diffraction data, another purification protocol was described to purify the wild type and mutant forms of the α2β2 StTS complex15. Purification was carried out with an initial precipitation using spermine and PEG 8,000 into the clarified cell lysate and a large bulky precipitate was removed by centrifugation. The supernatant fraction containing high amounts of α2β2 StTS complex was stored for 16-48 h at 4 °C until yellow crystals precipitated. Crystals were washed and extensively dialyzed against different buffers. Protein complex was recrystallized in buffer containing ammonium sulfate and dialyzed15. Although, protein crystallization depends on protein and precipitant concentrations in solution, it is difficult to monitor, predict, and reproduce purification for other mutant forms of α2β2 StTS complex in solution. This protocol has the advantage that it does not use any chromatographic methods; however, the disadvantages are the long purification time necessary to crystallize, dialyze, and recrystallize, typically requiring 5-7 days. To obtain crystals suitable for X-ray data collection, more than 600 crystallization conditions were evaluated using a combination and variation of protein concentration, temperature, precipitants (PEG 4,000, 6,000, and 8,000), and additives (CaCl2, MnCl2, ZnCl2, cadaverine, putrescine, spermine, or spermidine)15. Crystals had a better crystalline form and grew faster in conditions containing 12% PEG 8,000 and 2 mM spermine. Crystallization was more favorable at 25 °C rather than at 4, 30, or 42 °C and grew to maximum dimensions within 3 days15. Several α2β2 StTS crystal structures were reported at that time (1996-1999)16,17,18,19,20,21 and many other structures have been published to date.
Here, the main purpose is to present alternative protocols to purify tryptophan synthase and optimize protein crystallization. The present work shows significant improvements to purify the wild type isolated α-subunit (αStTS), isolated β-subunit (βStTS), reconstituted α2β2 StTS complex from the isolated subunits, and wild type and mutant forms of the α2β2 StTS complex. The advantages over past protocols are considerable since purification time was reduced significantly and crystallization and cryoprotection were optimized. Mutant forms of α2β2 StTS complex engineered in this work have crystallized near the same condition used for the wild type form. However, fine crystallization optimization was necessary to obtain large single crystals of sufficient quality for structure determination at near atomic resolution. To date, there are 134 tryptophan synthase crystal structures deposited in the Protein Data Bank (PDB), accounting 101, 31 and 2 crystal structures, respectively, for bacteria, archaea and eukaryote. Nicely, 73 structures belong to S. enterica serovar typhimurium and 5 crystal structures of the α2β2 StTS complex have resolution limits higher than 1.50 Angstroms. Not surprisingly, 4 out 5 were prepared in our research group (PDB IDs:5CGQ at 1.18 Å, 4HT3 at 1.30 Å, 4HPJ at 1.45 Å, 6DZ4 at 1.45 Å resolution). The refined crystal structures of mutant form of α2β2 StTS complex are anticipated to provide new insights into the mechanism and roles played by essential amino acid residues involved in L-Tryptophan synthesis.
1. Fast protocol to purify the α- and β-subunit and the recombined α2β2 StTS complex
2. Purification of the wild type or mutant form of the α2β2 StTS complex
3. Optimized crystallization for wild type and mutant form of the α2β2 StTS complex
NOTE: The initial crystallization condition for the α2β2 StTS complex was previously reported in conditions containing 12% PEG 8,000 and 2 mM spermine22.
4. X-ray diffraction data collection and α2β2 StTS complex structure solution
NOTE: Prior to X-ray diffraction data collection, prepare cryoprotectant solution for each crystal in advance. Use the specific reservoir solution to prepare 3 aliquots containing increasing concentrations of dimethyl sulfoxide in solution (10, 20, and 30% v/v) and specific ligand (s). Dimethyl sulfoxide was found to be a better cryoprotectant than glycerol, ethylene glycol, and PEG 200-300.
Purification of the α- and β-subunits of the tryptophan synthase
The α-subunit (αStTS) and the β-subunit (βStTS) of the Salmonella typhimurium tryptophan synthase were subcloned in the modified pET SUMO vector. Figure 1A shows representative SDS-PAGE results of two strong bands corresponding to the His6-SUMO-αS...
We have successfully engineered mutant form α2β2 βQ114A, α2β2 βK167T, and α2β2 βS377A StTS complexes for structure-function correlation studies. Initially, we have tried to purify the mutants using a previous purification protocol22, which requires α2β2 StTS complex crystallization with PEG 8000 and spermine during purification. Although c...
The authors have nothing to disclose and declare no competing financial interests.
This work was supported by the US National Institute of Health (GM097569).
Name | Company | Catalog Number | Comments |
15 mL 10 kDa filter | MilliporeSigma | UFC901024 | centrifugal filter unit |
15 mL 100 kDa filter | MilliporeSigma | UFC910024 | centrifugal filter unit |
2 mL cryogenic vials | Corning | CLS430489 | Cryogenic vials |
2 mL microcentrifuge tubes | Fisher Scientific | 05-408-141 | microcentrifuge tubes |
24-well Cryschem Plate | Hampton Research | HR3-158 | 24-well sitting drop plates |
2-mercaptoethanol | Fisher Scientific | O3446I-100 | Chemical |
50 mL centrifuge conical tubes | Thermo Scientific | 12-565-270 | centrifuge conical tubes |
AB15 ACCUMET Basic | Fisher Scientific | 13-636-AB15 | pH meter |
Agarose | Fisher Scientific | BP1356-100 | Agarose gel |
ammonium sulfate | Fisher Scientific | A702-500 | Chemical |
Ampicillin | Fisher Scientific | BP1760-5 | Antibiotic |
Bacterial incubator | Fisher Scientific | S35836 | incubator. |
BamHI | New England Biolabs | R0136S | Restriction enzyme |
bicine | Fisher Scientific | BP2646100 | Chemical |
Branson 450 Digital Sonifier | Brason | B450 | Cell disruptor |
Cesium chloride | Fisher Scientific | BP210-100 | Chemical |
Cesium hydroxide | Acros Organics | AC213601000 | Chemical |
Chloramphenicol | Fisher Scientific | BP904-100 | Antibiotic |
dimethyl sulfoxide | Fisher Scientific | D1391 | Chemical |
dithiothreitol | Fisher Scientific | BP172-5 | Chemical |
DNA Polymerase | Thermo Scientific | F530S | HF polymerase |
dNTP Set | Invitrogen | 10-297-018 | dNTPs set |
EcoRI | New England Biolabs | R0101S | Restriction enzyme |
Ethylenediaminetetraacetic acid | Fisher Scientific | S311-100 | Chemical |
Excella E25R Orbital Shaker | Eppendorf New Brunswick | M1353-0004 | Orbital incubator |
GE AKTA Prime Plus | GE Healthcare | 8149-30-0004 | FPLC |
Gel Extraction Kit | Invitrogen | K210012 | DNA purification kit |
Glycerol | Fisher Scientific | G33-500 | Chemical |
HindIII | New England Biolabs | R0104S | Restriction enzyme |
His-Trap columns | GE Healthcare | GE17-5255-01 | 5 mL Histrap column |
imidazole | Fisher Scientific | O3196-500 | Chemical |
IPTG | Thermo Fisher Scientific | R0392 | Inducer |
Kanamycin | Fisher Scientific | BP906-5 | Antibiotic |
Kelvinator Series-100 | Kelvinator | discontinued | Ultra low freezer |
LB broth | Fisher Scientific | BP1426-500 | Liquid broth |
Luria Bertani agar | Fisher Scientific | BP1425-2 | Solid broth |
NaCl | Fisher Scientific | S271-500 | Chemical |
NcoI | New England Biolabs | R0193S | Restriction enzyme |
Ni-NTA affinity beads | Thermo Fisher Scientific | R90115 | Ni-NTA agarose beads |
PEG 8000 | Fisher Scientific | BP233-100 | Chemical |
phenylmethylsulfonyl fluoride | Fisher Scientific | 44-865-0 | Chemical |
pyridoxal phosphate | Acros Organics | AC228170010 | Chemical |
S-200 HR | Cytiva | 45-000-196 | Size exclusion column |
SacI | New England Biolabs | R0156S | Restriction enzyme |
Sodium hydroxide | Fisher Scientific | S318-100 | Chemical |
Sorvall RC-5B centrifuge | Sorvall | 8327-30-1004 | Floor cetrifuge |
Spermine | Acros Organics | AC132750010 | Chemical |
Superdex 200 prep grade | Cytiva | 45-002-491 | Size exclusion column |
T4 DNA ligase | New England Biolabs | M0202S | DNA liagse |
Tris | Fisher Scientific | BP152-500 | Chemical |
Ubl-specific protease 1 | Thermo Scientific | 12588018 | SUMO Protease |
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