This work was supported by the US National Institute of Health (GM097569).

<ol><li>Miles, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tryptophan+synthase%3A+a+multienzyme+complex+with+an+intramolecular+tunnel%5BTitle%5D)+AND+%22Chemical+Record%22%5BJournal%5D)">Tryptophan synthase: a multienzyme complex with an intramolecular tunnel.</a> Chemical Record. 1 (2), 140-151 (2001).</li>
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The authors have nothing to disclose and declare no competing financial interests.

We have successfully engineered mutant form &#945;2&#946;2 &#946;Q114A, &#945;2&#946;2 &#946;K167T, and &#945;2&#946;2 &#946;S377A StTS complexes for structure-function correlation studies. Initially, we have tried to purify the mutants using a previous purification protocol22, which requires &#945;2&#946;2 StTS complex crystallization with PEG 8000 and spermine during purification. Although crystallization rate depends on the mutant form and on the concentration of the complex in solution, being difficult to predict when crystals appear in a large solution volume. Crystallization could be achieved either after long periods (48-96 h) or being necessary the addition of extra amounts of PEG 8000 after crystallization initiation15.
Unfortunately, mutant forms of &#945;2&#946;2 StTS complex presented in this work were not successfully purified using this protocol since they failed to crystallize during the initial steps of the protocol, impairing crystallographic studies. Therefore, we have created a simple and efficient purification protocol comprising ammonium sulfate fractionation and size exclusion chromatography, which give high yields of wild type and mutant form of &#945;2&#946;2 StTS complex. This protocol is faster (1-2 days) and reproducible when compared with the previous protocol (5-7 days)15,22, since there is no crystallization requirements and protocol troubleshooting along purification. In addition, we have created new expression constructs and protocol to purify high amounts of the &#945;-subunit, &#946;-subunit and the reconstitute &#945;2&#946;2 StTS complex from the isolates.
Future application includes the recombination of wild type and mutant sub-units to perform functional and structural studies. Mutant &#945;2&#946;2 &#946;Q114A, &#945;2&#946;2 &#946;K167T, and &#945;2&#946;2 &#946;S377A complex crystallized in conditions containing higher concentrations of spermine (4-8 mM) when compared with the wild type form (2 mM). Therefore, it is worthwhile spending time on improving the quality of protein crystals by varying the concentration of precipitants and buffer pH. Single large crystal forms randomly grew in bicine buffered solution (pH 7.6-8.0) containing 6-11% PEG 8,000. The methods described in this work will be used to prepare crystal structures of the wild type and mutant forms of the &#945;2&#946;2 complex with different ligands within the &#945;- and &#946;-active sites, mimicking different intermediates and transition states involved in the conversion of indole and serine to tryptophan. The crystal structures of these mutants are anticipated to provide new insights into the mechanism and roles played by key residues in L-Tryptophan synthesis.

The tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2) 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 &#945;2&#946;2 TS complex (&#945;2&#946;2 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 &#945;-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 &#946;-catalytic site contains a pyridoxal 5&#8242;-phosphate (PLP) cofactor molecule bound to &#946;-Lys87 via a Schiff base, which functions as an electron sink in the course of the reactions at the enzyme &#946;-subunit3,7. The &#946;-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 &#945;- and &#946;-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 (&#946;-Gln114Ala, &#946;-Lys167Thr, and &#946;-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 &#946;-subunit.
Detailed research on the catalytic mechanism of &#945;2&#946;2StTS was initiated by the research group of Edith W. Miles. Early studies with native Escherichia coli &#945;2&#946;2 TS complex have focused on the purification and characterization of the isolated &#945;-subunit8,9, isolated &#946;-subunit10,11 and the reconstitution of the &#945;2&#946;2 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 &#945;2&#946;2 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 &#945;2&#946;2 TS complex were purified and crystalized14,15. The recombinant &#945;2&#946;2 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 &#945;2&#946;2 StTS complex were reported14. However, long and thin needle like &#945;2&#946;2 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 &#945;2&#946;2 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 &#945;2&#946;2 StTS complex was stored for 16-48 h at 4 &#176;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 &#945;2&#946;2 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 &#176;C rather than at 4, 30, or 42 &#176;C and grew to maximum dimensions within 3 days15. Several &#945;2&#946;2 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 &#945;-subunit (&#945;StTS), isolated &#946;-subunit (&#946;StTS), reconstituted &#945;2&#946;2 StTS complex from the isolated subunits, and wild type and mutant forms of the &#945;2&#946;2 StTS complex. The advantages over past protocols are considerable since purification time was reduced significantly and crystallization and cryoprotection were optimized. Mutant forms of &#945;2&#946;2 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 &#945;2&#946;2 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 &#197;, 4HT3 at 1.30 &#197;, 4HPJ at 1.45 &#197;, 6DZ4 at 1.45 &#197; resolution). The refined crystal structures of mutant form of &#945;2&#946;2 StTS complex are anticipated to provide new insights into the mechanism and roles played by essential amino acid residues involved in L-Tryptophan synthesis.

<table><tbody><tr><td>15 mL 10 kDa filter</td><td>MilliporeSigma</td><td>UFC901024</td><td>centrifugal filter unit</td></tr><tr><td>15 mL 100 kDa filter</td><td>MilliporeSigma</td><td>UFC910024</td><td>centrifugal filter unit</td></tr><tr><td>2 mL cryogenic vials</td><td>Corning</td><td>CLS430489</td><td>Cryogenic vials</td></tr><tr><td>2 mL microcentrifuge tubes</td><td>Fisher Scientific</td><td>05-408-141</td><td>microcentrifuge tubes</td></tr><tr><td>24-well Cryschem Plate</td><td>Hampton Research</td><td>HR3-158</td><td>24-well sitting drop plates</td></tr><tr><td>2-mercaptoethanol</td><td>Fisher Scientific</td><td>O3446I-100</td><td>Chemical</td></tr><tr><td>50 mL centrifuge conical tubes</td><td>Thermo Scientific</td><td>12-565-270</td><td>centrifuge conical tubes</td></tr><tr><td>AB15 ACCUMET Basic</td><td>Fisher Scientific</td><td>13-636-AB15</td><td>pH meter</td></tr><tr><td>Agarose</td><td>Fisher Scientific</td><td>BP1356-100</td><td>Agarose gel</td></tr><tr><td>ammonium sulfate</td><td>Fisher Scientific</td><td>A702-500</td><td>Chemical</td></tr><tr><td>Ampicillin</td><td>Fisher Scientific</td><td>BP1760-5</td><td>Antibiotic</td></tr><tr><td>Bacterial incubator</td><td>Fisher Scientific</td><td>S35836</td><td>incubator.</td></tr><tr><td>BamHI</td><td>New England Biolabs</td><td>R0136S</td><td>Restriction enzyme</td></tr><tr><td>bicine</td><td>Fisher Scientific</td><td>BP2646100</td><td>Chemical</td></tr><tr><td>Branson 450 Digital Sonifier</td><td>Brason</td><td>B450</td><td>Cell disruptor</td></tr><tr><td>Cesium chloride</td><td>Fisher Scientific</td><td>BP210-100</td><td>Chemical</td></tr><tr><td>Cesium hydroxide</td><td>Acros Organics</td><td>AC213601000</td><td>Chemical</td></tr><tr><td>Chloramphenicol</td><td>Fisher Scientific</td><td>BP904-100</td><td>Antibiotic</td></tr><tr><td>dimethyl sulfoxide</td><td>Fisher Scientific</td><td>D1391</td><td>Chemical</td></tr><tr><td>dithiothreitol</td><td>Fisher Scientific</td><td>BP172-5</td><td>Chemical</td></tr><tr><td>DNA Polymerase</td><td>Thermo Scientific</td><td>F530S</td><td>HF polymerase</td></tr><tr><td>dNTP Set</td><td>Invitrogen</td><td>10-297-018</td><td>dNTPs set</td></tr><tr><td>EcoRI</td><td>New England Biolabs</td><td>R0101S</td><td>Restriction enzyme</td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Fisher Scientific</td><td>S311-100</td><td>Chemical</td></tr><tr><td>Excella E25R Orbital Shaker</td><td>Eppendorf New Brunswick</td><td>M1353-0004</td><td>Orbital incubator</td></tr><tr><td>GE AKTA Prime Plus</td><td>GE Healthcare</td><td>8149-30-0004</td><td>FPLC</td></tr><tr><td>Gel Extraction Kit</td><td>Invitrogen</td><td>K210012</td><td>DNA purification kit</td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>G33-500</td><td>Chemical</td></tr><tr><td>HindIII</td><td>New England Biolabs</td><td>R0104S</td><td>Restriction enzyme</td></tr><tr><td>His-Trap columns</td><td>GE Healthcare</td><td>GE17-5255-01</td><td>5 mL Histrap column</td></tr><tr><td>imidazole</td><td>Fisher Scientific</td><td>O3196-500</td><td>Chemical</td></tr><tr><td>IPTG</td><td>Thermo Fisher Scientific</td><td>R0392</td><td>Inducer</td></tr><tr><td>Kanamycin</td><td>Fisher Scientific</td><td>BP906-5</td><td>Antibiotic</td></tr><tr><td>Kelvinator Series-100</td><td>Kelvinator</td><td>discontinued</td><td>Ultra low freezer</td></tr><tr><td>LB broth</td><td>Fisher Scientific</td><td>BP1426-500</td><td>Liquid broth</td></tr><tr><td>Luria Bertani agar</td><td>Fisher Scientific</td><td>BP1425-2</td><td>Solid broth</td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>S271-500</td><td>Chemical</td></tr><tr><td>NcoI</td><td>New England Biolabs</td><td>R0193S</td><td>Restriction enzyme</td></tr><tr><td>Ni-NTA affinity beads</td><td>Thermo Fisher Scientific</td><td>R90115</td><td>Ni-NTA agarose beads</td></tr><tr><td>PEG 8000</td><td>Fisher Scientific</td><td>BP233-100</td><td>Chemical</td></tr><tr><td>phenylmethylsulfonyl fluoride</td><td>Fisher Scientific</td><td>44-865-0</td><td>Chemical</td></tr><tr><td>pyridoxal phosphate</td><td>Acros Organics</td><td>AC228170010</td><td>Chemical</td></tr><tr><td>S-200 HR</td><td>Cytiva</td><td>45-000-196</td><td>Size exclusion column</td></tr><tr><td>SacI</td><td>New England Biolabs</td><td>R0156S</td><td>Restriction enzyme</td></tr><tr><td>Sodium hydroxide</td><td>Fisher Scientific</td><td>S318-100</td><td>Chemical</td></tr><tr><td>Sorvall RC-5B centrifuge</td><td>Sorvall</td><td>8327-30-1004</td><td>Floor cetrifuge</td></tr><tr><td>Spermine</td><td>Acros Organics</td><td>AC132750010</td><td>Chemical</td></tr><tr><td>Superdex 200 prep grade</td><td>Cytiva</td><td>45-002-491</td><td>Size exclusion column</td></tr><tr><td>T4 DNA ligase</td><td>New England Biolabs</td><td>M0202S</td><td>DNA liagse</td></tr><tr><td>Tris</td><td>Fisher Scientific</td><td>BP152-500</td><td>Chemical</td></tr><tr><td>Ubl-specific protease 1</td><td>Thermo Scientific</td><td>12588018</td><td>SUMO Protease</td></tr></tbody></table>

pcr mutagenesis, cloning, expression, fast protein purification protocols and crystallization of the wild type and mutant forms of tryptophan synthase

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 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 &#945;- and isolated &#946;-subunit allowed the purification of high amounts of pure subunits and &#945;2&#946;2 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 &#946;-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant &#945;2&#946;2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of &#945;2&#946;2 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 &#945;2&#946;2 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 &#945;2&#946;2 StTS complexes.

Purification of the &#945;- and &#946;-subunits of the tryptophan synthase 
The &#945;-subunit (&#945;StTS) and the &#946;-subunit (&#946;StTS) 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-&#945;StTS (lane &#945;ON) and His6-SUMO-&#946;StTS (lane &#946;ON) fusion protein. The purification protocol described in this work allowed purification of both subunits individually within 2 days. The first day was used to purify each protein by Ni-NTA affinity chromatography, ammonium sulfate precipitation followed by His-SUMO-tag cleavage, removal of His-SUMO-tag traces, and protein concentration. Figure 1B and 1C show representative SDS-PAGE results of the &#945;-subunit and the &#946;-subunit purification, respectively. On the second day, the concentrate &#945;-subunit, &#946;-subunit, and the &#945;2&#946;2 StTS complex from the &#945;- and &#946;-subunits were loaded on a size exclusion chromatography column. Figure 1D shows a typical elution profile of &#945;StTS, &#946;StTS, and &#945;2&#946;2StTS complexes on a S-200 HR size exclusion column. Figure 1E shows a representative SDS-PAGE result of the collected peak fractions. The purest peak fractions were pooled, concentrated, and the &#945;2&#946;2 StTS complex was used for protein crystallization studies.
Purification of the wild type and mutant &#945;2&#946;2 StTS complex 
Another rapid and efficient protocol to purify the wild type and mutant form of the &#945;2&#946;2 S. typhimurium tryptophan synthase complex is described in this work. Figure 2 shows a representation of the pEBA-10 construct containing the wild type translationally coupling gene (trpA and trpB) encoding for the &#945;- and &#946;-subunits22. The two-step PCR mutagenesis protocol to generate mutant forms of the &#945;2&#946;2 StTS complex is depicted in Figure 3.
The coding regions of mutant &#945;2&#946;2 StTS complex in pEBA10 were confirmed by DNA sequencing and used to transform E. coli strain CB149 cells26. The wild type and mutant form of the &#945;2&#946;2 StTS complex were overexpressed and the recombinant proteins were purified successfully within 1-2 days. Ammonium sulfate fractionation at room temperature readily removed most of the contaminant proteins from the heterologous expression system (Figure 4A, lanes 20P, 30P and 40S). A representative elution profile with relative elution position of &#945;2&#946;2 StTS (143.06 kDa) complex on a HiPrep 16/60 Sephacryl S-200 HR size exclusion chromatography column is shown in Figure 4B. The purity of the excluded peak fractions was SDS-PAGE analyzed before pooling (Figure 4C).
Optimization of wild type and mutant &#945;2&#946;2 tryptophan synthase complex crystallization 
Aliquots of wild type and mutant &#945;2&#946;2 StTScomplex at 15 mg ml-1 were used to set up 24-well sitting drop plates. Typically, droplets consisting of 5 &#181;L protein solution and the equivalent volume of reservoir solution were equilibrated against 500 &#181;L of reservoir solution (Figure 5). Spermine is required to crystallize the wild type and mutant &#945;2&#946;2 StTS complex22. While the final concentration of spermine to crystallize the wild type is 2 mM, the concentration of spermine to crystalize the mutant complex in this work showed to be slightly higher (4-8 mM).
Large single crystals were obtained through a fine crystallization optimization, varying PEG 8000 (6-11%) and bicine buffer pH. Crystals with different morphologies appeared in 2-5 days and crystals grew to full size within two weeks (Figure 6). Prior to X-ray diffraction data collection, crystals were soaked in cryoprotectant solution (reservoir buffer containing up 30% dimethyl sulfoxide). The optimized process resulted in quality crystals suitable for X-ray diffraction measurements at near atomic resolution.
X-ray diffraction data analysis 
A crystal structure of the wild type &#945;2&#946;2 StTS complex was prepared with methods described in this article and X-ray diffraction data was collected at near atomic resolution. The crystal was soaked in cryoprotective solution containing F9 inhibitor (2- ({[4- (Trifluoromethoxy)Phenyl]Sulfonyl}Amino)Ethyl Dihydrogen Phosphate) and L-Tryptophan.
A complete X-ray diffraction data set was collected on the SIBYLS synchrotron beamline 12.3.1 at the Advanced Light Source (Berkeley-CA) by rotating the crystal 360&#176; in increments of 0.5&#176;. X-ray diffraction intensities were processed, and data-collection statistics are summarized in Table 1. Symmetry analysis indicates that the crystal belonged to the monoclinic space group C2. The unit-cell parameters are a = 182.55, b = 59.30, c = 67.37&#197;, &#945; = 90.00, &#946; = 94.82, &#947; = 90.00&#176;. The calculated value of the Matthews coefficient (Vm = 2.57 &#197;3 Da-1) suggests the presence of one TS heterodimer molecule (&#945;&#946; StTS) in the asymmetric unit of the crystal with a solvent content of 52.08%35,36. All X-ray data were collected at low temperatures (100 K) to improve the diffraction quality and decrease the radiation decay. The &#945;2&#946;2 StTS crystal structure in complex was solved by the molecular replacement method using the wild type &#945;&#946; StTS model in complex with the inhibitor F9 at the &#945;-site and cesium ion at the metal coordination site (PDB ID code: 4HT3). The final coordinate file and the structure factors were deposited in the PDB with accession code 5CGQ (Figure 7A). The crystal structure of the wild type &#945;2&#946;2 StTS complex with inhibitor F9 at the enzyme &#945;-site (Figure 7B), cesium ion at the metal coordination site (Figure 7C), the cofactor pyridoxal 5&#39;-phosphate covalently bonded to &#946;Lys87 (Figure 7D), and the product L-tryptophan at the enzyme &#946;-site (Figure 7E) was solved at 1.18 Angstrom resolution. Model 5CGQ is the highest resolution &#945;2&#946;2 StTS crystal structure deposited in the PDB to date.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61839/61839fig1v2.jpg" /> 
Figure 1: Purification of the &#945;- and &#946;-subunits and the &#945;2&#946;2StTS complex. (A) Recombinant protein expression. 12% SDS-PAGE gel of the overnight expression profile of SUMO-&#945;StTS (&#945;ON) and SUMO-&#946;StTS (&#946;ON) after IPTG induction at 30 &#176;C (&#945;/&#946;0 prior IPTG induction). (B, C) Ni-NTA affinity chromatography followed by ammonium sulfate precipitation (60% saturation), (S) clarified crude extract (FT1) Ni-NTA column pass through sample (W) column wash sample (E) eluate sample (60S) and (60P) supernatant and precipitate fractions after high-speed centrifugation, respectively (D) SUMO-protease digestion product (FT2) Ni-NTA column pass through sample containing the tag-less &#945;StTS or &#946;StTS subunit. (D) elution profile of &#945;StTS subunit (28.67 kDa), &#946;StTS subunit (42.86 kDa), and &#945;2&#946;2 StTS complex (143.06 kDa) with a HiPrep 16/60 Sephacryl S-200 HR size exclusion chromatography column. Each run was performed separately. (E) SDS-PAGE gels of the excluded peak fractions from each individual chromatography. While 15% SDS-PAGE gels were prepared to analyze &#945;2&#946;2 StTS complex and &#945;StTS subunit, a 12% SDS-PAGE gel was prepared to analyze the &#946;StTS subunit. Lane MW, molecular-weight markers in kDa. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61839/61839fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61839/61839fig2v2.jpg" /> 
Figure 2: Representation of the construct pEBA-10.&#160;(A) Representation of the wild type translationally coupling gene (trpA and trpB) encoding the &#945;- and &#946;-subunits of the tryptophan synthase from bacterium Salmonella enterica serovar typhimurium (Yang, Ahmed et al. 1996). (B) The vector contains an ampicillin resistance (amp) gene, a replication origin (ori), a lacIq gene to better&#160;shutdown a lac promoter&#160;in absence of IPTG inducer, and the LacI-repressed promoter. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61839/61839fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61839/61839fig3v2.jpg" /> 
Figure 3: Overall representation of the two-step PCR mutagenesis protocol.&#160;The pEBA-10 vector was used as a DNA template. The first round of PCR was prepared with primers TS-FW-NcoI and MUT-REV (a reverse primer containing a mutation) to generate the first fragment and primers TS-Rev-SacI and MUT-FW (a forward primer containing a mutation) to generate the second fragment). Fragments were gel purified and equimolarly combined, heat denatured, and annealed. The recombinant strands were extended with polymerase and deoxyribonucleotides. The second round of PCR was prepared with primers TS-FW-NcoI and TS-Rev-SacI. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61839/61839fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61839/61839fig4v2.jpg" /> 
Figure 4: Purification of wild type and mutant form of &#945;2&#946;2 StTS complex.&#160;(A) 12% SDS-PAGE gel of samples collected along the ammonium sulfate precipitation using 20, 30, 40 and 50% ammonium sulfate saturation at room temperature: (CE) crude extract (S) and (P) supernatant and precipitate fractions after high-speed centrifugation. (B) Elution profile of &#945;2&#946;2 StTS (143.06 kDa) complex with a HiPrep 16/60 Sephacryl S-200 HR size exclusion chromatography column. (C) 12% SDS-PAGE gel picture of the excluded peak fractions. Lane MW, molecular-weight markers in kDa (Precision Plus Protein Unstained Standards, Bio-Rad). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61839/61839fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61839/61839fig5v2.jpg" /> 
Figure 5: Crystallization optimization for wild type and mutant form of &#945;2&#946;2 StTS complex.&#160;Crystals were grown in 50 mM Bicine-CsOH buffer containing 50 mM CsCl2. The concentration of polyethylene glycol 8000 (6-11%) and spermine (2-8 mM) were varied to obtain single large crystals forms to perform structural studies by X-ray protein crystallography. (A) Bicine-CsOH, pH 7.6. (B) Bicine-CsOH, pH 7.8. (C) Bicine-CsOH, pH 8.0. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61839/61839fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61839/61839fig6v2.jpg" /> 
Figure 6: Photomicrograph of crystals of wild type and mutant form of &#945;2&#946;2 StTS complex.&#160;Crystals differ in morphology, but they belong to the space group C2. The crystals grew to their full dimensions in the final conditions after two weeks. Crystals of approximately 0.20 x 0.15 x 0.10 mm in size. (A-D) PLP holo-crystals in complex with cesium ion at the metal coordination site of the wild type form (column A), mutant form &#945;2&#946;2 &#946;Q114A (column B), &#945;2&#946;2 &#946;K167T (column C), and &#945;2&#946;2 &#946;S377A (column D). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61839/61839fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61839/61839fig7v2.jpg" /> 
Figure 7: Overall visualization of crystal structure and validation of electron density maps obtained after crystal structure refinement.&#160;(A) crystal structure of the wild type &#945;2&#946;2 StTS complex with inhibitor F9 at the enzyme &#945;-site (yellow colored), cesium ion at the metal coordination site (blue colored), the cofactor pyridoxal 5&#39;-phosphate covalent bonded to &#946;Lys87 (green colored), and the product L-tryptophan at the enzyme &#946;-site (cyan colored) at 1.18 Angstrom resolution. While the &#945;-subunit is colored in light blue, the &#946;-subunit is colored in salmon. (B-E) Electron density maps contoured at 1.0 r.m.s. level around (B) inhibitor F9 (C) cesium ion (D) pyridoxal-5&#8242;-phosphate, and (E) L-Tryptophan. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61839/61839fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Data collection and processing</td>
		</tr>
		<tr>
			<td>X-ray source / Beam line</td>
			<td>ALS Beamline 12.3.1</td>
		</tr>
		<tr>
			<td>Wavelength (&#197;)</td>
			<td>10,000</td>
		</tr>
		<tr>
			<td>Resolution (&#197;)</td>
			<td>40.00 - 1.18 (1.24 - 1.18)</td>
		</tr>
		<tr>
			<td>Total number of reflections</td>
			<td>2151280 (252941)</td>
		</tr>
		<tr>
			<td>Total number unique reflections</td>
			<td>231646 (32187)</td>
		</tr>
		<tr>
			<td>Space group for indexing, scaling and merging</td>
			<td>C 1 2 1</td>
		</tr>
		<tr>
			<td colspan="2">Cell dimensions</td>
		</tr>
		<tr>
			<td>a, b, c (&#197;)</td>
			<td>182.55, 59.30, 67.37</td>
		</tr>
		<tr>
			<td>&#945;, &#946;, &#947; (&#176;)</td>
			<td>90.00, 94.82, 90.00</td>
		</tr>
		<tr>
			<td>Mosaicity</td>
			<td>0.61</td>
		</tr>
		<tr>
			<td>Matthews volume VM (&#197;3 Da-1)</td>
			<td>2.57</td>
		</tr>
		<tr>
			<td>Rmeas (%)</td>
			<td>8.6 (93.0)</td>
		</tr>
		<tr>
			<td>&#60;I/&#963;(I)&#62;</td>
			<td>14.7 (3.0)</td>
		</tr>
		<tr>
			<td>CC1/2 (%)</td>
			<td>0.999 (0.778)</td>
		</tr>
		<tr>
			<td>Completeness (%)</td>
			<td>98.6 (94.2)</td>
		</tr>
		<tr>
			<td>Multiplicity</td>
			<td>9.3 (7.9)</td>
		</tr>
		<tr>
			<td colspan="2">Refinement statistics</td>
		</tr>
		<tr>
			<td>Rwork/Rfree (%)</td>
			<td>14.04 / 16.05</td>
		</tr>
		<tr>
			<td>RMSD bond length (&#197;)</td>
			<td>0.0120</td>
		</tr>
		<tr>
			<td>RMSD bond angle (&#176;)</td>
			<td>14,059</td>
		</tr>
		<tr>
			<td>Ramachandran favored</td>
			<td>515 (96.44%)</td>
		</tr>
		<tr>
			<td>Ramachandran allowed</td>
			<td>16 (3.00%)</td>
		</tr>
		<tr>
			<td>Ramachandran disallowed</td>
			<td>3 (0.56%)</td>
		</tr>
	</tbody>
</table>
Table 1: Data collection and processing. Values in parentheses are for the outer shell.

Watch this Scientific Journal Video about PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase at JoVE

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

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 (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand ...

pcr-mutagenesis-cloning-expression-fast-protein-purification

Nanyang Technological University

Research

JoVE Journal

Biochemistry

4.3K Views.  University of California-Riverside. Dit artikel presenteert een reeks opeenvolgende methoden voor de expressie en zuivering van Salmonella typhimurium tryptofaan synthase comp dit protocol een snel systeem om het eiwitcomplex in een dag te zuiveren. Behandelde methoden zijn site-gerichte mutagenese, eiwitexpressie in Escherichia coli,affiniteitschromatografie, gelfiltratiechromatografie en kristallisatie.

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

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion cofactors triggering a dimer-dodecamer transition. Upon metal ion binding, several structural modifications occur in the active site and at the interaction interface, shaping dimers to promote the self-assembly. To observe such modifications, stable oligomers must be isolated prior to structural study. Reported here is a method that allows the purification of stable dodecamers and dimers of TmPep1050, an M42 aminopeptidase of T. maritima, and their structure determination by X-ray crystallography. Dimers were prepared from dodecamers by removing metal ions with a chelating agent. Without their&#160;cofactor, dodecamers became less stable and were fully dissociated upon heating. The oligomeric structures were solved by the straightforward molecular replacement approach. To illustrate the methodology, the structure of a TmPep1050 variant, totally impaired in metal ion binding, is presented showing no further breakdown of dimers to monomers.

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

This protocol has been developed to study the dimer-dodecamer transition of TmPep1050, an M42 aminopeptidase, at the structural level. It is a straightforward pipeline starting from protein purification to X-ray data processing. Crystallogenesis, data set indexation, and molecular replacement have been emphasized through a case of study, TmPep1050H60A H307A variant.

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Bioengineering

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Biology

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Immunology and Infection

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins in less than 24 h using cell-free expression. After receiving DNA from a vendor, the gene fragment is PCR-amplified, cut, circularized, and cryo-banked. A small amount of the banked DNA is then diluted and amplified significantly (up to 106x) using isothermal rolling circle amplification (RCA). RCA can yield microgram quantities of the minimal expression template from picogram levels of starting material (mg levels if all starting synthetic fragment is used). In this work, a starting amount of 20 pg resulted in 4 &#181;g of the final product. The resulting RCA product (concatemer of the minimal template) can be added directly to a cell-free reaction with no purification steps. Due to this method being entirely PCR-based, it may enable future high-throughput screening efforts when coupled with automated liquid handling systems.

The study describes a protocol for creating large (&#181;g-mg) quantities of DNA for protein screening campaigns from synthetic gene fragments without cloning or using living cells. The minimal template is enzymatically digested and circularized and then amplified using isothermal rolling circle amplification. Cell-free expression reactions could be performed with the unpurified product.

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins ...

Purification of a High Molecular Mass Protein in Streptococcus mutans

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has been disrupted. Loss of function following gene disruption is subsequently restored by exogenous addition of the product of the disrupted gene. This helps to determine the function of the gene. A method previously described involves generating a gtfC gene-disrupted Streptococcus mutans strain. Here, an undemanding method is described for purifying the gtfC gene product from the newly generated S. mutans strain following the gene disruption. It involves the addition of a polyhistidine-coding sequence at the 3&#8242; end of the gene of interest, which allows simple purification of the gene product using immobilized metal affinity chromatography. No enzymatic reactions other than PCR are required for the genetic modification in this method. The restoration of the gene product by exogenous addition after gene disruption is an efficient method for determining gene function, which may also be adapted to different species.

Purification of a High Molecular Mass Protein in Streptococcus mutans

Described here is a simple method for the purification of a gene product in Streptococcus mutans. This technique may be advantageous in the purification of proteins, especially membrane proteins and high molecular mass proteins, and can be used with various other bacterial species.

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has ...

Cell-Type Specific Protein Purification and Identification from Complex Tissues Using a Mutant Methionine tRNA Synthetase Mouse Line

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol describes in vivo labeling and subsequent purification of newly synthesized proteins using an engineered mouse line to direct protein labeling to specific cellular populations. It is an inducible line by Cre recombinase expression of L274G-Methionine tRNA synthetase (MetRS*), enabling azidonorleucine (ANL) incorporation to the proteins, which otherwise will not occur. Using the method described here, it is possible to purify cell-type-specific proteomes labeled in vivo and detect subtle changes in protein content due to sample complexity reduction.

This protocol describes how to perform cell-type-specific protein labeling with azidonorleucine (ANL) using a mouse line expressing a mutant L274G-Methionine tRNA synthetase (MetRS*) and the necessary steps for labeled cell-type-specific proteins isolation. We outline two possible ANL administration routes in live mice by (1) drinking water and (2) intraperitoneal injections.

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

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