A frozen culture of Salmonella typhimurium LT2 was a generous gift from Dr. Lucia Freytag. Rabbit anti-LamB polyclonal antibody was a generous gift from Dr. Thomas Silhavy. Endonucleases (NdeI and XhoI) were obtained from New England Biolabs, Ipswich, MA. Culturing media, Teknova Brilliant Blue R-250 and most chemicals were obtained from Fisher Scientific, Hercules, CA. All polyethylene glycols, lauroyl sarcosine (LS), kanamycin, dithiothreitol (DTT), 4-chloro-1-naphthol (4CN), and goat anti-rabbit immunoglobulin G (IgG) were obtained from Sigma-Aldrich, St. Louis, MO. Rabbit anti-YshA antisera were produced by Covance, Denver, PA. Streptavidin-bound horseradish peroxidase (SHRP) was obtained from Jackson ImmunoResearch, West Grove, PA. PCR master mix with Taq polymerase and T4 Ligase were obtained from Promega, Madison, WI. QIAPrep Miniprep and QIAQuick gel extraction kits were obtained from QIAGEN, Valencia, CA. Site-directed mutagenesis Quik-Change kit was obtained from Stratagene, La Jolla, CA. All oligonucleotides were synthesized by Integrated DNA technologies, Coralville, IA. Proteinase K, all 4–12% BisTris polyacrylamide gels, nitrocellulose membranes, molecular weight standards, electrophoresis materials, buffers and apparatus were obtained from Invitrogen, Carlsbad, CA. Detergents tetraoxyethylene monooctyl ether (C8E4) and n-dodecyl-β-D-maltopyranoside (DDM) were obtained from Anatrace, Maumee, OH. Isopropyl-β-D-1-thiogalactopyranoside (IPTG) was obtained from Inalco Spa, Milano, Italy. Palmitoyl-oleoyl-phosphatidyl-choline (POPC), palmitoyl-oleoyl-phosphatidyl-glycerol (POPG), and lissamine rhodamine B-palmitoyl-oleoyl-phosphatidyl-ethanolamine (RhoPE) were obtained from Avanti Polar Lipids, Alabaster, AL.
2.2 Cloning and mutagenesis of gene yshA
The cells from a 3 mL culture of S. typhimurium LT2 grown to OD600 = 1 were harvested and lysed using the QIAPrep Miniprep kit alkaline cell lysis protocol. Proteinase K was added to the cell lysate to a final concentration of 0.1 mg/ml. The cell lysate was centrifuged to pellet cellular debris and the genomic DNA was extracted from the aqueous phase by the addition of phenol: chloroform: isoamyl alcohol (25:24:1) solution and then washed with chloroform: isoamyl alcohol (24:1) solution. All washing took place in the phase-lock gel tubes supplied with the QIAPrep Miniprep kit which allowed the liquid to be removed by centrifugation and the DNA to remain in the upper compartment. After two washes, the genomic DNA was precipitated from the extract by the addition of 3 M sodium acetate and pure ethanol, and the solvent was evaporated at room temperature. The DNA was washed twice with 70% ethanol and dried at room temperature again. The DNA was subsequently re-dissolved in a standard Tris-EDTA (ethylenediamine tetraacetic acid), pH 8.0.
Gene yshA, which encodes a putative outer membrane, was cloned from the isolated genomic DNA of Salmonella typhimurium LT2 by PCR using PCR Master Mix with Taq polymerase and the following oligonucleotide primers: forward primer 5′-GCC CCT GTA GCC CCG CAT ATG AAA TCT CTG AAT C-3′ and reverse primer 5′-CCA GTC CCC ATT AGC CCT CGA GTC AGA AGA AAT ACT TCG-3′. Gene yshA was inserted into the multiple-cloning site of pET24b between the NdeI and XhoI sites using the standard techniques of digesting the amplicon and the vector with the aforementioned endonucleases for 1 h at 37 °C, subsequently purifying the products by agarose gel electrophoresis and extraction using the QIAQuick gel extraction kit, and finally ligating the purified amplicon and vector with T4 ligase overnight at 14 °C following the manufacturers’ protocols. The resulting vector was named pTF01. The plasmid was purified from the ligase-reaction mixture using the QIAPrep Miniprep kit, amplified by transformation and culturing in E. coli, followed by harvesting via Miniprep kit, and sequencing to verify successful cloning.
The sequence encoding the N-terminal export signal peptide, a domain typically absent in mature outer membrane proteins, was deleted from the gene so that the signal peptide could not interfere with protein refolding. The signal peptide was predicted using the SignalP server (http://www.cbs.dtu.dk/services/SignalP/
) which uses neural networks and Hidden Markov Models to predict an export signal peptide and the position at which a signal peptidase will cleave the signal peptide from the protein precursor. The signal peptide of YshA was predicted to be cleaved C-terminal to Ala20. The plasmid vector carrying the cloned yshA
gene, pTF01, was subjected to site-directed mutagenesis to delete bases 4–60. These bases encoded amino acid residues 2-20, and their deletion resulted in the plasmid pTF01Δ2-20. Site-directed mutagenesis was performed using a Quik-Change kit, following the manufacturer’s protocols, using the following oligonucleotide primers: forward primer 5′-CTC TAG AAA TAA TTT TGT TTA ACT TTA AGA AGG AGA TAT ACA TAT GGG CGC TTA TGT AGA AAA CCG TGA GGC CTA-3′ and the antisense reverse primer 5′-TAG GCC TCA CGG TTT TCT ACA TAA GCG CCC ATA TGT ATA TCT CCT TCT TAA AGT TAA ACA AAA TTA TTT CTA GAG-3′. Plasmids were transformed into competent E. coli
DH5αF′ using standard techniques. The cells were plated on LB agar supplemented with 30 μg/mL of kanamycin for selective growth. Individual colonies were then selected and cultivated in LB broth supplemented with 30 μg/mL kanamycin overnight (approx. 14 h) at 37°C. The cells were pelleted by centrifugation, and the plasmids were harvested using the QIAPrep Miniprep kit.
2.3 Protein expression
The plasmids pTF01, pTF01Δ2-20, and pET24a (empty vector), were transformed into competent E. coli BL21(DE3) using standard techniques and grown in 1 L of LB broth with 30 μg/mL kanamycin at 37 °C until they reached an OD600 = 0.6. At this optical density protein expression was induced by the addition of 1 mM (final concentration) IPTG for 3 h. There were instances where IPTG was not added to cells transformed with either pTF01 or pET24a, and in these cases the cultures were grown to an OD600 = 1.0 before harvesting. The protein was expressed even in the absence of IPTG in this bacterial expression strain because it lacks control over basal levels of transcription and subsequent expression; this was desirable in order to more easily distinguish membrane-bound protein from inclusion body proteins. All cells were harvested by centrifugation at 5000 rpm for 20 min. at 4°C in a Sorvall RC-3C Plus centrifuge using a H6000A rotor. The supernatant was removed, and the pellets were resuspended in 10 mL of 50 mM sodium phosphate buffer, pH 7.6. These suspensions were then lysed via two passes through a cold French Pressure Cell. Lysates of cultures that were induced to express protein were processed for isolation of protein from the inclusion bodies, and the lysates of cultures that were not induced were processed for isolation of protein from membrane fractions.
2.4 Antibody production
To produce anti-YshA polyclonal antibodies, E. coli BL21 (DE3) transformed with pTF01 was induced to overexpress YshA, and the cells were harvested, and lysed as described above. To the protein denatured in 8 M urea was added an equal volume of 2X LDS loading dye with 0.1 M DTT, which was then boiled for 10 min. The sample was loaded onto a one-well preparative polyacrylamide gel (8×8 cm) and electrophoresed for 30 min at 200V. The protein bands were detected by Coomassie staining. The band corresponding to the approximate MW of YshA (27 kDa) was cut from the gel. The protein in the gel slice was electroeluted from the slice using the S&S Elutrap starter kit 46170 (Schleicher & Schuell) for 16 h following the manufacturer’s protocol. Approximately 2 mg of the purified protein was lyophilized and sent to Covance Research Products, Inc., where polyclonal antibodies were produced in NZW (New Zealand White) rabbits.
2.5 Subcellular fractionation
The cytosol, inner membrane, and outer membrane fractions were separated to identify where YshA localizes when expressed in vivo. The cellular lysates of the cultures transformed with pET24a and pTF01 grown as described above (not induced) were centrifuged at 10,000 × g at 4°C for 12 min to pellet the unbroken cells and inclusion bodies. The supernatant was then removed and centrifuged at 100,000 × g for 2 h at 4°C to obtain the cellular membranes. The total membrane pellet was resuspended by sonication and incubated with 25 mM LS (lauroyl sarcosine), 10 mM sodium phosphate buffer, pH 7.6 at room temperature for 45 min and then centrifuged at 100,000 × g to obtain the outer membrane pellet. The outer membrane pellet was resuspended in 150 mM C8E4, 50 mM sodium phosphate, pH 7.6 and stirred gently at room temperature for 1.5 h. The outer membrane extracts were then centrifuged at 100,000 × g for 1 h to remove detergent-insoluble material.
2.6 SDS-PAGE and western blot analysis
Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using either 10-well or 15-well PAGs. The samples were mixed 1:1 with 2x LDS loading dye with DTT and either boiled or not boiled (for electrophoretic mobility shift assay) before loading. Gels (8×8 cm) were electrophoresed either at 200 V (single gel) or 175 V (dual gels) for 30 to 40 min. Gels were stained with Teknova Brilliant Blue R-250 and destained with 40% methanol, 7% acetic acid. For western blots, dual gels were electrophoresed and one was transferred to nitrocellulose membranes using the Invitrogen Mini-Blot transfer module following the manufacturer’s protocol. The dried membranes were rewet with PBS with 0.5% TWEEN-20 (PBS-T) for 5 min. Next, the membranes were incubated in blocking buffer (5% powdered milk, PBS-T) for 30 min. Nitrocellulose membranes were then covered in blocking buffer containing either anti-LamB polyclonal antibodies diluted 1:750 or anti-YshA polyclonal antisera diluted 1:15,000 for 1 h followed by multiple rinses with PBS-T. Then the membranes were incubated with goat anti-rabbit IgG diluted 1:500 in blocking buffer for 1 h followed by rinsing. Next, the membranes were incubated in blocking buffer with 2 μg/ml SHRP for 1 h, and then rinsed. Finally, antibody-protein complexes were detected by immersing the membranes in 30% methanol, 0.03% hydrogen peroxide, and 0.6 mg/ml 4CN in PBS for less than 1 min, followed by rinsing with distilled deionized water.
2.7 Purification of YshA from inclusion bodies
The expression of the signal peptide deletion mutant, YshAΔ2-20, was induced by IPTG. Because the protein lacked a signal peptide and was so abundantly expressed, all of it was deposited into inclusion bodies. The cultures were grown, harvested, and lysed as described above. The cellular lysate was centrifuged at 12,500 × g for 30 min. at 4 °C to pellet inclusion bodies and other cellular debris. The supernatant was removed and the pellet was resuspended in 10 ml of 50 mM sodium phosphate, 25 mM LS, 1% (W/V) Triton X-100, pH 7.6 and rocked at room temperature for 1 hour. The detergent-insoluble material was pelleted and washed as above three more times. After the final detergent wash the pellet was washed twice with 50 mM sodium phosphate buffer, pH 7.6. The pellet was denatured with either 8 M urea or 250 mM SDS in 50 mM sodium phosphate buffer, pH 7.6 at room temperature by brief sonication, and then centrifuged at 100,000 × g for 1h at 4 °C to remove any remaining insoluble material.
The crude YshAΔ2-20 preparation from inclusion bodies was subjected to RP-HPLC using a Waters 600e controller and pump with Rheodyne 7725i injector. Various volumes of the crude prep (1–2 mL) were injected into a 2 ml sample loop. The proteins were resolved on either a VP 250×10 mm Nucleosil 100-7 C2 column (Macherey-Nagel) with a Trident high pressure inline filter (Restek) using a flow rate of 3 ml/min at ambient temperature or a Dynamax 250×10 mm Microsorb 300-5 C4 column (Varian) under the same conditions. The following solvents were used for the mobile phase 0.1% trifluoracetic acid (TFA) (solvent A), 0.1% TFA in acetonitrile (solvent B) and 0.1% TFA, 8 M urea (solvent C). The C2 column was equilibrated with 30% B, 70% solvent C for 5 min, followed by 70% solvent A, 30% solvent B for 10 min. Next, solvent B was linearly increased from 30% to 100% over 30 min. Then 100% solvent B flowed through the column for 5 min, followed by a linear decrease of solvent B to 30% (70% solvent A) over 5 min. The column was reequilibrated with 30% solvent B, 70% solvent C. Protein peaks were detected with a Waters 2487 Dual Absorbance Detector at 295 nm (to measure absorbance of tryptophan) and 220 nm (to measure absorbance of peptide backbone).
2.8 Protein folding
YshAΔ2-20 was refolded using a modified version of a two-step folding technique previously used to fold a mitochondrial porin [18
]. HPLC-purified YshAΔ2-20 was divided into approximately 1-mg aliquots prior to dehydration under vacuum. YshAΔ2-20 was rehydrated overnight in 300 μL of 250 mM SDS, 50 mM sodium phosphate, at pH 7.6. The protein solution was diluted with phosphate buffer until the SDS concentration was 112 mM and then concentrated to a volume of 0.25 mL using a Microcon YM-10 centrifugal filter unit (Millipore; Billerica, MA). To SDS-solubilized YshAΔ2-20 was added 122.5 mg powdered DDM, followed by dilution with 0.75 mL of 50 mM sodium phosphate and rotated end-over-end overnight at room temperature. The final concentration of stock YshAΔ2-20 in SDS/DDM was 40 μM YshAΔ2-20 in 28 mM SDS, 240 mM DDM, and 50 mM phosphate buffer pH 7.6. This stock was diluted appropriately for various experiments.
2.9 Liposome and proteoliposome preparation
Liposomes and proteoliposomes containing 90% POPC and 10% POPG (unlabeled samples) and 89.5% POPC, 10% POPG and 0.5% RhoPE (fluorescent-labeled samples) were prepared as follows. Lipid films were prepared by mixing chloroform-dissolved lipids in a glass vessel and evaporating the chloroform under a stream of gaseous N2. The lipid films were further dried under vacuum overnight. Lipid films were rehydrated in 50 mM sodium phosphate, pH 7.6 for 10–15 min by intermittent vortexing. The resuspended lipids were then subjected to 10 freeze-thaw cycles (freezing in a dry ice/ethanol bath for 3–4 min. and thawing in a ~60 °C water bath) to make multi-lamellar vesicles (MLVs). The MLVs were passed through a Lipidex extruder with successive membrane pore sizes of 0.4, 0.2 and finally 0.1 μm 10–20 times to make large unilamellar vesicles (LUVs).
Proteoliposomes were prepared by mixing 5 μM refolded YshAΔ2-20 prepared as described above and LUVs containing ~2.5 mM total lipid. The detergent was removed by adsorption onto BioBeads, a hydrophobic polystyrene resin, which allowed the lipids to spontaneously aggregate and reform liposomes [22
]. BioBeads were measured (1 g BioBeads/5 mL solution) and poured into a disposable 10 mL syringe with a filter needle threaded onto the Luer-Lok fitting. The beads were washed three times with full syringe volumes of methanol and five times with phosphate buffer. The protein/LUV solution was aspirated into the syringe slowly to avoid making bubbles, then rotated at room temperature for 1.5–2 h, and then ejected slowly. Proteoliposomes were then freeze-thawed as above, except thawing was performed in a water bath between 25 and 37 °C. Proteoliposomes were then extruded as above.
2.10 Circular Dichroism
Circular dichroism (CD) measurements were performed with a JASCO J-810 spectropolarimeter with a Pelltier-type thermal controller. Measurements were taken in the range of 185 nm to 250 nm for far-UV measurements and 250–350 for near-UV measurements. The samples were measured in either 0.1 or 1 cm cuvettes. The CD (ε; in mdeg) measured for each protein sample was converted to mean residue ellipticity (θ; deg*cm2
) using the following equation:
Where NA is the number of amino acids in the protein, [S] is the molar concentration of the protein in the sample, and l is the path length of the cuvette in cm.
2.11 Liposome flotation assay
Proteoliposomes were prepared with fluorescent-labeled LUVs as described above. Total lipid concentration was 5 mM, and YshAΔ2-20 was at a final concentration of 5 μM. Liposomes were prepared to the same lipid concentrations without protein, and detergent-refolded YshAΔ2-20 was diluted to 5 μM for control experiments. The assay was performed as described by Hong and Tamm with some modifications [23
]. A sucrose step gradient was prepared from bottom to top by carefully layering 30 μL of 30% sucrose, 40 μL each of 25%, 20%, and finally 10% sucrose in an Ultra Clear 8×20 mm centrifuge tube (Beckman). Sucrose solutions were made in either 50 mM sodium phosphate, pH 7.6 (non-denaturing sucrose) or 6 M urea (denaturing sucrose). Liposomes, detergent-solubilized YshAΔ2-20, and proteoliposomes were each mixed 1:1 with 60% either non-denaturing or denaturing sucrose. Samples applied to the denaturing sucrose gradients were also mixed with solid urea to a final concentration of 6 M and heated in a 95 °C water bath for 5–10 min to ensure complete protein denaturation. All samples were left on the bench to equilibrate at room temperature for at least 30 min, and then 50 μL of each sample was carefully pipetted at the bottom of a given gradient. The samples were then centrifuged for 1 h at approximately 100,000 × g in an Airfuge table-top centrifuge equipped with an A-95 rotor (Beckman) set to an air pressure of 30 psi. After centrifugation, 34 μL aliquots were taken from bottom to top and mixed 1:1 with sodium phosphate buffer. The aliquots were then transferred to a black 96-well plate (Corning) and fluorescence measurements were taken in a Synergy 2 (Biotek) fluorescent plate reader. Lissamine rhodamine fluorescence emission was measured at 590 nm after excitation at 540 nm. The tryptophan fluorescence emission of YshAΔ2-20 was measured at 360 nm after excitation at 284 nm.
2.12 Liposome swelling assay
Liposomes and proteoliposomes were prepared as above with liposome compositions of 90% POPC and 10 % POPG. For this assay 1 mM PEG 8000 was entrapped in the liposomes by adding it prior to the freeze-thaw cycles of the proteoliposome preparation. Total lipid concentration was 4 mM, and the final YshAΔ2-20 concentration was 2.5 μM. The samples were mixed in a 1 cm quartz cuvette with 1 mM of either arabinose, PEG 200, PEG 400, or PEG 600, and absorbance at 440 nm was measured in a Cary 50 UV-Vis spectrophotometer (Varian) using the kinetic measurement mode for at least 8 min. At the end of each measurement the pore-forming peptide melittin was added to a final concentration of 5 μM and the sample’s absorbance was measured again over the same time period.