This work was supported by the Canadian Institute of Health Research. CSC was funded by a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada. FD is a Tier II Canada Research Chair.

Overall Reconstitution Process
 The reconstitution process starts by mixing the membrane scaffold protein (MSP) with the purified MalFGK2 complex in the presence of detergent-solubilized phospholipids. The step is followed by the slow removal of the detergent by an adsorbent polystyrene material called Bio-Beads or Amberlite (Figure 1). The auto-assembly process occurs most likely because of the apolar interactions between the hydrophobic phospholipids, the MalFGK2 complex and the surface of the MSP amphipathic protein. The final product is a discoid particle made of two molecules of MSP wrapping around the MalFGK2 complex. The particles are separated from the adducts and aggregates by ultra-centrifugation and analytical size-exclusion chromatography. The particles are characterized by native-gel electrophoresis and dynamic light scattering spectroscopy.
 1. Preparation of the Membrane Scaffold Protein, MSP
 <ol>
 <li>The his-tagged MSP (version MSP1D11) is produced from plasmid pMSP1D1 in E. coli BL21 (DE3) induced at OD600~0.5 with 0.5 mM isopropyl &beta;-D-1-thiogalactopyranoside for 3 hr at 37 &deg;C.</li>
 <li>The cells are harvested by centrifugation at 5,000 x g for 10 min at 4 &deg;C and resuspended in TSG10 buffer containing 100 &mu;M phenylmethanesulfonyl fluoride. </li>
 <li>The cells are lysed with a French press (3x) at 8,000 psi and the insoluble material is removed by centrifugation at 5,000 x g for 10 min at 4 &deg;C. The soluble fraction containing the MSP is isolated by ultra-centrifugation at 125,000 x g for 45 min. </li>
 <li>The MSP is purified by nickel-chelating chromatography using ~1.5 ml Ni Sepharose HP in TSG10 buffer. Contaminants are washed away with TSG10 buffer containing 50 mM imidazole. The MSP is eluted with TSG10 buffer containing 600 mM imidazole. The purified protein is dialyzed in TSG10 buffer and stored in -70 &deg;C at a protein concentration of ~10-15 mg/ml.</li>
 </ol>
 2. Preparation of the MalFGK2 Complex
 <ol>
 <li>The MalFGK2 complex, his-tagged at the C-terminus of MalK, is expressed from plasmid pBAD22-FGK in E. coli BL21 (DE3) and induced at OD600~0.5 with 0.2% L-arabinose for 3 hr at 37 &deg;C.</li>
 <li>Following cell lysis and centrifugation as in step 1.3, the membrane pellet is resuspended in TSG20 buffer at a final concentration of 5 mg/ml. The material is solubilized with 1% w/v n-dodecyl-&beta;-maltopyranoside (DDM) for 3 hr at 4 &deg;C with gentle shaking.</li>
 <li>The insoluble material is removed by ultra-centrifugation as in step 1.3 and the supernatant containing the solubilized MalFGK2 complex is collected and purified as in step 1.4, but without dialysis.</li>
 <li>Further purification is achieved by gel filtration chromatography in TSGD buffer on a Superdex 200 HR 10/300 column at a flow rate of 0.5 ml/min.</li>
 </ol>
 3. Preparation of Phospholipids
 <ol>
 <li>An E. coli total lipid extract dissolved in chloroform is separated into 1,000 nmol aliquots in screw cap microfuge tubes. The solvent is evaporated under a gentle stream of nitrogen and dried further overnight in a vacuum desiccator.</li>
 <li>The lipid film is dissolved in TS buffer at 5 nM final concentration and vortexed vigorously and sonicated. The dissolved lipids will appear slightly opaque due to their general insolubility in aqueous TS buffer, but they should remain in suspension. DDM is added to a final concentration of 0.5% (~10 mM), at which the solution becomes clear.</li>
 <li>The lipid mixture is placed in a water bath and pulse sonicated for 5 times for ~5 sec. The lipid mixture is stored at 4 &deg;C for a maximum of 1 week.</li>
 </ol>
 4. Preparation of Bio-Beads
 <ol>
 <li>Approximately 10-15 ml (dry volume) Bio-Beads is placed into a 50 ml tube.</li>
 <li>The beads are successively washed with 50 ml of 100% methanol, 95% ethanol, milliQ H2O, and finally TS buffer (twice each).</li>
 <li>The washed beads are stored at 4 &deg;C in ~10 ml TS buffer.</li>
 </ol>
 5. Nanodisc Reconstitution
 <ol>
 <li>The MSP is diluted in TSGD buffer at the final concentration of ~7 mg/ml (~0.3 mM).</li>
 <li>~2 nmol of purified MalFGK2 complex are mixed at a protein:MSP:lipid ratio of 1:3:60 or 1:3:400 in TSGD buffer. The final concentration is 6 &mu;M MalFGK2, 18 &mu;M MSP and 360 &mu;M lipids (1:3:60) or 2.4 mM lipids (1:3:400). The final volume is 300 &mu;l. The final DDM concentration is 0.08% (~1.6 mM). The final concentration of glycerol depends on the amount of lipid added but remains around 5-10% v/v.</li>
 <li>~50 &mu;l of Bio-Bead suspension is added to the tube and the mixture is incubated overnight on a rocking table at 4 &deg;C.</li>
 <li>The beads are sedimented by gravity and the solution is pipetted through a narrow tip to avoid as much Bio-Beads as possible.</li>
 <li>Large precipitates are removed by ultra-centrifugation at 100,000 x g for 20 min. The discs are purified by gel filtration chromatography as in step 2.4 in TSG10 buffer. Fractions containing the discs are pooled and an aliquot should be re-injected on the same gel filtration column to test the stability of the preparation. </li>
 <li>The purified discs can stored at -70 &deg;C for an extended period of time. Following thawing on ice, the discs should be subjected to ultra-centrifugation as in step 5.5 to remove potential precipitates. An aliquot should be analyzed by gel filtration chromatography to ensure that significant aggregation or precipitation did not occur during the thawing process. </li>
 </ol>
 6. Native Gel Electrophoresis
 <ol>
 <li>1 &mu;M of nanodiscs (~0.2 mg/ml) are analyzed by native PAGE6, 7 (Tris-HCl pH 8.8, 4-12%) to assess the quality of the reconstitution (Figure 3A).</li>
 <li>The binding of MalE (1 &mu;M) to the MalFGK2-complex reconstituted in nanodiscs is also assessed by native-PAGE (Figure 3A).</li>
 <li>After electrophoresis, the gel is stained with Coomassie blue for 10 min, and destained for ~1 hr.</li>
 </ol>
 7. Dynamic Light Scattering (DLS)
 <ol>
 <li>Nanodiscs are purified by gel filtration chromatography as in step 2.4 in TSG10 buffer using a Superdex 200 HR 10/300 column at a flow rate of 0.1 ml/min. Fractions containing nanodiscs are pooled and concentrated to ~10 mg/ml with an Amicon centrifugal filter.</li>
 <li>The sample is filtered twice (0.22 &mu;m filter) prior to analysis by DLS using a DynaPro nanostar instrument (Wyatt Technology) in a 1 &mu;l inner volume quartz cuvette. Data is fitted using the DYNAMICS software (Wyatt Technology) to estimate the diameter and molecular weight of the particles.</li>
 </ol>
 8. ATPase Measurements
 <ol>
 <li>The ATPase activity of MalFGK2-reconstituted nanodiscs is determined using a colorimetric assay8.</li>
 <li>1 &mu;M of purified discs and 1 mM ATP are mixed together with increasing amounts of MalE and incubated at 37 &deg;C for 20 min. The release of inorganic phosphate is measured at 660 nm.</li>
 <li>The amount of phosphate released is compared to a standard curve generated with a Phosphorous Standard solution.</li>
 </ol>
 9. Representative Results
 The nanodiscs are purified by gel filtration chromatography (Figure 2A, left). The chromatogram shows that the majority of the reconstituted discs (black trace) elute as a single peak, whereas discs made with excess lipids (red trace) elute in the void volume and as a series of broad peaks. The quality of the nanodiscs is further analyzed by native gel electrophoresis and dynamic light scattering spectroscopy (DLS). Properly reconstituted discs migrate as a sharp band on the gel whereas those reconstituted in the presence of an excess of lipids migrate as a smear (Figure 2A, right). Analysis by DLS shows that the disc population is homogeneous with an average diameter of 11.4 nm (Figure 2B). The reconstituted discs have an apparent molecular weight of 215 kDa based on the DLS approximation. Samples reconstituted in the presence of excess lipids display widely distributed radii around 100 nm, which is typical for non-homogeneous samples. 
 The quality of the MalFGK2 complex is assessed by native-gel electrophoresis and its activity by ATPase measurements (Figure 3). The maltose binding protein MalE binds with a high-affinity to the MalFGK2 transporter9, 10. Using non-denaturing gel electrophoresis, it is possible to detect a complex between MalE and MalFGK2 (Figure 3A). The stimulation of the MalK2 ATPase activity by MalE is shown in Figure 3B.
 <img src="/files/ftp_upload/3910/3910fig1.jpg" alt="Figure 1"/> 
 Figure 1. Typical flowchart for the reconstitution protocol. 
 <img src="/files/ftp_upload/3910/3910fig2.jpg" alt="Figure 2"/> 
 Figure 2. Quality control of the nanodisc preparation. A. Gel filtration analysis (Superdex 200 HR 10/300 column) of the nanodiscs reconstituted at low lipid ratio (1/3/60; black trace) or high lipid ratio (1/3/400; red trace). Native gel electrophoresis of the same disc preparation. Molecular weight markers in kDa are indicated. B. Dynamic light scattering analysis of the same disc preparation. <a href="https://www.jove.com/files/ftp_upload/3910/3910fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
 <img src="/files/ftp_upload/3910/3910fig3.jpg" alt="Figure 3"/> 
 Figure 3. Analysis of the MalFGK2-nanodisc particles. A. Gel shift analysis of MalE incubated with increasing amounts of MalFGK2-nanodisc particles. B. ATPase activity of the MalFGK2-nanodisc particles as a function of MalE concentration.

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No conflicts of interest declared.

We describe a simple procedure for the reconstitution of the maltose transporter into nanodiscs. The transporter is ATPase active and the interaction with the soluble binding partner MalE can be recreated (Figure 3). The successful reconstitution of the transporter into nanodiscs open the way for additional biophysical and biochemical analysis. Of particular interest will be the systematic analysis the MalK ATPase and maltose transport activity in detergent, liposome and nanodiscs. ABC transporters c...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Amicon Ultra-4 50K centrifugal filter</td>
 <td>Millipore</td>
 <td>UFC805008</td>
 <td>Follow manufacturer's protocol for proper use </td>
 </tr>
 <tr>
 <td>Bio-Beads SM-2 Adsorbent</td>
 <td>Bio-Rad</td>
 <td>152-3920</td>
 <td></td>
 </tr>
 <tr>
 <td>E. coli total lipids</td>
 <td>Avanti Polar Lipids</td>
 <td>100500C</td>
 <td>Dissolved in chloroform, handle as appropriate for an organic solvent</td>
 </tr>
 <tr>
 <td>Ni sepharose HP resin</td>
 <td>GE Healthcare</td>
 <td>17-5268-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphorous standard solution</td>
 <td>Sigma-Aldrich</td>
 <td>P3869</td>
 <td></td>
 </tr>
 <tr>
 <td>pMSP1D1</td>
 <td>Addgene</td>
 <td>20061</td>
 <td></td>
 </tr>
 <tr>
 <td>Superdex 200 HR 10/300</td>
 <td>GE Healthcare</td>
 <td>17-5172-01</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table I. Specific reagents.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>
 <table border="1">
 <tbody>
 <tr>
 <td>Name </td>
 <td>Composition</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>DDM stock</td>
 <td>10% w/v DDM</td>
 <td>Resuspend in milliQ water and store at -20 &deg;C</td>
 </tr>
 <tr>
 <td>MalFGK2 stock</td>
 <td>1-2 mg/ml 
 50 mM Tris-HCl, pH7.9 
 100 mM NaCl 
 10% v/v glycerol 
 0.03% w/v DDM</td>
 <td>Store at -70 &deg;C after purification</td>
 </tr>
 <tr>
 <td>MSP stock</td>
 <td>10-15 mg/ml 
 50 mM Tris-HCl, pH7.9 
 100 mM NaCl 
 10% v/v glycerol</td>
 <td>Store at -70 &deg;C after purification in &lt;1 ml aliquots and avoid excessive freeze/thaw cycles</td>
 </tr>
 <tr>
 <td>Phospholipid stock</td>
 <td>5 nM E. coli total lipids 
 0.5% w/v (10 mM) DDM 
 50 mM Tris-HCl, pH 7.9 
 50 mM NaCl</td>
 <td>Store at 4 &deg;C for 1 week</td>
 </tr>
 <tr>
 <td>TS buffer</td>
 <td>50 mM Tris-HCl, pH 7.9 
 50 mM NaCl</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>TSG10 buffer</td>
 <td>50 mM Tris-HCl, pH7.9 
 100 mM NaCl 
 10% v/v glycerol</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>TSG20 buffer</td>
 <td>50 mM Tris-HCl, pH8 
 100 mM NaCl 
 20% v/v glycerol</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>TSGD buffer</td>
 <td>50 mM Tris-HCl, pH7.9 
 100 mM NaCl 
 10% v/v glycerol 
 0.03% w/v DDM</td>
 <td>Store at 4 &deg;C and add DDM just before use</td>
 </tr>
 <tr>
 <td colspan="3">Table II. Solution recipes.</td>
 </tr>
 </tbody>
 </table>
 </td>
 </tr>
 </tbody>
 </table>

a step-by-step method for the reconstitution of an abc transporter into nanodisc lipid particles

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane. In this visual protocol, we present a step-by-step reconstitution of a well characterized bacterial ABC transporter, the MalE-MalFGK2 complex. The formation of the disc is a self-assembly process that depends on hydrophobic interactions taking place during the progressive removal of the detergent. We describe the essential steps and we highlight the importance of choosing a correct protein-to-lipid ratio in order to limit the formation of aggregates and larger polydisperse liposome-like particles. Simple quality controls such as gel filtration chromatography, native gel electrophoresis and dynamic light scattering spectroscopy ensure that the discs have been properly reconstituted.

Watch this Scientific Journal Video about A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles at JoVE.com

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

Nanodiscs are small discoid particles that incorporate membrane proteins into a small patch of phospholipid bilayer. We provide a visual protocol that shows the step-by-step incorporation of the MalFGK2 transporter into a disc.

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

a-step-step-method-for-reconstitution-an-abc-transporter-into

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Biology

25.3K 조회수.  University of British Columbia. Nanodiscs는 인지질 이중층의 작은 패치로 막 단백질을 포함 작은 원반 모양의 입자입니다. 우리는 디스크에 MalFGK2 수송의 단계별 설립을 보여줍니다 시각적 프로토콜을 제공합니다.

비디오: A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...