We thank Cecilia de Agrela Pinto, Tom&#225;s de Garay, and Katharina H&#228;u&#223;ermann for their assistance with mass photometry (iSCAT) experiments; Arjen Jakobi and Wiel Evers for their help with TEM; Lucia Baldauf for her assistance with TIRF; Pascal Verdier-Pinard for his advice concerning native electrophoresis;&#160;Agata Szuba and Marjolein Vinkenoog for their help in setting up the Drosophila septin purification efforts, and the Cell and Tissue Imaging (PICT-IBiSA), Institut Curie, member of the French National Research Infrastructure France-BioImaging (ANR10-INBS-04).&#160;This research received funding from the Netherlands Organization for Scientific Research (NWO/OCW) through the &#39;BaSyC-Building a Synthetic Cell&#39; Gravitation grant (024.003.019)&#160;and from the Agence Nationale pour la Recherche (ANR grants ANR-17-CE13-0014: &#8220;SEPTIMORF&#8221;; ANR-13-JSV8-0002-01: &#8220;SEPTIME&#8221;; and ANR-20-CE11-0014-01: &#8220;SEPTSCORT&#8221;).

The authors declare no competing or financial interests.

The method described here allows for the robust purification and quality control of pre-formed septin hetero-oligomers. Some of the key issues to consider for the correct application of the method are as follows. During the elution steps in the chromatographic separations, it is important to use the recommended (or lower) flow rate to minimize the dilution of the septin complexes. Additionally, to maximize the recovery of protein during the final concentration step, the concentrator column is oriented in such a way that the solution is not pushed against the filter (when there is only a filter on one side). If the solution goes directly to the filter, the protein sticks much more to it, substantially decreasing the final yield. It is also important to consider that the concentration step is not always necessary. Picking fractions only from a narrow range around the peak in the chromatogram usually gives a high enough stock concentration (&#62;3,000 nM) for many reconstitution applications (which usually operate between 10-300 nM). Finally, for the quality control of the functionality of the septin complexes by fluorescence microscopy, it is important to correctly passivate the surface of the microscopy slides, since septin complexes avidly stick to glass. Passivation of the glass slides can be done either via PLL-PEG functionalization or by the formation of neutral (100% DOPC) supported lipid bilayers11,32.
Compared to the original purification protocol first described in Iv et al.10, there is a change in the buffer compositions (Table 2). The concentration of MgCl2 has been reduced from 5 mM to 2 mM, and the concentration and pH of Tris-HCl have been reduced from 50 mM to 20 mM and from 8.0 to 7.4, respectively. These changes were made to make the buffer conditions compatible with studies of the interactions of human septins with lipid bilayers, actin filaments, and microtubules10,11,32. This is because the authors formed supported lipid bilayers and polymerized actin in the F-buffer, whose composition is identical to that of darkSPB, apart from the presence of ATP in the F-buffer. The buffer change did not produce any changes in the quality or lifetime of the purified septins compared to the original buffers.
This method of purification still has several limitations. First, different purification attempts can vary in yield (0.5-1 mL of 2-5 &#956;M septin complexes) and functional quality, as checked by the bundle formation ability of the purified septin complexes. That is why it is very important to consistently perform the quality checks described in this paper. Controlling very well the times of expression and the optical density of the bacterial culture can help mitigate the difference in yield. Second, this purification pipeline cannot distinguish between trimers and hexamers or between tetramers and octamers (Figure 1B). However, the quality control experiments can be used to prove that the majority of septin complexes are in their long oligomer form. In case an even narrower oligomer size distribution is required, size exclusion chromatography can be inserted in between step 1.6. and step 1.7. of the purification protocol. This optional step, however, dramatically decreases the yield, and it is not recommended unless it is strictly needed. A last, more fundamental, limitation comes from the use of E. coli as an expression system for recombinant septin complexes. Naturally, this system does not allow for post-translational modifications (PTMs), which have been reported in animal cells, such as phosphorylation, acetylation, and sumoylation6,51,52,53. These posttranslational modifications could be added by implementing a similar purification strategy in insect or human cells. Furthermore, this paper has only discussed the reconstitution of septins by themselves, but studies in cells indicate that regulatory proteins such as proteins from the Borg family54,55 and anillin24,25,56 can have substantial yet poorly understood effects on the assembly and functions of septins and are, therefore, important to eventually incorporate in in vitro studies. Protocols for the purification of Borg proteins and anillin have been reported54,57.
The septin purification protocol reported here offers a standardized way to purify septins in their oligomer form with the correct subunit stoichiometry, offering an important advance over many earlier in vitro studies relying on single septin subunits. Even though some septins in specific contexts can act as a single subunit2, the current body of literature strongly suggests that, in animal cells, septins mostly function in complexes9,58. Therefore, the use of pre-formed hetero-oligomers, such as the ones described in this paper and others10,11,18,32,35,36,37, is of great importance to study the structural and biophysical properties of septins via in vitro reconstitution to dissect their functions in the cell. Furthermore, septins are self-assembling proteins with many interaction partners, including the membrane and the cytoskeleton, which makes them of great interest for bottom-up synthetic biology59,60,61 and studies of protein-induced changes in membrane biophysical properties such as curvature42,62,63.

The cytoskeleton has been classically described as a three-component system consisting of actin filaments, microtubules, and intermediate filaments1, but recently, septins have been acknowledged as a fourth component of the cytoskeleton1. Septins are a family of GTP-binding proteins that are conserved in eukaryotes2. Septins are involved in many cellular functions such as cell division3, cell-cell adhesion4, cell motility5, morphogenesis6, cellular infection7, and the establishment and maintenance of cell polarity8. Despite their important functions, how septins are involved in such processes is poorly understood.
The septin family of proteins is subdivided into several subgroups (four or seven, depending on the classification) based on protein sequence similarity2. Members of different subfamilies can form palindromic hetero-oligomeric complexes, which are the building blocks of filaments and which, in turn, assemble into higher-order structures such as bundles, rings, and meshworks1,9,10,11,12. Further molecular complexity arises from the presence of different splice variants, an example being human SEPT9, where there is evidence for specific functions of different splice variants13,14,15. Additionally, the length of the hetero-oligomers depends on species and cell type. For instance, Caenorhabditis elegans septins form tetramers16, Drosophila melanogaster septins form hexamers17 (Figure 1A), Saccharomyces cerevisiae septins form octamers18, and human septins form both hexamers and octamers19 (Figure 1A). The ability of septin isoforms, splice variants, and post-translationally modified septins from the same subfamily to substitute each other in the complex and the (co-)existence of differently sized hetero-oligomers have made it difficult to delineate the cellular functions of different hetero-oligomeric complexes12.
Another interesting ability of septins is their ability to interact with many binding partners in the cell. Septins bind the plasma membrane and membranous organelles during interphase and cell division20,21,22. In dividing cells, septins cooperate with anillin23,24,25 and actin and myosin during cytokinesis26,27. At the late stages of cytokinesis, septins seem to regulate the endosomal sorting complexes required for the transport (ESCRT) system for midbody abscission28. Additionally, there is also evidence of septin located on the actin cortex and actin stress fibers of cells in interphase cells29,30,31. In specific cell types, septins also bind and regulate the microtubule cytoskeleton32,33.
All of these features make septins a very interesting protein system to study, but also a challenging one. The combination of the large number of septin subunits (13 genes in humans without counting splice variants2) with the potential of septin subunits from the same subfamily to substitute each other and form differently sized hetero-oligomers makes it difficult to draw a conclusion on the cellular function of a specific septin by genetic manipulation. Furthermore, the multiple interactions of septins make interpreting the effects of common research tools such as drugs34 directed at cytoskeletal or membrane components a hard task.
A way to overcome this situation is to complement research in cells with in vitro (cell-free) reconstitution of septins. In vitro reconstitution allows for the isolation of a single type of septin hetero-oligomers with a specific subunit composition and length18,35,36,37. This complex can then be studied in a controlled environment, either alone to discover the basic structural and physicochemical properties of septins38,39,40, or in combination with desired partners such as model biomembranes11,41,42, actin filaments10,27, or microtubules32,36 to decipher the nature of their interactions.
Therefore, a reliable method to purify different septin complexes efficiently is vital for septin research. However, even using the same protocol, different purifications can give proteins with different activity/functionality or even integrity. For commercially available proteins such as enzymes, the functionality and enzymatic activity are carefully validated43. Implementing careful quality control for cytoskeletal or structural proteins such as septins can be challenging, but it is essential to make experiments across labs comparable.
This paper describes a robust method to purify high-quality recombinant septins in their hetero-oligomeric form based on the simultaneous expression of two vectors containing mono- or bi-cistronic constructs (Table 1) in Escherichia coli cells. The method consists of a two-step affinity chromatography approach to capture septin hetero-oligomers containing both a his6-tagged septin and a Strep-II-tagged septin (Figure 1B,C). This protocol, first described in Iv et al.10, has been used to purify Drosophila septin hexamers11,27,35, human septin hexamers10, and several human septin octamers containing different native (isoform 1, 3, and 5)10,32 or mutated SEPT9 isoforms32. Furthermore, a description of a set of techniques to assess the quality of the purified septins is given. First, the integrity and correct stoichiometry of the septin subunits is checked using denaturing electrophoresis and transmission electron microscopy (TEM). Then, the presence of hetero-oligomers of the correct molecular mass and the presence of monomers or smaller oligomers indicative of complex instability are examined by native electrophoresis and mass photometry via interferometric scattering microscopy (iSCAT). Finally, the last step consists of the assessment of the polymerizing activity of the septins using fluorescence microscopy and TEM.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63871/63871fig01v2.jpg" /> 
Figure 1: Purification strategy. (A) Schematics of the septin hetero-oligomers that exist in human (left) and Drosophila (right) cells. Numbers denote septin subunits from the indicated groups, and P denotes Peanut. Human SEPT9 can be any of its isoforms. The septin subunits have an asymmetric shape and are longitudinally associated with two distinct interfaces, the NC:NC and the G:G interface, as denoted by NC and G, respectively, on top of the human hexamer. (B,C) Schematic illustration of the two-step chromatography strategy, shown for (B) human septin hexamers and (C) octamers. H indicates the his-tags, while S indicates the Strep-II-tags. <a href="https://www.jove.com/files/ftp_upload/63871/63871fig01v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>488nm laser combiner iLAS2</td><td>Gataca</td><td></td><td>TIRF microscope</td></tr><tr><td>488nm Sapphire laser lines</td><td>Coherent</td><td></td><td>Confocal microscope</td></tr><tr><td>4k X 4k F416 CMOS camera</td><td>TVIPS</td><td></td><td>For JEM-1400plus</td></tr><tr><td>4x sample buffer nativePAGE</td><td>Thermo Fisher scientific</td><td>BN2003</td><td></td></tr><tr><td>6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (TROLOX)</td><td>Sigma-Aldrich</td><td>238813</td><td>To prevent blinking</td></tr><tr><td>AKTA pure 25 M1</td><td>GE healthcare</td><td>1680311</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>A9518-25G</td><td></td></tr><tr><td>Carbon Type-B, 300 mesh EM grid</td><td>Ted pella</td><td>01813-F</td><td></td></tr><tr><td>Carbon Type-B, 300 mesh EM grid</td><td>Electron micoscopy sciences</td><td>CF300-Cu</td><td></td></tr><tr><td>Cover glass #1.5H</td><td>Thorslabs</td><td>CG15KH</td><td></td></tr><tr><td>CSU-X1-M1 confocal unit</td><td>Yokogawa</td><td></td><td>Confocal microscope</td></tr><tr><td>Desthiobiotin</td><td>Sigma-Aldrich</td><td>D1411-1G</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma-Aldrich</td><td>D9779</td><td></td></tr><tr><td>DNAse</td><td>Sigma-Aldrich</td><td>10104159001</td><td></td></tr><tr><td>DOPC</td><td>Avanti Polar Lipids</td><td>850375C</td><td></td></tr><tr><td>Eclipse Ti2-E</td><td>Nikon instruments</td><td></td><td>Confocal microscope</td></tr><tr><td>EDTA-free protease inhibtor cocktail</td><td>Roche</td><td>481761</td><td></td></tr><tr><td>HisTrap HP, 5 mL</td><td>GE healthcare</td><td>29-0588-3</td><td></td></tr><tr><td>iLAS2 azimuthal TIRF illumination system&amp;nbsp;</td><td>Gataca</td><td></td><td>TIRF microscope</td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>1202-1KG</td><td></td></tr><tr><td>InstantBlue Protein Gel Stain</td><td>Westburg Life Sciences</td><td>EP ab119211</td><td></td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside (IPTG)</td><td>Thermo Fisher scientific</td><td>10849040</td><td></td></tr><tr><td>iXon Ultra 888 EMCCD camera</td><td>Andor</td><td></td><td>Confocal microscope</td></tr><tr><td>iXon Ultra 897 EM-CCD&amp;nbsp;</td><td>Andor</td><td></td><td>TIRF microscope</td></tr><tr><td>JEM-1400plus</td><td>JOEL</td><td></td><td>TEM microscope TUDelft</td></tr><tr><td>kappa-cassein&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C0406</td><td></td></tr><tr><td>LB broth</td><td>Sigma-Aldrich</td><td>L3022-6X1KG</td><td></td></tr><tr><td>Lyzozyme</td><td>Sigma-Aldrich</td><td>62971-10G-F</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Sigma-Aldrich</td><td>M8266-100G</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Sigma-Aldrich</td><td>746452-1KG</td><td></td></tr><tr><td>Methylecllulose</td><td>Sigma-Aldrich</td><td>8074844</td><td></td></tr><tr><td>MilliQ system (Integral 10)</td><td>Merck-Millipore</td><td></td><td>I-water dispenser</td></tr><tr><td>Mini protean TGX gels</td><td>BIORAD</td><td>4561086</td><td></td></tr><tr><td>NativeMark unstained protein standard</td><td>Invitrogen</td><td>LC0725</td><td>For iSCAT and Native gels</td></tr><tr><td>NativePAGE 4-16% GELS</td><td>Thermo Fisher scientific</td><td>BN1002BOX</td><td></td></tr><tr><td>NativePAGE Running Buffer kit</td><td>Thermo Fisher scientific</td><td>BN2007</td><td></td></tr><tr><td>Nikon Ti2-E&amp;nbsp;</td><td>Nikon instruments</td><td></td><td>TIRF microscope</td></tr><tr><td>Nr. 1 Menzel coverslips</td><td>Thermo Fisher scientific</td><td>11961988</td><td></td></tr><tr><td>parafilm&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P7668</td><td></td></tr><tr><td>Plan Apo &amp;times;100/1.45 NA oil immersion objective</td><td>Nikon instruments</td><td></td><td>Confocal microscope</td></tr><tr><td>PMSF</td><td>Sigma-Aldrich</td><td>10837091001</td><td></td></tr><tr><td>Poly(L-lysine)-graft-biotinylated PEG (PLL-PEG)</td><td>SuSoS</td><td>CHF560.00</td><td></td></tr><tr><td>Poly-L-lysine solution 0.01%</td><td>Sigma-Aldrich</td><td>P4832</td><td>For iSCAT glass slides</td></tr><tr><td>Pottassium Chloride</td><td>Sigma-Aldrich</td><td>P9541-1KG</td><td></td></tr><tr><td>Power supply for native gels</td><td>CONSORT</td><td>S/N 71638</td><td></td></tr><tr><td>POWERPAC UNIVERSAL</td><td>BIORAD</td><td>042BR31206</td><td></td></tr><tr><td>Protocatechuate 3,4-Dioxygenase (PCD)</td><td>Sigma-Aldrich</td><td>P8279-25UN</td><td>oxygen scavenger - enzyme</td></tr><tr><td>Protocatechuic acid (PCA)</td><td>Sigma-Aldrich</td><td>03930590-50MG</td><td>oxygen scavenger - reagent</td></tr><tr><td>Q500 Sonicator</td><td>Qsonica</td><td>Q500-110</td><td></td></tr><tr><td>Quemesa camera</td><td>Olympus</td><td></td><td>For Tecnai Spirit</td></tr><tr><td>Refeyn OneMP</td><td>Refeyn</td><td></td><td></td></tr><tr><td>Sample buffer, laemmli 2x concentrate</td><td>Sigma-Aldrich</td><td>S3401-10vl</td><td></td></tr><tr><td>Silicon gaskets</td><td>Sigma-Aldrich</td><td>GBL103250-10EA</td><td></td></tr><tr><td>Slide-A-Lyzer Dialysis cassettes 30k MWCO 3mL</td><td>Thermo Fisher scientific</td><td>66381</td><td></td></tr><tr><td>Spectinomycin</td><td>Sigma-Aldrich</td><td>PHR1441-1G</td><td></td></tr><tr><td>StrepTrap HP, 1 mL</td><td>GE healthcare</td><td>28-9075-46</td><td></td></tr><tr><td>Tecnai Spirit microscope</td><td>Thermo Scientific, FEI</td><td></td><td>TEM microscope Institute Curie</td></tr><tr><td>Terrific broth</td><td>Sigma-Aldrich</td><td>T0918-1KG</td><td></td></tr><tr><td>Tris/Glyine/SDS buffer</td><td>BIORAD</td><td>1610772</td><td></td></tr><tr><td>Tris-HCl</td><td>Sigma-Aldrich</td><td>T5941-1KG</td><td></td></tr><tr><td>Ultrasonic cleaner</td><td>Branson</td><td>CPX2800H-E</td><td></td></tr><tr><td>Vivaspin 6, 30,000 MWCO PES</td><td>Sartorius</td><td>VS0622</td><td></td></tr></tbody></table>

purification and quality control of recombinant septin complexes for cell-free reconstitution

Septins are a family of conserved eukaryotic GTP-binding proteins that can form cytoskeletal filaments and higher-order structures from hetero-oligomeric complexes. They interact with other cytoskeletal components and the cell membrane to participate in important cellular functions such as migration and cell division. Due to the complexity of septins' many interactions, the large number of septin genes (13 in humans), and the ability of septins to form hetero-oligomeric complexes with different subunit compositions, cell-free reconstitution is a vital strategy to understand the basics of septin biology. The present paper first describes a method to purify recombinant septins in their hetero-oligomeric form using a two-step affinity chromatography approach. Then, the process of quality control used to check for the purity and integrity of the septin complexes is detailed. This process combines native and denaturing gel electrophoresis, negative stain electron microscopy, and interferometric scattering microscopy. Finally, a description of the process to check for the polymerization ability of septin complexes using negative stain electron microscopy and fluorescent microscopy is given. This demonstrates that it is possible to produce high-quality human septin hexamers and octamers containing different isoforms of septin_9, as well as Drosophila septin hexamers.

As mentioned in the protocol, 5 L of E. coli cells co-transformed with the two septin-expressing plasmids were grown, and the expression of septins was induced by adding IPTG. After 3 h, the cells were collected by centrifugation, resuspended in lysis buffer, and lysed by sonication. The lysate was then clarified by centrifugation, and the clarified solution was applied to a HisTrap column (Figure 2A). After the first purification, the septin-containing fractions were pooled and applied onto a StrepTrap column (Figure 2B). This typically yields around 3-5 mL of ~1 &#956;M septin complex. Before pooling the septin-containing fractions, denaturing gel electrophoresis can be used to check for the integrity of the septin subunits and the equimolar stoichiometric ratio between the different septin subunits forming the complex. (Figure 3A). If the gel shows similarly intense bands corresponding to the molecular weights (Table 3) of the septin subunits, the protocol can be continued. If not, it is recommended to start the protocol again. In the example shown for human septin octamer with SEPT9_i1, Figure 3A clearly shows bands corresponding to SEPT9_i1, SEPT6, SEPT7, and SEPT2 (in the order from top to bottom) with similar intensities; the 99% confidence interval of the normalized intensity was 1.128 &#177; 0.048 for SEPT2, 1.092 &#177; 0.034 for SEPT6, 1.108 &#177; 0.040 for SEPT7, and 1.067 &#177; 0.029 for SEPT9. If SEPT2 is tagged with msfGFP, it will shift up very close below SEPT9_i1. Depending on the electrophoresis system used and the presence of the C-terminal TEV-Strep tag for SEPT7 (which makes it migrate more slowly than untagged SEPT7), the SEPT7 and SEPT6 bands sometimes merge due to their comparable molecular weights. The next step is to pool the fractions and dialyze them against septin buffer with DTT. After the dialysis, if the concentration is too low (&#60;2 &#956;M) or a higher concentration is needed for the experiments, a concentration step can be included, as described in the protocol. Concentrations below 1 &#956;M usually indicate a bad functional quality of the septins. A final septin complex concentration between 3.5 &#956;M and 7 &#956;M works well for most in vitro assays. These concentrations are usually obtained when the volume after concentration reaches 0.5-1 mL.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63871/63871fig02.jpg" /> 
Figure 2: Example chromatograms corresponding to the purification of dark human septin octamers_9i1. (A) HisTrap column chromatogram. After the septin elution peak, the absorbance does not go back to zero, likely due to the presence of imidazole in the buffer. The pooled fraction went from the start of the elution peak until the absorbance stabilized at around 250 mL. (B) StrepTrap column chromatogram. The pooled fraction went from the start of the elution peak until the absorbance went back to around 0 at 50 mL. <a href="https://www.jove.com/files/ftp_upload/63871/63871fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To continue with the quality control, native electrophoresis, as described in the protocol, was performed (Figure 3B). In the gels, a major band corresponding to the intact hetero-oligomers and, usually, a minor band corresponding to dimers can be observed. Human hexamers are found a little bit above the 242 kDa marker band, while octamers are found above the 480 kDa band, above their calculated molecular mass. The location of these bands was checked by western blot analysis of eukaryotic cell extracts32. Tagging with msfGFP couples each SEPT2 with an msfGFP protein. This causes an increase in the molecular weight of septin complexes of 53.4 kDa (26.7 kDa/msGFP molecule). Nevertheless, on the native electrophoresis gel, the apparent molecular weight of the msfGFP-tagged complexes is indistinguishable from that of the untagged complexes.
A complementary technique to test whether the septin complexes are intact is mass photometry by iSCAT microscopy. iSCAT monitors the light scattering of molecules landing on a glass slide amplified by interference with reference light, typically the reflection of the laser on the bottom of the glass slide. Then, a background subtraction approach is used to give contrast to the particles. Due to this correction, the signal shows positive and negative values depending on whether the particles land on the glass or move away from it49. The detected signal is directly proportional to the molecular weight of proteins50. Therefore, a signal-to-mass calibration with a mass standard can determine the mass of the sample proteins. Figure 3C shows an example of human septin octamers containing SEPT9_i1. Most of the detected single particles (~50%) are of a molecular weight expected for complete octamers containing SEPT9_i1 (423 kDa) (Figure 3C). There are also particles with masses between 150-300 kDa, but no clear peak is observed, indicating the possible presence of other septin species in low abundance. Similarly, most of the detected single particles for mEGFP-tagged Drosophila hexamers are of a molecular weight expected for intact hexamers (361 kDa) (Figure 3D). An additional clear peak at 241 kDa indicates the presence of stable tetramers containing two peanut proteins, one DSep1 and one mEGFP-DSep2. Finally, both the human and the fly septin complexes show a peak around 80 kDa that could be a mix of monomers and dimers, possibly amplified by a trace of DTT or any other small molecule that aggregates, showing a peak in the positive side of the plot45.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63871/63871fig03v2.jpg" /> 
Figure 3: Examples of results of the oligomer quality control. (A) Example of denaturing gel showing different fractions of the elution peak from the purification of dark human septin octamers_9i1. (B) Example of native electrophoresis of different septin complexes. (C,D) Different examples of histogram results of mass photometry at 12.5 nM of septin complexes: (C) dark human septin octamers_9i1 and (D) DSep1-msfGFP Drosophila septin hexamers. Lines are Gaussian fits. (E) TEM image of 25 nM dark human septin octamers_9i1 in septin buffer. Scale bar = 200 nm. (F) Class average image of SEPT2-msfGFP human septin octamers_9i1. The msfGFP tags are visible as fuzzy densities on the two ends. Scale bar = 10 nm. Panels (E) and (F) are the copyright of The Company of Biologists and have been adapted from Iv et al.10 with permission. <a href="https://www.jove.com/files/ftp_upload/63871/63871fig03v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Given that both native gels and iSCAT provide ensemble-averaged information only, class averaging of transmission electron microscopy images of single septin oligomers was used to check the integrity and purity of the complexes by direct visualization. In TEM images of septin complexes in septin buffer, rods of 24 nm (hexamers) or 32 nm (octamers) in length can be observed. An example of a human septin octamer containing SEPT9_i1 can be seen in Figure 3E. When class averaging them, each of the subunits can be observed and counted, as seen for the msfGFP-tagged human octamer with SEPT9_i1 in Figure 3F. In case the oligomer is fluorescently labeled, extra densities that correspond to the SEPT2-msfGFP can be observed at the end of the rods (Figure 3F).
The combination of the above techniques proves that octamers (or hexamers) with the correct stoichiometric ratio and high purity can be purified using the described protocol. Finally, the last quality control check is for the functionality of the septin complexes in terms of their polymerization ability. In the presence of low salt concentration (&#60;150 mM KCl with the described buffer9), if septins are not in the presence of other proteins or negatively charged lipid membranes, they self-assemble into bundles9. Septins are prevented from polymerization by keeping them in the storage buffer, which has a high (300 mM) KCl concentration. The septin hetero-oligomers are then diluted at a 1:6 volume ratio in a buffer of the same composition but without KCl to achieve a final KCl concentration of 50 mM. To do fluorescence imaging, this buffer is complemented with an oxygen scavenging system to protect from photobleaching and with a blinking suppressor. In TIRF microscopy, small clusters of proteins can be observed within the shallow TIRF field (~100 nm; Figure 4A,B). On a confocal microscope, large clusters of filamentous structures can be seen floating higher up in solution (Figure 4C). Finally, with TEM, small bundles of septin (Figure 4D), corresponding to the clusters observed by TIRF, and large bundles (Figure 4E), corresponding to the structures observed by confocal microscopy, can be observed. The insets of Figure 4D,E reveal that both types of structures consist of long, thin filaments that run in parallel, forming bundles with tapered ends. Together, the fluorescence and TEM images prove that the purified septin complexes can polymerize into filaments, which in turn self-assemble into bundles.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63871/63871fig04.jpg" /> 
Figure 4: Examples of results of the polymerization ability quality control. (A) TIRF image of 300 nM human septin hexamers (10% msfGFP-labelled hexamers) in fluoSPB. (B) TIRF image of 300 nM human septin octamers containing SEPT9_i1 (10% msfGFP-labelled octamers9_i1) in fluoSPB. (C) Confocal maximum-intensity projections of Z-stacks across ~30 &#956;m with 0.5 &#956;m of spacing of 300 nM human septin octamers_9i3 in fluoSPB. (A-C) Scale bar = 10 &#956;m and inverted grayscale. (D,E) Example TEM images of (D) small and (E) large bundles of human septin octamers_9i1 in darkSPB. Insets show regions where clear filaments running parallel within the bundle can be observed. Scale bars = 500 nm. Panels (C-E) are copyright of The Company of Biologists and have been adapted from Iv et al.10 with permission. <a href="https://www.jove.com/files/ftp_upload/63871/63871fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: List of plasmids. Plasmids to purify septin oligomers following this protocol. All plasmids have been deposited in Addgene (first column). <a href="https://www.jove.com/files/ftp_upload/63871/Table 1_V2.zip" target="_blank">Please click here to download this Table.</a>
Table 2: List of buffers. Buffer compositions used for the purification and quality control of septin oligomers. <a href="https://www.jove.com/files/ftp_upload/63871/Table 2(1).xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Molecular weights and extinction coefficients. List of molecular weights (MW) and optical extinction coefficients (&#949;) at a wavelength of 280 nm calculated with ProtParam based on the sequences of the complex, assuming linear fusion of the septin subunits, the different septin complexes, and the unique septin subunits (only MW) that can be purified with the plasmids listed in Table 1. <a href="https://www.jove.com/files/ftp_upload/63871/Table 3.xlsx" target="_blank">Please click here to download this Table.</a>

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Purification and Quality Control of Recombinant Septin Complexes for Cell-Free Reconstitution

In vitro reconstitution of cytoskeletal proteins is a vital tool to understand the basic functional properties of these proteins. The present paper describes a protocol to purify and assess the quality of recombinant septin complexes, which play a central role in cell division and migration.

Septins are a family of conserved eukaryotic GTP-binding proteins that can form cytoskeletal filaments and higher-order structures from hetero-oligomeric ...

purification-quality-control-recombinant-septin-complexes-for-cell

Research

JoVE Journal

Biochemistry

2.0K Views.  Delft University of Technology. Dit protocol maakt het mogelijk om hoogwaardige septinecomplexen te zuiveren, en dit is essentieel om de vele rollen van septines in de cel te bestuderen. Het belangrijkste voordeel van deze aanpak is de synthese van een eenvoudige procedure om hoogwaardige septine te zuiveren met behulp van Addgene-plasmiden. Septines zijn moeilijk te bestuderen in cellen vanwege hun vele interacties.Zwavelreconstitutie helpt ons deze complexiteit te ontwarren door ze in een eenvoudig systeem te bestu...

Video: Purification and Quality Control of Recombinant Septin Complexes for Cell-Free Reconstitution

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.

This protocol describes the reconstitution of nucleosomes containing differentially isotope-labeled sister histones. At the same time, asymmetrically post-translationally modified nucleosomes can be generated after using a premodified histone copy. These preparations can be readily used to study modification crosstalk mechanisms, simultaneously on both sister histones, by using high-resolution NMR spectroscopy.

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

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.

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 ...

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...

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

Recombinant Human Myosin-7a Production and Functional Characterization Protocol

MultiBac System-Based Purification and Biophysical Characterization of Human Myosin-7a

Myosin-7a is an actin-based motor protein vital for auditory and visual processes. Mutations in myosin-7a lead to Usher syndrome type 1, the most common and severe form of deaf-blindness in humans. It is hypothesized that myosin-7a forms a transmembrane adhesion complex with other Usher proteins, essential for the structural-functional integrity of photoreceptor and cochlear hair cells. However, due to the challenges in obtaining pure, intact protein, the exact functional mechanisms of human myosin-7a remain elusive, with limited structural and biomechanical studies available. Recent studies have shown that mammalian myosin-7a is a multimeric motor complex consisting of a heavy chain and three types of light chains: regulatory light chain (RLC), calmodulin, and calmodulin-like protein 4 (CALML4). Unlike calmodulin, CALML4 does not bind to calcium ions. Both the calcium-sensitive, and insensitive calmodulins are critical for mammalian myosin-7a for proper fine-tuning of its mechanical properties. Here, we describe a detailed method to produce recombinant human myosin-7a holoenzyme using the MultiBac Baculovirus protein expression system. This yields milligram quantities of high-purity full-length protein, allowing for its biochemical and biophysical characterization. We further present a protocol for assessing its mechanical and motile properties using tailored in vitro motility assays and fluorescence microscopy. The availability of the intact human myosin-7a protein, along with the detailed functional characterization protocol described here, paves the way for further investigations into the molecular aspects of myosin-7a in vision and hearing.

Author Spotlight: Unraveling the Role of Myosin-7a and Usher Proteins in Hearing and Human Disease

This protocol details the procedures for recombinantly producing the human myosin-7a holoenzyme using the MultiBac Baculovirus system and for studying its motility using a tailored in vitro filament gliding assay.

Myosin-7a is an actin-based motor protein vital for auditory and visual processes. Mutations in myosin-7a lead to Usher syndrome type 1, the most common ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Biology

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for isolation of protein complexes under physiological conditions. Fusion proteins containing two copies of Strep-tag II, designated twin-Strep-tag or SIII-tag, have the advantage of higher affinity for Strep-Tactin compared to those containing only a single Strep-tag, thus allowing more efficient protein purification. However, this advantage is offset by the fact that elution of twin-Strep-tagged proteins with biotin may be incomplete, leading to low protein recovery. The recovery can be dramatically improved by using denaturing elution with sodium dodecyl sulfate (SDS), but this leads to sample contamination with Strep-Tactin released from the resin, making the assay incompatible with downstream proteomic analysis. To overcome this limitation, we have developed a method whereby resin-coupled tetramer of Strep-Tactin is first stabilized by covalent cross-linking with Bis(sulfosuccinimidyl) suberate (BS3) and the resulting cross-linked resin is then used to purify target protein complexes in a single batch purification step. Efficient elution with SDS ensures good protein recovery, while the absence of contaminating Strep-Tactin allows downstream protein analysis by mass spectrometry. As a proof of concept, we describe here a protocol for purification of SIII-tagged viral protein VPg-Pro from nuclei of virus-infected N. benthamiana plants using the Strep-Tactin polymethacrylate resin cross-linked with BS3. The same protocol can be used to purify any twin-Strep-tagged protein of interest and characterize its physiological binding partners.

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3

A method is described for efficient purification of twin-Strep-tagged fusion proteins and their specific complexes on modified streptavidin (Strep-Tactin) resin covalently cross-linked with Bis(sulfosuccinimidyl) suberate (BS3). The method has the advantages of fast speed, good target protein recovery and high purity, and is compatible with subsequent analysis by mass spectrometry.

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for ...

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

Purification and Quality Control of Recombinant Septin Complexes for Cell-Free Reconstitution

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