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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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 &#946;-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&#160;</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&#160;</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&#160;</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&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P7668</td>
			<td></td>
		</tr>
		<tr>
			<td>Plan Apo &#215;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>

Watch this Scientific Journal Video about Purification and Quality Control of Recombinant Septin Complexes for Cell-Free Reconstitution at JoVE.com

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 ...

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Biochemistry

1.9K Views.  Delft University of Technology. 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. 

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 ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

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

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

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

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts prepared from Xenopus laevis eggs have been powerful in dissecting biochemical networks underlying the cell-cycle progression, their ensemble average measurement typically leads to a damped oscillation, despite each individual oscillator being sustained. This is due to the difficulty of perfect synchronization among individual oscillators in noisy biological systems. To retrieve the single-cell dynamics of the oscillator, we developed a droplet-based artificial cell system that can reconstitute mitotic cycles in cell-like compartments encapsulating cycling cytoplasmic extracts of Xenopus laevis eggs. These simple cytoplasmic-only cells exhibit sustained oscillations for over 30 cycles. To build more complicated cells with nuclei, we added demembranated sperm chromatin to trigger nuclei self-assembly in the system. We observed a periodic progression of chromosome condensation/decondensation and nuclei envelop breakdown/reformation, like in real cells. This indicates that the mitotic oscillator functions faithfully to drive multiple downstream mitotic events. We simultaneously tracked the dynamics of the mitotic oscillator and downstream processes in individual droplets using multi-channel time-lapse fluorescence microscopy. The artificial cell-cycle system provides a high-throughput framework for quantitative manipulation and analysis of mitotic oscillations with single-cell resolution, which likely provides important insights into the regulatory machinery and functions of the clock.

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions.

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial purification of biologically important RNA/protein complexes. However, this strategy suffers from one major disadvantage that limits its broader utilization: the biotin/streptavidin interaction can be broken only under denaturing conditions that also disrupt the integrity of the eluted complexes, hence precluding their subsequent functional analysis and/or further purification by other methods. In addition, the eluted samples are frequently contaminated with the background proteins that nonspecifically associate with streptavidin beads, complicating the analysis of the purified complexes by silver staining and mass spectrometry. To overcome these limitations, we developed a variant of the biotin/streptavidin strategy in which biotin is attached to an RNA substrate via a photo-cleavable linker and the complexes immobilized on streptavidin beads are selectively eluted to solution in a native form by long wave UV, leaving the background proteins on the beads. Shorter RNA binding substrates can be synthesized chemically with biotin and the photo-cleavable linker covalently attached to the 5&#39; end of the RNA, whereas longer RNA substrates can be provided with the two groups by a complementary oligonucleotide. These two variants of the UV-elution method were tested for purification of the U7 snRNP-dependent processing complexes that cleave histone pre-mRNAs at the 3&#39; end and they both proved to compare favorably to other previously developed purification methods. The UV-eluted samples contained readily detectable amounts of the U7 snRNP that was free of major protein contaminants and suitable for direct analysis by mass spectrometry and functional assays. The described method can be readily adapted for purification of other RNA binding complexes and used in conjunction with single- and double-stranded DNA binding sites to purify DNA-specific proteins and macromolecular complexes.

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial ...

Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro enzymatic synthesis of a flavonol. In this method, Atf3h and Atfls1, two key genes in the biosynthetic pathway of the flavonols, are cloned and overexpressed in Escherichia coli. The recombinant enzymes are purified via an affinity column and then a bienzymatic cascade is established in a specific synthetic buffer. Two flavonols are synthesized in this system as examples and determined by TLC and HPLC/LC/MS analyses. The method displays obvious advantages in the derivation of flavonols over other approaches. It is time- and labor-saving and highly cost-effective. The reaction is easy to be accurately controlled and thus scaled up for mass production. The target product can be purified easily due to the simple components in the system. However, this system is usually restricted to the production of a flavonol from a flavanone.

The derivation of a flavonol is crucial for its application in healthcare and the food industry. Here, we provide a detailed protocol for the biosynthesis of a flavonol from a flavanone and discuss the crucial steps and its advantages over other approaches.

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

Preparation of High-Quality Fermented Fish Product

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures were: (1) pretreatment of farmed sturgeon including decapitation, evisceration, skinning-off, cleaning and cutting; (2) marinating fish cubes in 6-12% (w/v) salt solution (1:1, fish cube mass to solution volume); (3) drying fish cubes to a water content of 50-60% by hot air (40-60 &#176;C) or by vacuum; (4) fermentation involving inoculating fish cubes with 0.4-1.6% (w/w) S. cerevisiae in flavor solution to fish cubes and fermenting at 25-35 &#176;C for 6-10 h; (5) sealing fish cubes in vacuum packages with marinating and fermenting solutions; (6) sterilizing at 115-121 &#176;C for 10-20 min. The sturgeon meat product prepared by this method has delicious taste which is mellow and thick, has various types and large amounts of volatile flavor compounds such as alcohols and esters which could mask musty and unpleasant odor from fish, has moderate salt content but good texture properties such as high springiness, gumminess and chewiness, and has bright russet color and attractive appearance. This new technique could also be applied in the processing of other fish to provide convenient fish snack foods which could be stored at room temperature. It is appropriate for both marine and freshwater fish.

The goal of the protocol is to provide an industrialized fish fermentation technique based on inoculation of Saccharomyces cerevisiae.

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures ...

Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug discovery because they are the targets of more than half of all drugs. An obstacle to conducting biochemical, biophysical, and structural studies of membrane proteins as well as antibody development has been the difficulty in producing large amounts of high-quality membrane protein with correct conformation and activity. Here we describe a &#8220;bilayer-dialysis method&#8221; using a wheat germ cell-free system, liposomes, and dialysis cups to efficiently synthesize membrane proteins and prepare purified proteoliposomes in a short time with a high success rate. Membrane proteins can be produced as much as in several milligrams, such as GPCRs, ion channels, transporters, and tetraspanins. This cell-free method contributes to reducing the time, cost and effort for preparing high-quality proteoliposomes, and provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

This protocol describes an efficient cell-free method for production of high-quality proteoliposome by bilayer-dialysis method using wheat cell-free system and liposomes. This method provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug ...