Name: purification and quality control of recombinant septin complexes for cell-free reconstitution
Uploaded: 2022-06-23T13:00:00
Duration: 11 min 50 s
Description: Watch this Scientific Journal Video about Purification and Quality Control of Recombinant Septin Complexes for Cell-Free Reconstitution at JoVE.com

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>

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			<td>488nm laser combiner iLAS2</td>
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			<td>Terrific broth</td>
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			<td>T0918-1KG</td>
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			<td>Tris/Glyine/SDS buffer</td>
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			<td>Tris-HCl</td>
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			<td>T5941-1KG</td>
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			<td>Ultrasonic cleaner</td>
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			<td>Vivaspin 6, 30,000 MWCO PES</td>
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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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