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

This protocol makes it possible to purify high-quality septin complexes, and this is essential to study the many roles of septins in the cell. The main advantage of this approach is the synthesization of a simple procedure to purify high-quality septin using Addgene plasmids. Septins are difficult to study in cells due to their many interactions.

Sulfur reconstitution helps us disentangle this complexity by studying them in a simple system. We recommend to do purification in a single day to prevent degradation. In our experience, this will increase the lifetime and the quality of the protein.

To begin, grow the transformed bacterial culture at 37 degrees Celsius in a shaker incubator until the optical density at a wavelength of 600 nanometers reaches two to three for unlabeled septins, or 0.6 to 0.8 for msfGFP or mEGFP labeled septins. After incubation, induce the protein expression by adding IPTG to a final concentration of 0.5 millimolar and incubate the cells expressing unlabeled septin hetero-oligomers at 37 degrees Celsius for three hours, or the cells expressing msfGFP labeled hetero-oligomers at 17 degrees Celsius overnight. Then pellet the cells at 4, 000 times G for 20 minutes at four degrees Celsius.

Discard the supernatant and dissolve the pellet in 100 milliliters of lysis buffer to lyse the cells. Next, use a tip sonicator at 30%amplitude to sonicate the cell. Clarify the cell lysate by centrifuging at 20, 000 times G for 30 minutes at four degrees Celsius and save the supernatant.

Optionally, take a sample for denaturing electrophoresis as described in the manuscript. For affinity chromatography of His-tagged septins, equilibrate a pre-packed nickel sepharose high performance chromatography column with all the required buffers. Load the clarified supernatant down to the column and start the chromatography system.

Then elute the septin complexes with 50%HisTrap elution buffer at a flow rate of one milliliter per minute. To yield an imidazole concentration of 250 millimolar, collect 0.5 milliliter fractions. Now monitor the optical absorbance of the eluate at 280 nanometers with a fast protein liquid chromatography system and pick the fractions containing septin complexes.

For affinity chromatography of Strep-II tagged proteins equilibrate a pre-packed StrepTactin sepharose high performance chromatography column with septin buffer. Load the septin containing fractions recovered from the nickel column at a rate of one milliliter per minute and start the chromatography system. Then wash the bound protein and elute the septin complexes with 100%StrepTrap elution buffer at a flow rate of one milliliter per minute.

To yield a concentration of 2.5 millimolar desthiobiotin, collect 0.5 milliliter fractions. Pick the fractions containing septin complexes as indicated by the optical absorbance of the eluate at 280 nanometers monitored online with a fast protein liquid chromatography system. After removing the desthiobiotin by dialysis aliquot the protein complexes into the desired aliquot size, snap freeze the aliquot, and store it at minus 80 degrees Celsius.

For interferometric scattering microscopy, wash glass slides number 1.5 by sonicating them in an ultrasonic cleaner for five minutes each in water, isopropanol, and again in deionized water. Dry two glass slides with a gentle stream of nitrogen gas. Then place a seven microliter drop of 0.01%Poly-L-Lysine solution on the center of one of the slides and place the center of the other slide on top of the Poly-L-Lysine drop, orienting the two slides orthogonally for easy separation.

After incubating for 30 seconds, wash the slide by immersing in a beaker containing deionized water once and directly applying a stream of water twice. Dry them with a nitrogen gas flow and these slides can be stored for around six weeks at room temperature in dry conditions. Prior to the experiment, cut a piece of two by two, three by two, or three by three gaskets and stick them on the Poly-L-Lysine treated part of a glass slide, avoiding the glass slide and the gaskets contacting any dirty surface by placing the slide on a light duty wiper tissue.

Now press the gaskets with a pipette tip to stick them with the protecting plastic present on the gaskets. Next, warm the septin buffer to room temperature. Thaw the proteins in hand and keep them on ice afterward.

Then place the slide with gaskets on the commercial mass photometry system containing 19 microliters of septin buffer and focus the microscope using the autofocus option. Refocus with the new settings, using the autofocus option. To create a project folder, click File followed by New Project, and to load a project folder for storing data, click File followed by Load Project.

Then pipette one microliter of sample to 19 microliters of septum buffer drop. Mix, and while mixing, avoid touching anything to prevent the movement of the slide. Then record a 6, 000 frame video by clicking on Record.

Analyze the videos using the manufacturer's software to obtain the protein mass distribution. If the peaks of different septin hetero-oligomer sizes overlap too much or too many events are detected decrease the final septin concentration and measure again. And when enough counts of single molecules are not measured, increase the septin concentration and measure again.

For septin analysis, prepare the 5X darkSPB and a septin mix consisting of 100%dark septin at six times higher concentration than the desired final concentration in septin buffer and one millimolar Dithiothreitol. Then polymerize the septin by mixing water, 20%5X darkSPB and 16.67%septin mix, in this specific order, and incubate for 30 minutes at room temperature. Add three to five microliters of sample to a glow discharged electron microscopy grid and incubate for one minute.

Now, wash the grid twice by adding a drop of darkSPB buffer and absorb the liquid with a filter paper. Wash once with water and incubate for 30 seconds with 2%uranyl acetate. Next, blot the stain and air dry the sample for a few minutes.

Then image the septin bundles at 120 kilovolts and magnifications between 5, 000X and 60, 000X with a defocus of one to two micrometers. The HisTrap and StrepTrap chromatogram showed that the pooled fraction went from the start of the elution peak until the absorbance stabilized at around 250 milliliters and 50 milliliters respectively. The septin bands showed similar intensities in denaturing gel electrophoresis, suggesting that the septins are intact.

In native electrophoresis, the major band corresponding to the intact hetero-oligomers and a minor band corresponding to trimors or tetramers were observed. The apparent molecular weight of the msfGFP tagged complexes is indistinguishable from that of the untagged complexes. Mass photometry detected intact octamers and hexamers of septins.

An additional peak at 241 kilodaltons indicated the presence of two peanut proteins, DSep1 and mEGFP-DSep2. Transmission electron microscopy images of septin complexes showed rods of hexamers and octamers. The msfGFP tags are visible as fuzzy densities on the two ends in septin 2 msfGFP human septin octamers_9i1.

Small clusters of proteins can be observed in the shallow TIRF field. The confocal microscopy revealed large clusters of floating filamentous structures. Transmission electron microscopy showed small and large septin bundles corresponding to the clusters observed by total internal reflection fluorescence microscopy and confocal microscopy.

We recommend to work on ice at all times during the purifications and to add fresh reagents that we specify in the protocol. We're interested in interactions of septins in the context of cytokinesis. We reconstituted septin together with modern membranes actin and microtubules to study them with microscopy and several biophysical assays.

We use the purified septins to study how septins interact with actin filaments, microtubules and lipids. And we also use the purified septins to build synthetic cells where the septins facilitates cell division.