Recombinant expression and purification of LwaCas13a
Introduction
Materials
SOC Outgrowth Medium 25 mL (New England BioLabs cat. no. B9020S)
Terrific Broth – TB Media (1 L, ampicillin): Reconstitute TB media by adding 50.8 g of TB powder to a 2 L flask, add 4 mL of 100% Glycerol, and fill up with deionized water to 1 L final volume. Heat at 50°C with repeated stirring to dissolve completely, then autoclave at 121.0°C for 15 minutes. Let cool for several hours, then add Ampicillin to 100 μg mL−1 final concentration. TB Media is stable at 4°C for 6 months, or for 1 month at room temperature.
Procedure
Thaw one vial of RosettaTM 2 (DE3) pLysS competent cells on ice for 30 minutes, then add 1 μL of 50 ng/μL LwaCas13 expression plasmid. Incubate on ice for 5 minutes.
Heat shock cells by placing the vial into a 42°C pre-heated water bath for 45 seconds, then cold-shock cells on ice for 2 minutes.
Add 200 μL of SOC media to cells, and plate 100 μL of cell suspension on a prewarmed LB-Agarose plate containing 100 μg/mL Ampicillin. Incubate the plate overnight in 37°C incubator. TROUBLESHOOTING
The next day, inoculate 25 mL of TB media containing 100 μg/mL Ampicillin with a single colony and incubate overnight at 37°C on rotary shaker at 300 RPM.
Inoculate 4–12 L TB media, containing 100 μg/mL Ampicillin, with 5 mL/L starter culture and determine the optical density (OD, 600nm). The amount of starter culture depends on the downstream expression scale. It is recommended to start with 5 mL of starter culture for every 1 L of large-scale culture. Then shake at 37°C, 300 RPM.
Monitor OD every hour until cells reach an optical density of 0.4 – 0.6, then transfer flasks to 4°C for 30 min to allow flasks to cool prior to induction. Take an aliquot of uninduced culture for SDS-PSGE analysis. Critical step: For optimal expression, it is important to strictly follow the indicated OD of 0.4–0.6 at the time-point of induction.
Induce expression by adding 1 mL/L 0.5M IPTG and shake cultures for 16 hours on rotary shaker at 300 RPM, in a pre-chilled 21°C incubator.
Harvest cells by spinning the culture down at 5000 RPM for 15 minutes at 4°C. Take a small aliquot and resuspend in 500 μL P1 Buffer (Qiagen). Run together with the uninduced culture aliquot on a Bolt™ 4–12% Bis-Tris Plus SDS-PAGE gel, in 1x Bolt™ MES Buffer for 20 minutes at 200V.
Pause point. Cells can be directly used for purification, or stored at −80°C for up to 1 year. Cells are routinely stored as spread paste in Ziploc bags, which enables future expression testing and aliquoting by breaking frozen paste.
Critical. Perform all steps at 4°C and do not let the resin run dry. In steps where a working environment of 4°C cannot be achieved, try to keep the sample near 4°C by cooling on ice.
Crush and resuspend frozen pellet in 4x (w/v) supplemented lysis buffer (e.g. 20g of pellet in 80mL of buffer) by stirring the mixture at 4°C for 30 minutes on a magnetic stir table.
Critical step. To ensure optimal lysis downstream, monitor resuspension progress until a homogenous mixture is obtained.
Lyse cells by passing the cell suspension once through a pre-chilled LM20 Microfluidizer system at 27k PSI. Alternatively, cells can be ruptured on ice using sonication, with an amplitude setting of 100% for 1 second on and 2 seconds off, in a total of 10-minute sonication time is recommended to avoid heat induced denaturation of lysed protein. Collect a 100 μL fraction for SDS-PAGE analysis.
Clear Lysate by centrifugation for 1 hour at 10k RPM at 4°C.
Decant cleared supernatant into a conical 250 mL tube and collect a 100 μL fraction for SDS-PAGE analysis. With a 1000 μL pipette tip, streak an aliquot of insoluble fraction and resuspend in 100 μL of lysis buffer for SDS-PAGE.
Add 5 mL of Superflow Streptactin resin to the supernatant. Batch-bind the recombinant protein to the resin for 2 hours by gentle shaking at 4°C.
Meanwhile, prepare a 50 mL Bio-Rad Glass Econo-Column® by washing the column with 2x 50 mL of cold lysis buffer, then add 20 mL of cold lysis buffer to equilibrate the column bed. Drain the column immediately prior to sample application.
Critical step. Do not use supplemented lysis buffer, as the presence of protease inhibitor will affect downstream cleavage of Streptactin-SUMO tag.
Pour the resin-sample suspension over the prepared column and collect the flowthrough. Collect a 100 μL fraction for SDS-PAGE analysis. Then wash the collected resin three times with 25 mL cold lysis buffer. With a 200 μL pipette tip, take a small aliquot of resin and resuspend in 100 μL lysis buffer for SDS-PAGE analysis.
Add 15 mL of SUMO protease cleavage solution to the resin and allow for SUMO protease cleavage overnight at 4°C under gentle shaking.
Critical step. Avoid vigorous shaking to prevent foam formation and extensive coating of the column glass surface with protein-bound resin.
The next day, drain the column and collect the cleavage solution into a separate 50-mL Falcon tube. Then wash the remaining sample three times with 5 mL of lysis buffer to ensure complete transfer of cleaved protein. Collect a 100 μL aliquot of cleaved fraction for SDS-PAGE analysis.
Critical step. The resin bound with Twin-Strep-SUMO tag will remain in the column, while the collected fraction should contain untagged, native LwaCas13a. To ensure cleavage is completed, take a small aliquot of resin with a 200 μL pipette and resuspend in 100 μL lysis buffer for SDS-PAGE analysis.
Perform SDS-PAGE analysis of collected protein fractions to assure successful cleavage by SUMO protease. To do so, add 10 μL of sample to 30 μL of SDSPAGE sample buffer, heat to 95°C for 5 minutes and run SDS-PAGE on a Bolt™ 4–12% Bis-Tris Plus SDS-PAGE gel, in 1x BoltTM MES Buffer for 20 minutes at 200V.
Using an ÄKTA-Pure or similar FPLC system, wash a 5 mL HP SP column with 5 column volumes (25 mL) of Buffer B followed by 5 column volumes of Buffer A (25 mL) using a system flow-rate of 5 mL/min. Then equilibrate the column with 5 column volumes of a mixture of 12.5% Buffer B in A (250 mM NaCl) at a flow rate of 5 mL/min.
Dilute the collected sample two-fold by adding 30 mL of Buffer A.
Critical step. The dilution step is important to lower the NaCl concentration to 250 mM, to efficiently bind the sample to the equilibrated column during sample application.
Start sample application and cation exchange chromatography by running below program at 5 mL/min.
Collect 100 μL of peak-containing fractions for SDS-PAGE analysis. Example FPLC chromatogram of the ion exchange chromatography result is shown in Figure 4b.
Pool fractions containing LwaCas13 protein into a 15-mL centrifugal spin filter (50 MWCO) and concentrate to <1 mL final volume by spinning for 15 min at 4000 g centrifugation.
CRITICAL STEP. Sample concentration is important to reduce the sample loading volume for down-stream size exclusion chromatography, which is done to ensure a high-resolution separation of potential protein aggregates and other proteins from monomeric LwaCas13.
Equilibrate an S200 Increase 10/300 GL size exclusion column with 5 column volumes of S200 buffer at 0.75 mL/min.
Apply sample and start size-exclusion chromatography by running below program at 0.75 mL/min
Collect 100 μL of peak-containing fractions for SDS-PAGE analysis.
Pool and concentrate fractions containing LwaCas13a by centrifugation at 4000 g for 15 min in a 15-mL (50 MWCO) centrifugal spin filter. Then add protein storage buffer to 15 mL into the same centrifugal filter and repeat the centrifugation step
Critical step. Buffer exchange into protein storage buffer is very important for storage and downstream SHERLOCK. Therefore, for optimal results, do not change the formulation of protein storage buffer.
Determine the final protein concentration, dilute to 2 mg/mL in protein storage buffer, and store as 5 μL aliquots at −80°C until use. Aliquots are stable for at least six months at −80°C.
Pause point. The purified protein should be high quality and ready for Cas13based nucleic acid detection. A 5 μL aliquot is sufficient for roughly 50 individual SHERLOCK reactions, or 12.5 individual SHERLOCK conditions with 4 technical replicates for each condition.
