REVOLVER: A low-cost automated protein purifier based on parallel preparative gravity column workflows

Abstract

Protein purification is a ubiquitous procedure in biochemistry and the life sciences, and represents a key step in the protein production pipeline. The need for scalable and parallel protein purification systems is driven by the demands for increasing the throughput of recombinant protein characterization. Therefore, automating the process to simultaneously handle multiple samples with minimal human intervention is highly desirable, yet there are only a handful of such systems that have been developed, all of which are closed source and expensive. To address this challenge, we present REVOLVER, a 3D-printed programmable protein purification system based on gravity-column workflows and controlled by Arduino boards that can be built for under $130 USD. REVOLVER takes a cell lysate sample and completes a full protein purification process with almost no human intervention and yields results indistinguishable from those obtained by an experienced biochemist when purifying a real-world protein sample. We further present and describe MULTI-VOLVER, a scalable version of the REVOLVER that allows for parallel purification of up to six samples and can be built for under $250 USD. Both systems can help accelerate protein purification and ultimately link them to bio-foundries for protein characterization and engineering.

Keywords: 3D printing; Automation; Recombinant protein purification.

Graphical abstract

Fig. 1

Typical protein purification protocol. We focus on protein purification from Escherichia coli modified to express large quantities of a recombinant target protein, highlighted in magenta. Step 1) an overnight culture of the E. coli strain is inoculated into a large volume of nutrient media and allowed to grow to medium density (O.D. 0.6–1.0). Step 2) an inducer chemical is added that triggers protein expression. Step 3) continue protein expression by allowing the cell to grow in the presence of the inducer, then harvest the cells by centrifugation. Step 4) use any combination of methods to physically/ chemically lyse the cells to release the target protein. Step 5) remove cellular debris by centrifugation, then apply the supernatant (i.e., lysate) to either 5a) a gravity column containing the appropriate affinity resin, or 5b) an FPLC system. In (5a), application of the lysate to the resin is followed by a wash to remove bound non-target proteins, then an elution step where changes in some solution condition (e.g., pH, salt, imidazole) leads to the target protein eluting from the column where it may be collected for further work. The FPLC automatically performs these steps with real-time monitoring of the solution condition and protein detection. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Design of the REVOLVER protein purification system. (a) Concept, (b) Overview of the bodies comprising one REVOLVER device, (c) Schematic of the REVOLVER system with a gravity column and a flow diffuser.

Fig. 3

Design of the MULTI-VOLVER. (a) Concept of MULTI-VOLVER with six REVOLVERs, (b) Top-down view, (c) Components specific to the distributor in the MULTI-VOLVER configuration. The 3D-printed column grippers allow for precise positioning of the individual gravity columns. Note the electronics box here is different from the REVOLVER version.

Fig. 4

REVOLVER and MULTI-VOLVER Communication Protocol. (a) The user interface with the REVOLVER is through a serial USB connection with a personal computer (PC), (b) The user interface with the MULTI-VOLVER is through a serial USB connection a PC and the Distributor, which is separately connected to the individuals REVOLVERs (viz., the Workers), (c) Algorithm for storing and parsing commands in the REVOLVER, (d) Algorithm for the MULTI-VOLVER’s Distributor and individual REVOLVERs (viz., the Workers). Commands are stored with an algorithm similar to that in panel (c). Blocks with the same color in the Distributor and Workers denote communication between the devices via I2C.

Fig. 5

Top-down view of unique parts for the REVOLVER. (a) Overview of all unique parts, (b) Parts related to liquids: 1) column, 2) a8 – flow distributor, 3) 12 mm M3 screw, 4) 6 mm M3 screw, 5) M3 nut, 6) 6 mm Phillips screw, 7) servo horn, 8) DC pumps with fittings for small tubing (cf. Supp.Fig. 1), 9) small tubing, 10) large tubing, 11) 1-way valve, 12) valve adaptor, 13) tube connector; (c) Electronics components: 14) alligators (M/F), 15) hookup wire, 16) jumper (MF), 17) jumper (MM), 18) jumper (FF), 19) stepper, 20) servo, 21) power supply, 22) nano, 23) PCB (soldered), 24) magnet, 25) Hall sensor, and (d) 3D-printed components: 26) a9 – column grip, 27) a5 – servo arm, 28) a1 – tower, 29) a3 – waste collector, 30) a6 – box top, 31) a2 – plate, 32) a7 – box bottom.

Fig. 6

Step-by-step guide to assemble the waste collector.

Fig. 7

Step-by-step guide to assemble the tube sensor.

Fig. 8

Step-by-step guide to assemble the motor tower.

Fig. 9

Step-by-step guide to assemble the major components of the REVOLVER.

Fig. 10

Step-by-step guide to combine the hardware with the firmware.

Fig. 11

Step-by-step guide to wire the hardware with the firmware.

Fig. 12

Fully-assembled REVOLVER wired for firmware and pumps connected. The third pump is not included in this image, but is used during the recording of demonstration videos.

Fig. 13

Design and characterization of the waste sensor. (a) Waste collector version 1, (b) Detection of liquid drops without a third pump attached to the waste collection version 1 and (c) with a third pump attached, (d) Waste collector version 2, (e) detection of self-flushing mechanism where no vacuum is required for waste collector version 2. Note: waste collector version is only discussed in Section 7.1. All other sections refer to waste collector version 1.

Fig. 14

Design and characterization of the tube sensor. (a) Detailed view of the tube sensor component, (b) Time to fill and (c) weight of each tube after adding 16 mL of buffer to the column. (d) Relative error in weight of each tube compared to the mean weight of all samples. (e) Estimated albumin content in tubes after dipping the servo sensor sequentially. Values for tubes 2–8 are below the lower limit (5 µg/mL) of the calibration curve. Protein concentrations were determined by a BCA assay (ThermoFisher #23225).

Fig. 15

SDS-PAGE analysis of CcSBPII protein purification using (a) an automated protocol using REVOLVER, and (b) manual protocol by hand. Ladder numbers adjancent to SDS-gel images demark the molecular weight of the proteins. L = Lysate (post-sonication). E1-5 = Elution Fractions collected in 2 mL aliquots. (c) Average purity comparison of CcSBPII in E1-5 between REVOLVER and Manual as determined by densitometry using ImageLab 6.0.1.