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

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.

Specifications table

Hardware in context

To understand living organisms at the molecular level, biomacromolecules are studied in isolation, in cellular systems, and in more complex networks (e.g., microbial communities or multicellular organisms). Specifically, proteins are one of the major categories of biomacromolecules and their isolation, termed protein purification, is a ubiquitous task required in any investigation seeking to learn what a protein looks like and how it works. This is especially true for uncharacterized proteins or those that have been mutated or engineered for some purpose, such as to study diseases or improve enzyme catalysis. After the success of the Human Genome Project and the consequent rise in accessible sequencing technologies by the late 1990 s, there existed a plethora of genes encoding for proteins with no structural data that would allow scientists to deeply understand the mechanisms by which these proteins functioned [1]. The field of structural genomics (SG) was thus born to address this grand challenge.

From 2000 to 2015, the field grew immensely with 51 SG facilities solving nearly 15,000 protein structures by X-ray crystallography or nuclear magnetic resonance [1]. To achieve this level of throughput, protein purification needed to become a more industrial endeavor through new technologies and techniques that were adopted during this time, which are still used to this day and will continue to be used in the foreseeable future. There were two features that were needed to purify proteins at scale: parallel workflows, and automation. Prior to SG, proteins were purified using glass gravity columns where a single protein can be purified at a time as described in Fig. 1 [2]. With the need to process more proteins regularly, SG scientists would manually purify proteins in parallel by simultaneously processing several gravity-columns with the appropriate resin packed inside (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 10, Fig. 11, Fig. 12, Fig. 13, Fig. 14, Fig. 15). There also existed fast protein liquid chromatography systems (FPLC), predominantly from ÄKTA (now owned by Cytiva). To leverage the FPLC’s ability to automatically load the resin with a single protein sample, wash, and elute the target protein (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 10, Fig. 11, Fig. 12, Fig. 13, Fig. 14, Fig. 15), SG scientists modified these systems (sometimes using existing laboratory equipment like peristaltic pumps) to improve their functionality [3], [4], [5], [6]. This type of work is continued to this day by scientists, as well as ÄKTA engineers that have developed newer versions [7], [8], [9]. While multiple FPLC systems could be run in parallel, this is an expensive alternative to manually processing several protein purifications by gravity column.

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.

In 2016, the Seattle Structural Genomics Center for Infectious Disease published their work developing the Protein Maker in partnership with automation company Emerald BioSystems (now sold by Protein BioSolutions). The Protein Maker combines both aforementioned features as it can automatically purify up to 24 proteins in parallel [10], [11]. The Protein Maker, however, is a commercially supplied solution for automated parallel protein purification that is patent protected (US Patent No. 6818060). Similar technologies include Profinia from Bio-Rad, which is a discontinued product; and the Fluent automated workstation by Tecan, which is extremely expensive. While both gravity columns and FPLC are used to this day, to our knowledge there does not exist an inexpensive open-source solution that achieves automatic and parallel protein purification. Therefore, we sought to design, build, and test a scalable system that can perform the basic functions of a paralleled FPLC system (i.e., the Protein Maker). Specifically, we want to process cell lysates by applying them to an affinity resin, washing the resin, and eluting the protein from the resin (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 10, Fig. 11, Fig. 12, Fig. 13, Fig. 14, Fig. 15). We call our inventions the REVOLVER and the MULTI-VOLVER.

Hardware description

In this section we first describe the design of REVOLVER, which is comprised of several components that can be independently 3D-printed and fitted with the firmware to form one functional device (Fig. 2, Section 2.1). Several REVOLVERs can be connected with additional components and firmware for automated parallel protein purification. We call this combination the MULTI-VOLVER, which we discuss in a subsequent section (Fig. 3, Section 2.3). Our printed circuit board (PCB) design is discussed in Section 2.3; the communication protocol between the Arduino Nano microcontrollers and their interaction with the user is discussed in Section 2.4. While we do our best to explain various concepts underlying these two inventions herein, we strongly suggest the reader first consider watching Supp.Video 1 – 4 to see the systems in action.

REVOLVER: single sample fraction collector

The objective of the REVOLVER (Fig. 2a) is to take the user’s cell lysate as input and produce small aliquots of purified protein in microfuge tubes as outputs with no human intervention. To do this, the REVOLVER must move various solutions through the resin packed into the gravity-column (herein termed ‘column’ for brevity). Specifically, after manual application of the protein sample to the resin (viz., users must first pour the cell lysate into the gravity-column), REVOLVER must detect when the cell lysate has finished flowing out of the column, then automatically wash the protein-bound resin and elute the target protein into a set of microfuge tubes for downstream processing. REVOLVER’s current hardware is designed specifically for these core steps using the following features: 1) flushed joints requiring minimal screws for simple assembly, 2) closed-loop functionality for automatic handling of solutions, and 3) flow reduction to limit disturbances in the packing of the resin.

Simplified assembly

First, REVOLVER’s bodies (Fig. 2b) are designed to fit into each other using joints (e.g., tongue-and-groove, double-lap butt). The waste collector fitted with the servo that holds the tube sensor is first combined with the motor tower, which is together inserted into the slits available on the top of the electronics box. If desired, hot glue is sufficient to lock these bodies together (e.g., the motor tower and electronics box). Screws are only needed to attach the motors to the bodies since these electronics make precise movements for which REVOLVER’s functionality depends on. An optional Hall sensor can be incorporated into the waste collector to auto-home (i.e., centre) the rotator plate. The plate is inserted onto the motor tower’s stepper. While not shown in Fig. 2, the gravity column can be held using a single-device column gripper that we have designed if the user does not have a metal rod-plate apparatus. These design features were intentional for the purpose of minimizing the number of parts and assembly actions. Detailed assembly instructions can be found in Section 5.0 (Fig. 7, Fig. 8, Fig. 9, Fig. 10, Fig. 11, Fig. 12, Fig. 13, Fig. 14, Supp. Figs. 1–4).

Closed-loop functionality

Second, REVOLVER boasts closed-loop features to address several inconveniences that come with manual protein purification using gravity columns. Extensively used columns can have slower flow rates than others due to poor column filter cleaning, or the presence of solid precipitates inside of the filter that block the passage of the solution. Even clean columns can have minor variations in flow rates based on how well the resin is packed and how much liquid is in the column (i.e., hydrostatic pressure). This means that the time spent in each operation is not always known beforehand. Consequently, the user must be vigilant in watching the column to ensure the resin does not dry during the wash steps, or that each fraction of many has finished eluting without overflowing and spilling precious sample. This can be an inefficient use of time and limits the number of samples to be processed at once depending on the skill and focus of the operator.

One alternative to address this issue was to have a peristaltic pump system to ensure a constant flow rate through the resin like in FPLC systems, which would allow for open-loop operation of the system since the time required for each operation can be predetermined. However, this would complicate the design and require expensive equipment. Another alternative was to attach a flow rate sensor to the exit hole of the column to calculate the amount of solution that had passed through the resin, yet these sensors are not cheap (>$50 CAD), inaccessible for low flow rates (<1 mL/min), and require custom fittings to adapt to columns. Rather than attempting to measure low flow rates, we designed a simple sensor to determine whether a conductive solution was flowing through the system. Specifically, we used a pair of pins that were physically close to one another (∼1–2 mm) but not touching, one connected to ground and the other to a digital input pin in the Arduino. The pair of pins act as a digital-like switch when there is liquid present between them to carry the current across, which is detectable by the firmware.

Two pairs of these liquid-detecting pins exist in REVOLVER: one pair inside the waste collector to monitor solution (i.e., cell lysate flow-through and wash) that would pass through the resin into waste, and the second mounted onto the servo motor to detect when each of the microfuge tubes were filled during the final elution step. The servo lifts the pins out of the microfuge tube to allow the plate to rotate and position the subsequent tube, and then lowers the pins to monitor the level of liquid again. Because all solutions regularly handled during protein purification were readily conductive (300 – 600 mM NaCl), our liquid sensors (the pair of pins) were effective at detecting the movement of solutions across the resin. Sensor testing is detailed in Section 7.1 and 7.2 (Figs. 15–17).

Flow reduction to minimize resin bed disturbance

Third, REVOLVER includes a hardware feature to reduce the flow of solutions into the column. High flow rates (>0.5 L/min) directly entering the column as a single stream can lift the packed resin from the base and expose the column’s filter. This results in the solution following the path of least resistance and avoiding paths through the resin; thus reducing protein binding, wash stringency, and target protein yields. We avoid high flow rates by first supplying the DC motor pumps with the minimum voltage required (6 V, a setting on the power source). We then use a 3D-printed adaptor we designed for the column’s entrance hole that dissipates the force of the solution entering the column and evenly distributes it onto the interior column wall to gently fill the column. This piece is called the flow diffuser (Fig. 2c).

MULTI-VOLVER: multiple sample fraction collector

The objective of the MULTI-VOLVER (Fig. 3a) is to take multiple cell lysates as inputs, and return multiple sets of purified proteins in microfuge tubes as outputs with no human intervention. One could manufacture multiple REVOLVERs each with their own pumps and power supplies for automated parallel protein purification. These components represent the largest fraction of the cost of REVOLVER, yet the pumps remain idle during most of the protein purification and the capacity of the power supply is underutilized. The MULTI-VOLVER is a cheaper solution that we devised to economize the use of a single set of pumps and one power socket instead. To be exact, six REVOLVERs with their own pumps and power supplies would cost $990 CAD. The MULTI-VOLVER brings this price down to $310 CAD – a 60% reduction for the same throughput.

This is accomplished through an additional central distributor device controlled by an Arduino that can service the “needs” of up to six REVOLVERs (i.e. the Workers) (Fig. 3b and 3c). “needs” here referring to completing a wash step and being ready for another round of wash to be pumped into the column, or for the elution of the protein to begin. The Distributor services the Workers through two additional functionalities: 1) an (optional) auto-homing mechanism that allows a distribution arm to find the location of the gravity columns, and 2) a distribution arm that rotates in circular fashion to guide the ends of the pumps towards the gravity column that is ready for the next solution to be pumped inside. Moreover, the Distributor coordinates the individual REVOLVERs and passes the instructions for executing the purification protocol; this communication protocol is detailed in Section 2.4. Since the MULTI-VOLVER is designed to have up to six REVOLVERs working together, arranged symmetrically (Fig. 3b), it benefits from the three design features previously described (Section 2.1.1. to 2.1.3.). Assembly details are described in Section 5.0.

Auto-homing to locate gravity columns

Unlike the REVOLVER (Fig. 2a), the lip of the gravity columns in the MULTI-VOLVER can be fitted with a special ring holding a Hall sensor that can be attached with hot glue. This allows the MULTI-VOLVER to automatically detect the position of the Workers’ columns (Fig. 3a). This ring can then be slid up and down such that it is beneath the distribution arm’s magnet when the distribution arm is rotated. When the position of the magnet is aligned directly on top of the Hall sensor during the auto-homing routine, the Distributor and Worker specific for that Hall sensor communicate to record the angular position of the distribution arm. This essentially provides the Distributor with the angular “address” for that Worker so in the future when solution is ready to be pumped into that Worker’s column, the Distributor knows which address to move the distributor arm to. It is also possible to manually rotate the distributor arm to store the address for each Worker. Details for automatic and manual homing of the distributor arm is further described in Section 6.1.

Distribution arm to dispense solutions

Since there is only one set of pumps, which includes one for the wash solution and the other for the elution solution, the Distributor has to move the distributor arm to the Workers’ addresses for the pumps to deliver the wash/elution solution to the right column. The distributor arm is controlled by a stepper motor, which is controlled by the Arduino that is housed inside the electronics box of the Distributor (Fig. 3c). To prevent tangling of the tubes for the pumps, the firmware restricts the distributor arm from making a full 360° rotation. More specifically, the firmware determines a set point where the distributor arm cannot cross (e.g., if the distributor arm is at Worker 1, and the Distributor needs to pump solution into left-adjacent Worker 6, and the set point is between Worker 1 and 6, it will rotate counter-clockwise ∼300° rather than clockwise 60°). The set point is automatically determined based on the distributor arm’s position when the MULTI-VOLVER is turned on.

Simplified wiring – PCB design

REVOLVER uses an Arduino Nano as a microcontroller but there are no off-the-shelf expansion boards to connect it to the components in our system. We therefore designed a custom printed circuit board (PCB) that would fit in the electronic boxes of REVOLVER. We ordered the PCB through JLCPB, although any other PCB manufacturer should be able to produce it.

The headers, sockets, and other electrical components (e.g., resistors, capacitors, diodes, transistors, screw block) were soldered on according to the system configuration (Fig. 6, Supp. Fig. 3). When completed, the PCB allows the user to simply plug the different components (e.g., motors and sensors) into the PCB for easy assembly of the hardware and electronics. The PCB includes extra headers for a 5 V line that can be controlled by the remaining analog and digital pins we did not use. Another line set to be the same voltage delivered by the power supply is set to is also provided (e.g., 1 – 20 V using our power supply). These were included in case the user wishes to modify the systems and power other useful features. While the PCB is a convenient feature of our design, it is not fully required and complete wiring diagrams can be found in Supp. Fig. 4.

Firmware and communication protocols

A requirement of our system was that it should be flexible enough to handle different purification protocols without having to update the firmware in each microcontroller. To achieve this, we made REVOLVER programmable by designing firmware that can take instructions and parameters from the user, and then execute them based on a predefined set of operations (e.g., wash, fill tubes).

The user interface with the REVOLVER is via serial USB connection with the user’s personal computer, specifically through the Arduino IDE software (Fig. 4a). Similarly, the user interface with the MULTI-VOLVER is via serial USB connection with the Arduino IDE on PC, except the connection exists with only the Distributor, not the Workers. The Workers are connected to the Distributor via an inter-integrated circuit (I2C) bus instead (Fig. 4b). In all cases each Arduino Nano microcontroller has to be uploaded (flashed) with the corresponding program for the REVOLVER and the MULTI-VOLVER’s Distributor and Workers before this serial USB/ serial USB + I2C bus user interface can be used, respectively.

For the Arduino Nano microcontroller configured for an independent REVOLVER, Fig. 4c depicts the main algorithm that takes inputs from the user through the Arduino IDE’s serial monitor. User commands take the form of “” where the “<” and “>” tell the program that a command is being made, and the “?” between the < > symbols represents a capitalized letter corresponding to a specific command the user wishes to run, plus the required arguments for that command. The firmware then executes the instruction based on a preset list of operations. A sequence of commands can be made using the notation in sequential form