A multi-column plate adapter allows chromatography columns to be interfaced with multi-well collection plates for parallel affinity or ion exchange purification providing an economical high throughput protein purification method. It can be used under gravity or vacuum yielding milligram quantities of protein via affordable instrumentation.
Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is also a key component in the development of new therapeutics. The evolving era of functional proteomics is fueling the demand for high-throughput protein purification and improved techniques to facilitate this. It was hypothesized that a multi column plate adaptor (MCPA) can interface multiple chromatography columns of different resins with multi-well plates for parallel purification. This method offers an economical and versatile method of protein purification that can be used under gravity or vacuum, rivaling the speed of an automated system. The MCPA can be used to recover milligram yields of protein by an affordable and time efficient method for subsequent characterization and analysis. The MCPA has been used for high-throughput affinity purification of SH3 domains. Ion exchange has also been demonstrated via the MCPA to purify protein post Ni-NTA affinity chromatography, indicating how this system can be adapted to other purification types. Due to its setup with multiple columns, individual customization of parameters can be made in the same purification, unachievable by the current plate-based methods.
Protein purification techniques to achieve milligram quantities of purified proteins are imperative to their characterization and analysis, especially for biophysical methods such as NMR. Protein purification is also central across other areas of study such as drug discovery processes and protein-protein interaction studies; however, achieving such quantities of pure protein can become a bottleneck for these techniques1,2,3. The principal method for protein purification is chromatography, which includes a variety of methods that rely on the individual characteristics of proteins and their tags. In affinity chromatography, proteins have an additional protein or peptide motif that works as a tag that has an affinity for a certain substrate on the chromatography resin4. The most common affinity method is immobilized metal affinity chromatography (IMAC) using His-tagged proteins, whereas another popular method is ion exchange chromatography that separates proteins based on their charge. For highest purity, a combination of affinity chromatography and ion exchange is frequently used together, usually requiring expensive lab equipment for high-throughput.
The evolving era of functional proteomics is fueling the demand for high-throughput techniques for purifying not singular proteins for specific analysis but large numbers of proteins simultaneously for comprehensive analysis and genome wide studies5. Immobilized metal affinity chromatography (IMAC) is one of the most widely used methods for high-throughput protein purification6,7 yet its automated systems are costly and unaffordable for smaller laboratories8. The more affordable plate-based alternatives that are currently available employ the use of accessible laboratory-based equipment, such as a vacuum. Although these methods are successful in improving the speed of purification, it can only achieve high-throughput purification on a smaller scale, only yielding protein in the microgram range. These limitations mean that the pre-packed 96 well filter plates (e.g., from GE Healthcare now owned by Cytiva) cannot be used before biophysical techniques9. Gravity chromatography is the most cost-efficient method of purification; however, setting up multiple columns is inconvenient and can be prone to error for multiple proteins.
A multi column plate adaptor (MCPA) has been developed and proven to successfully and conveniently run parallel affinity chromatography columns at once to purify His-tagged yeast SH3 domains10. The MCPA offers a cost-efficient high-throughput purification method that does not depend on costly instrumentation. Its flexible design can effectively purify milligrams of protein by multiple affinity chromatography columns under gravity or vacuum manifold. Furthermore, resin type, volume, and other parameters can be adjusted for each individual column for faster optimization. This study demonstrates that ion exchange chromatography by the MCPA can be used in conjunction with affinity chromatography by the MCPA to enhance the purification of the Abp1 SH3 domain. Additionally, up to 24 different proteins can be separated in parallel using these methods.
1. Denaturing Ni-NTA chromatography
2. Ion Exchange – Single protein purification
3. Ion Exchange – Simultaneous purifications of 24 different proteins
NOTE: See steps 2.1-2.3 for details on making buffers, a series of salt concentrations and preparing samples
As an example, the MCPA has successfully purified 14 AbpSH3 mutants in denaturing conditions via Ni-NTA (Figure 2A). A small contaminant ~ 25 kDa can be seen, however the protein is still largely pure. This contaminant is believed to be YodA, a common co-purified protein found in E. coli11. Figure 2B shows the purification of 11 different SH3 domains under native conditions. The small contaminant seen in denaturing conditions is removed in native conditions. This shows that the MCPA can be used for comparison of purifications composed of native or denaturing buffers as listed in Table 1.
Representative data for the purification of a lysate via IEX MCPA are shown in Figure 3. This suggests that AbpSH3 can be separated from the majority of the contaminants as it elutes later between 425 mM to 700 mM. The concentration of salt needed to elute the protein from the column relates to the strength of electrostatic interaction between the protein and the resin. The majority of bacterial proteins have low pIs; however the protein of interest is very negative and appears to have a lower pI. Good yields of considerably pure AbpSH3 protein were recovered with some various higher molecular weight contaminants AbpSH3 seen as bands at ~ 5 kDa. IEX via MCPA can therefore be used as the first step in a purification protocol as it can isolate sufficient quantities of protein from the present contaminants in a lysate.
Further purification by IEX using the MCPA has been successfully demonstrated on fractions eluted from an Ni-NTA MCPA run (Figure 4). Noticeably, two main peaks have been resolved with maxima at 400 and 700 mM salt, which may correspond to separating an N-terminal truncated version of this protein. Through further DSF data analysis it was made apparent that peak 1 was slightly less stable and had a slightly lower Tm relative to peak 2. In comparison to the IEX run of the lysate, the fractions overall are much cleaner and show the benefit of running a Ni-NTA step before IEX. Although there is slight contamination with proteins of a higher molecular weight, the fractions are still largely pure and have yielded good biophysical data using NMR and thermal/chemical denaturation assays.
Figure 1: A front view of the MCPA instrument. (A) Front view of the MCPA instrument with 24 columns attached. Columns are spaced out evenly within the 96 well sealing mat to guide the elutions into a 96, 48 or open collection plate. (B) Top view of the sealing mat. (C) Bottom view of the same sealing mat. (D) Front view of the MCPA with columns and syringe plungers attached. This figure has been modified from Dominguez et al.10. Please click here to view a larger version of this figure.
Figure 2: An example of a 1 x 24 column configuration, the purification of various SH3 mutants under denaturing and native conditions. (A) Denaturing purification of various AbpSH3 mutants. A slight contamination can be spotted of ~ 25 kDa across each lane.(B) Native purification of 11 different yeast SH3 domains. The contaminants have been removed from each lane and no longer visible on gel. This figure has been modified Dominguez et al.10. Please click here to view a larger version of this figure.
Figure 3: Purification of lysate using IEX via MCPA. (A) Absorbance readings of all elutions from the ion-exchange (IEX) were measured by an LVis plate (BMG). The readings are plotted against the corresponding NaCl concentration of the elution. The peak with the highest protein concentration is seen at 700 mM NaCl. (B) SDS-PAGE analysis of the IEX salt elutions presented in (A). The molecular weight marker is shown on the left of the gel. Please click here to view a larger version of this figure.
Figure 4: Purification of VJM2 Pool (post Ni-NTA) using IEX via MCPA system.
(A) Absorbance readings of the collected elutions from the ion-exchange (IEX) were measured using a NanoDrop. The readings are plotted against the corresponding NaCl concentration of the elution.(B) SDS-PAGE analysis of the IEX salt elutions presented in (A). The molecular weight marker is shown on the left of the gel. Please click here to view a larger version of this figure.
Denaturing Buffer Composition | Native Buffer Composition | |
Lysis Buffer | 10 mM NaH2PO4, 10 mM Tris, 6 M GuHCl, 10 mM imidazole, pH 8.0 | 20 mM Tris, 300 mM NaCl, 10 mM imidazole, pH 8.0 |
Wash Buffer | 10 mM NaH2PO4, 10 mM Tris, 6 M GuHCl, 20 mM imidazole, pH 8.0 | 20 mM Tris, 300 mM NaCl, 20 mM imidazole, pH 8.0 |
Elution Buffer | 10 mM NaH2PO4, 10 mM Tris, 6 M GuHCl, 0.2 M acetic acid, pH 3.0 | 20 mM Tris, 300 mM NaCl, 250 mM imidazole, pH 8.0 |
Buffer Composition | ||
High Salt | 5 mM Tris acid, 5 mM Tris base, pH 8.1, 4 M NaCl | |
Low Salt | 5 mM Tris acid, 5 mM Tris base, pH 8.1 |
Table 1: Composition of buffers, Ni-NTA denaturing and native purifications and Ion exchange low and high salt buffers.
The method is robust and simple to use for relatively inexperienced protein biochemists, however there are a few considerations to bear in mind.
Caution about overfilling collection plates
The 48-well collection plate itself only holds 5 mL per well while each 96-well only holds 2 mL. This needs to be kept in mind when adding buffer and running sample through the column as there is the risk of overfilling the wells. In particular, care needs to be taken when transferring the larger samples to the chromatography column for purification. In cases where there is excess supernatant, divide in parts, sufficient to fill the column, which should be allowed to run through before adding the next part to prevent any overfill and loss of sample. After each addition of supernatant, column should be mixed with a small plastic loop, to increase the likelihood of protein binding to the beads, before turning on the pump. To keep track of the plate's orientation and therefore content of the wells, ensure that the labelled corner 'A1' is always at the top left corner of the plate before starting the purification.
Eluting Protein
When eluting in the IEX and affinity step, the vacuum is used on the lowest setting to pull the elution buffer through the column. This speeds up the flow rate compared to gravity although if the protein concentration is high, it can lead to the protein solution to froth and potentially denature. If this is the case, an alternative is to use a syringe plunger on top of the open column to push the buffer through the column. In this case, the syringe plunger should be gently (not forcefully) pushed down into the column to push the liquid through and into the collection plate beneath. Care should be taken when removing the collection plate to ensure any elution drops remaining on the MCPA do not spill into neighboring wells, causing contamination.
Maintenance of resin and columns
A critical step in this protocol is the regeneration of the Ni resins and columns. Columns should be regenerated at either the start or end of the purification protocol. If regeneration occurs at the end, the resin should be stored in 20% ethanol at 4 °C. The chromatography column filters may become "blocked" causing the sample to flow through the column at a much slower rate than it should. If this is the case, columns need to be replaced or filters removed and cleaned by soaking in denaturing buffer overnight and rinsing with water.
As demonstrated under step 3, the diversity of the MCPA instrument can be exploited for the purification of multiple proteins as effectively as a single sample. The ion exchange protocol can be tailored to suit the needs of the experiment, adapting to the number of different samples and number of salt concentrations being used. For example, if less than 12 samples are simultaneously purified, it would involve any combination of moving the columns after each elution within the same plate and/or swapping collection plates for each elution. If for example, only 4 proteins were being purified in parallel, the columns can be moved across one collection plate to collect elutions of up to 4 salt concentrations before changing plates. MCPA is capable of purification using various resins – affinity and ion exchange have already been discussed but hydrophobic interaction chromatography is also possible and there is further potential for immunoaffinity chromatography12. Although this method has focused on multiple small-scale purifications, the MCPA can be used for just a single large chromatography column for purification from several liters of bacterial culture, without the need for a sample pump or an expensive FPLC.
Using the MCPA for high-throughput ion exchange as discussed would require multiple, up to 24, separate collection plates. This could be impractical and require a lot of space on a standard laboratory bench top and increased risk of human error. Furthermore, measuring protein absorbance of elutions from 24 different samples may be challenging. In this situation a multi-channel pipette would be beneficial and would make the transfer of multiple samples quicker and easier. For small volumes, consider using an LVis plate (BMG) containing 16 microdrops as it enables measurement of the concentrations directly, without the need to use any other reagents such the Bradford assay reagent.
While the use of a vacuum pump allows for a 3x quicker purification speed than what is achieved using just gravity10, without compromising the integrity of the resin, it does create some other issues. Maintaining the strength of the vacuum during the purification, for example, requires all the columns to be blocked with a 10 mL syringe plunger which needs to be taken out before inserting the column packed with resin. Taking the plungers out one by one is also a timely process and the resistance of the vacuum can make them difficult to remove from the column.
Details of a multiple protein IEX purification using the MCPA are given in the protocol. This higher-throughput method is time efficient and controllable, all parameters for each purification can be manipulated by the user. However, in most protein biochemistry labs, including industry, fast protein liquid chromatography is the preferred method of protein purification, which is superior in terms of throughput, reproducibility and method transfer and robustness. These systems such as the Protein Maker by Protein Biosolutions and the AKTA FPLC biomolecule purification system can alleviate the purification bottleneck problem with great success. Despite these systems obtaining superior results, the separation we see using the MCPA system is still good enough to obtain high purity protein. Interestingly, our lab also uses the AKTA start FPLC to perform ion exchange chromatography and although the resolution may be higher with a linear gradient capable with this machine, it is notably more time consuming to run multiple samples and it is much more challenging to train inexperienced students on this system.
Other significantly cheaper plate-based purification alternatives exist. For example, GE Healthcare life sciences (now Cytiva) and Sigma Aldrich sell pre-packaged 96 well filter plates and cartridges with specific purification resins. These filter plates offer small scale high-throughput purification but only purifying in the microgram yield range. Furthermore, the QIAvac 24 Plus from QIAGEN uses spin columns under vacuum however, it is not practical for collecting flow through or washes.
The flexible design of the MCPA allows for parallel protein purification, although manually moving columns and plates using the MCPA method potentially increases human errors compared to standard FPLC systems. However, manually loading the samples onto columns is more reliable for inexperienced users than loading samples onto columns using standard FPLCs, where mistakes can be more easily made as it involves switching valves and pumps that requires more extensive training. It is clear that fully automated systems for protein purification are better suited than the MCPA for purification groups in industry and academic labs which routinely work on purification. However, for small laboratories which cannot afford the expensive equipment and upkeep and want to avoid extensive training or only occasionally work on protein purification, the MCPA offers an effective alternative system which still obtains good separation and is cheap and easy to set up.
The MCPA consists of simple and inexpensive instrumentation which permits multiple columns to be interfaced for simultaneous parallel purification to produce milligram quantities of proteins. Furthermore, this technique allows modularity of the individual columns increasing the throughput. This is unique to this method and cannot be achieved using current plate-based purification kits.
Protein purification will remain essential in the study and characterization of proteins and the development of therapeutics. Biophysical techniques such as NMR and protein crystallography rely on milligram quantities of pure protein, therefore the current expression and purification systems need further development to improve the cost and time of achieving this2,13,14. As discussed, automated purification systems have many advantages over un-automated methods however they remain too costly for smaller scale laboratories requiring expensive instrumentation and training. The MCPA is considerably cheaper with a starting cost of $4510. Additionally, this MCPA does not need extensive training or continuous maintenance and should any problems arise these can be easily solved. Corrosive buffers such as the denaturing buffers used for the Ni-NTA can corrode purification systems if they are not cleaned properly. However, the flexible design of the MCPA allows for quick cleaning, repairing and changing of compartments if necessary. In conclusion, the MCPA will facilitate effective, higher-throughput protein purification for smaller laboratories until more affordable automated systems are established10.
The authors have nothing to disclose.
Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103451 and an internal research grant from the University of Liverpool.
2 mL/ well collection plate | Agilent technologies | 201240-100 | |
5 mL/ well collection plate | Agilent technologies | 201238-100 | |
12 mL chromatography columns | Bio-Rad | 7311550 | |
96 well long drip plate | Agilent technologies | 200919-100 | Come with 0.25 um filters which are to be removed. |
96 well plate seal/mat | Agilent technologies | 201158-100 | Should be peirceable |
His60 Ni Superflow Resin | Takara Bio | 635660 | |
HiTrap Q HP anion exchange column | GE Healthcare (Cytiva) | 17115301 | |
Lvis plate reader | BMG LABTECH | Compatible with FLUOstar Omega plate reader | |
Male leur plugs | Cole-Parmer | EW-45503-70 | |
PlatePrep 96 well Vacuum Manifold Starter kit | Sigma-Aldrich | 575650-U | |
Reservoir collection plate | Agilent technologies | 201244-100 | |
The Repeater Plus | Eppendorf | 2226020 | With 5 mL and 50 mL syringes |
VACUSAFE vacuum | INTEGRA | 158 320 | The vacusafe vacuum has a vacuum range from 300 mBar to 600 mBar and a 4 L waste collection bottle |