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Materials

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Biochemistry

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

Published: May 21st, 2021

DOI:

10.3791/62075

1School of Life Sciences, University of Liverpool, 2Health Sciences Center, Texas Tech University

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 prote....

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1. Denaturing Ni-NTA chromatography

  1. Preparing buffers
    NOTE: See Table 1 for details of all buffers.
    1. Make up 500 mL of 0.5 M NaOH, making sure to add the Milli-Q water first and then adding the NaOH slowly whilst the beaker is on a stirrer. Filter using a 0.22 µm filter.
    2. Prepare 100 mL of 0.1 M nickel sulphate and filter using a 0.22 µm filter.
    3. Prepare 50 mL of 2 M imidazole and filter using a 0.22 µm filter.
    4. Prepare and 0.22 &.......

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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 .......

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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 t.......

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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.

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Name Company Catalog Number Comments
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

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