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

Abstract

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.

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

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 from Dominguez et al

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.

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.