Affinity Purification Followed by Buffer Exchange Using INtip dSPE on Automated Liquid Handler for High Throughput Protein Purifications

Introduction

Pipette-based dispersive solid phase extraction (dSPE), or INtip dSPE, expands the functionality of many liquid handlers beyond traditional liquid transfer functions. Incorporation of INtip dSPE reduces the need for ancillary equipment, such as centrifuge, vacuum manifolds, or magnetic plates with grippers to support the sample extraction process. Here, we demonstrate two different chromatography techniques (affinity and size exclusion) on multiple protein targets on an automated liquid handler. The automated workflow starts with affinity extraction of proteins from crude lysates, followed by rapid buffer exchange on a size exclusion pipette. The flexibility of the system allows for multiple chromatography resins, designing different protein purification strategies by screening buffers, and assessing new formulations for micro- and milligrams of protein quantities in early protein discoveries.

Methods

All experiments were performed on Hamilton Microlab STAR. Purified green fluorescent protein (GFP) was fortified into bacterial cell lysates at various concentrations and purified utilizing affinity scripts. GFP eluates (150 µL) were loaded on SizeX₁₅₀ to remove imidazole salts, and the script was adjusted based on GFP recoveries. The final script was further tested using two other enzymes with polyhistidine tags to determine the workflow efficiencies for proteins with larger molecular weights (sulfatase with ~60 kDa and glucuronidase with estimated molecular weight of ~280 kDa as a tetrameric enzyme). The purified samples were further characterized using NanoDrop for UV absorbance, and Bradford assay. Standard deviations were derived from 8 samples and statistical comparison was using ANOVA test.

Preliminary Data

The script is a turn-key solution to a common protein purification process, where lysates containing the protein of interest were loaded onto the liquid handler and purified, desalted proteins were ready upon completion of the script. The advantages are the use of a single liquid handling system, rather than two separate platforms or two different programs.

< !·        Automated method achieves comparable recovery of target protein across multiple protein concentrations for both affinity and       buffer exchange process in comparison to the manual spin column method.

< !·        Increase of 20-30% in overall throughput in comparison to manual spin column method.

< !·        Performed 96 affinity purifications followed by buffer exchange with one method with a processing time of 1 hour.

Novel Aspect

This fully automated method incorporates two different sample preparation processes using tip-based chromatography on one liquid handling platform for the purification and buffer exchange of target proteins.