doi:10.1016/j.pep.2005.12.010
Copyright © 2006 Elsevier Inc. All rights reserved.
High-throughput protein purification using an automated set-up for high-yield affinity chromatography
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Johanna Steen, Mathias Uhlén, Sophia Hober and Jenny Ottosson
, 
School of Biotechnology, Division of Molecular Biotechnology, AlbaNova University Center, KTH, Royal Institute of Technology, Roslagsvägen 30 B, 106 91 Stockholm, Sweden
Received 2 June 2005;
revised 14 December 2005.
Available online 26 January 2006.
Abstract
One of the key steps in high-throughput protein production is protein purification. A newly developed high-yield protein purification and isolation method for laboratory scale use is presented. This procedure allows fully automated purification of up to 60 cell lysates with milligram yields of pure recombinant protein in 18.5 h. The method is based on affinity chromatography and has been set up on an instrument that utilizes positive pressure for liquid transfer through columns. A protocol is presented that includes all steps of equilibration of the chromatography resin, load of sample, wash, and elution without any manual handling steps. In contrast to most existing high-throughput protein purification procedures, positive pressure is used for liquid transfer rather than vacuum. Positive pressure and individual pumps for each liquid channel contribute to controlled flow rates and eliminate the risk of introducing air in the chromatography resin and therefore ensure stable chromatography conditions. The procedure is highly reproducible and allows for high protein yield and purity.
Keywords: High-throughput; Protein purification; IMAC; Recombinant protein; Proteomics
Fig. 1. The instrument set-up. (A) Front view of pump system and working table with sample racks 1–5. (B) Top view of working table. Each of the four syringe pumps are connected to the system buffer and a steel-probe mounted on a robotic arm above the working table. The different buffers are collected by the probes from reservoirs behind the racks (not shown in figure). Racks 1 and 5 holds thermostated blocks for 60 tubes for sample (1) and eluate (5). Sliding column trays above a waste and eluate collect zone constitutes the middle racks on which the trays are moved into position with the robotic arm. Rack 2 is displayed without the column tray, 3 with the column tray in waste position, and 4 in eluate collect position.
Fig. 2. High-yield protein purification of His-tag proteins under denaturing (24 samples) and native (4 samples) conditions. Clarified lysate (L) and eluate (E) pair-wise. 15 μL of each sample is analyzed and the lysate is a 1:20 dilution and the eluate a 1:10 dilution to give comparable data. (A–C) Denaturing conditions; (D) native conditions. The A-X naming of the proteins correspond to the accession numbers in Table 2.
Fig. 3. Western blot analysis of sample-to-sample contamination carry-over. Five samples of His6ABP were purified according to the protocol, with an additional water fraction sampled from the steel probes after the system wash. 10 μL of the additional fraction and the purified protein were analyzed on western blot and compared to a His6ABP standard of known concentration. Lane 1, 80 μg/mL; lane 2, 40 μg/mL; lane 3, 20 μg/mL; lane 4, 10 μg/mL; lane 5, 5 μg/mL; lane 6, 1 μg/mL. Lanes 7–16, pair-wise eluate sample (diluted 1:100) and corresponding test sample (undiluted). No sample carry-over could be detected.
Table 1.
Protocol for protein purification on ASPEC XL4 workstation
Table 2.
Results from automated IMAC protein purification under denaturing and native conditions of His-tag proteins
The amount of protein was determined by using the bicinchoninic acid (BCA) kit (Pierce), micro assay protocol, which utilizes a bovine serum albumin standard. The purity of the samples was analyzed by Bioanalyzer protein 50 chip (Agilent Technologies). Subscript ‘n’ for proteins O, S, T, and X denote purification under native conditions.
a Accession numbers from Uniprot.
b Molecular weight of the protein fragments including the His
6ABP-tag (17.7 kDa).
c Mean values ± SD from five purifications with identical cell lysate samples.
Corresponding author. Fax: +46 8 55378481.
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