Automation of protein purification for structural genomics

Abstract

A critical issue in structural genomics, and in structural biology in general, is the availability of high-quality samples. The additional challenge in structural genomics is the need to produce high numbers of proteins with low sequence similarities and poorly characterized or unknown properties. 'Structural-biology-grade' proteins must be generated in a quantity and quality suitable for structure determination experiments using X-ray crystallography or nuclear magnetic resonance (NMR). The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. The purification procedure must yield a homogeneous protein and must be highly reproducible in order to supply milligram quantities of protein and/or its derivative containing marker atom(s). At the Midwest Center for Structural Genomics we have developed protocols for high-throughput protein purification. These protocols have been implemented on AKTA EXPLORER 3D and AKTA FPLC 3D workstations capable of performing multidimensional chromatography. The automated chromatography has been successfully applied to many soluble proteins of microbial origin. Various MCSG purification strategies, their implementation, and their success rates are discussed in this paper.

Figure 1

Strategy for automation of protein purification steps for proteins expressed in E. coli.

Figure 2

Example of chromatograms (as part of a results file) of IMAC-I and buffer-exchange steps using AKTA EXPLORER 3D for a six-protein (APC35594, APC35601, APC35609, APC35617, APC35624, APC35625) run. A: The chromatogram showing the progress of sample loading and column wash of six proteins with buffer A. (B, C) The chromatograms showing the first two proteins, (a) wash with buffer A containing 20 mM imidazole, (b) elution of His₆-tagged target proteins with buffer A containing 250 mM imidazole, (c) His₆-tagged target proteins after buffer exchange. Target protein names are indicated as APC numbers. In each chromatogram, UV absorbance at 280 nm is plotted versus milliliters of buffer solution flow. (d) Progress of the step gradient is indicated by the curve of %B, in green.

Figure 3

Example of chromatogram (as part of a results file of a four-protein run) of IMAC-II and buffer exchange using AKTA FPLC 3D. Shown here is one protein (APC36103). (a) Sample loading and column wash with buffer A. (b) Elution of cleaved His₆-tags, His₇-tagged TEV protease, and uncleaved target protein with buffer A containing 250 mM imidazole. (c) Cleaved target protein after buffer exchange. In the chromatogram, UV absorbance at 280 nm is plotted versus milliliters of buffer solution flow.

Figure 4

SDS-PAGE of 30-kDa target protein (APC234), purified by the process described in Figure 1: lane 1 – crude extract; lanes 2 and 3 – IMAC-I flow through; lane 4 – IMAC-I elution; lane 5 – after TEV protease cleavage and IMAC-II; lane 6 – low-molecular-weight markers (Amersham Biosciences), which run with apparent molecular weights of 97, 66, 45, 30, 20.1, and 14.4 kDa.

Figure 5

Distribution of protein production levels using the automated chromatography process. Total number of proteins was 253. The numeral on top of each column corresponds to the number of proteins purified in the amount indicated below the column (in milligrams).