High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease

Abstract

The establishment of an efficient and reliable protein-purification pipeline is essential for the success of structural genomic projects. The SSGCID Protein Purification Group at the University of Washington (UW-PPG) has established a robust protein-purification pipeline designed to purify 400 proteins per year at a rate of eight purifications per week. The pipeline was implemented using two ÄKTAexplorer 100 s and four ÄKTAprimes to perform immobilized metal-affinity chromatography (IMAC) and size-exclusion chromatography. Purifications were completed in a period of 5 d and yielded an average of 53 mg highly purified protein. This paper provides a detailed description of the methods used to purify, characterize and store SSGCID proteins. Some of the purified proteins were treated with 3C protease, which was expressed and purified by UW-PPG using a similar protocol, to cleave non-native six-histidine tags. The cleavage was successful in 94% of 214 attempts. Cleaved proteins yielded 2.9% more structures than uncleaved six-histidine-tagged proteins. This 2.9% improvement may seem small, but over the course of the project the structure output from UW-PPG is thus predicted to increase from 260 structures to 318 structures. Therefore, the outlined protocol with 3C cleavage and subtractive IMAC has been shown to be a highly efficient method for the standardized purification of recombinant proteins for structure determination via X-ray crystallography.

Figure 1

Flowchart of the UW-PPG protein-purification protocol. Eight SSGCID targets were purified per week utilizing two research scientists, two ÄTKAexplorer 100s and four ÄTKAprimes (GE Healthcare, Piscataway, New Jersey, USA). Following initial immobilized metal-affinity chromatography (IMAC) of the soluble lysates, the polyhistidine tag was removed from the recombinant protein using 3C protease. The cleaved protein was separated from the 3C protease, the His-tag peptide, uncleaved protein and any Ni-binding contaminants through subtractive IMAC. Size-exclusion chromatography (SEC) was then used as a final purification step and SDS–PAGE was used to determine the fractions to pool. The pooled protein was concentrated to 20–30 mg ml⁻¹ and stored at 193 K. In our group, the procedures were carried out on the days noted in the upper right-hand corner of each box.

Figure 2

A GelCode Blue-stained (Thermo Scientific, Rockford, Illinois, USA) SDS–PAGE of samples from a typical purification, represented in this case by recombinant HAD-superfamily hydrolase from Ehrlichia chaffeensis. Lanes are labelled as follows: M, molecular-weight standards; T, total protein; S, soluble fraction; FT, flowthrough (nonbound) from the first IMAC column; P, purified protein after first IMAC column; B4–C4, successive size-exclusion chromatography (SEC) fractions from peak (see Fig. 2 ▶), the dotted fractions were pooled for final concentration; 3C+P, protein after overnight cleavage with 3C protease; FT, unbound protein from second IMAC column after dialysis with 3C protease; W, protein from second IMAC column that eluted in the wash fractions; E, protein eluted from the second IMAC column with 500 mM imidazole. The identity of the DnaK protein band was determined by gel extraction, trypsin digest and mass-spectrometric analysis.

Figure 3

Structure success rate for uncleaved versus cleaved proteins. An increase of 2.9% is seen in the structure success rate of cleaved proteins over uncleaved proteins. This is likely to be a consequence of the removal of contaminating Ni-binding E. coli proteins.