The Protein Maker: an automated system for high-throughput parallel purification

Abstract

The Protein Maker is an automated purification system developed by Emerald BioSystems for high-throughput parallel purification of proteins and antibodies. This instrument allows multiple load, wash and elution buffers to be used in parallel along independent lines for up to 24 individual samples. To demonstrate its utility, its use in the purification of five recombinant PB2 C-terminal domains from various subtypes of the influenza A virus is described. Three of these constructs crystallized and one diffracted X-rays to sufficient resolution for structure determination and deposition in the Protein Data Bank. Methods for screening lysis buffers for a cytochrome P450 from a pathogenic fungus prior to upscaling expression and purification are also described. The Protein Maker has become a valuable asset within the Seattle Structural Genomics Center for Infectious Disease (SSGCID) and hence is a potentially valuable tool for a variety of high-throughput protein-purification applications.

Figure 1

The Protein Maker instrument with syringe valves, liquid-handling sample manifold and deep-well plate deck (a). In the depicted configuration, 15 of the 24 syringe valves are at the front, with the remaining nine at the back (not shown). Also depicted is a schematic drawing of the plumbing for each individual nine-port valve (b) and a close-up image of the sample-load and primary purification manifolds with 24 × 1.0 ml purification columns in place (c). All 24 valves are individually lined and independently operated, thus allowing up to 24 sample-uptake lines and purification columns in a single run.

Figure 2

SDS–PAGE results for InvaB.07055.c (lanes 2–5) and InvaC.07055.b (lanes 7–­10) during nickel-chelate chromatography purification on the Protein Maker. Lanes 1 and 6, molecular-weight markers (labeled on the left in kDa); lanes 2 and 7, pooled protein from Nickel 1; lanes 3 and 8, flowthrough of cleaved protein in buffer A from Nickel 2; lanes 4 and 9, buffer A wash from Nickel 2; lanes 5 and 10, removal of 6×His-Smt tag, 6×His-tagged protease and uncleaved protein with buffer B from Nickel 2.

Figure 3

SDS–PAGE analysis of 12 different cell-lysis buffer conditions described in Table 2 ▶ for CYP51A1. Lanes correspond to the load (L), wash (W) and elution (E) cycles conducted in parallel on the Protein Maker, with the same molecular-weight standards throughout (labeled on the left in kDa). Cell-lysis buffer scouting resulted in two conditions with optimal yields after affinity chromatography (red boxed bands).