Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification

Abstract

His-tag affinity purification is one of the most commonly used methods to purify recombinant proteins expressed in E. coli. One drawback of using the His-tag is the co-purification of contaminating histidine-rich E. coli proteins. We engineered a new E. coli expression strain, LOBSTR (low background strain), which eliminates the most abundant contaminants. LOBSTR is derived from the E. coli BL21(DE3) strain and carries genomically modified copies of arnA and slyD, whose protein products exhibit reduced affinities to Ni and Co resins, resulting in a much higher purity of the target protein. The use of LOBSTR enables the pursuit of challenging low-expressing protein targets by reducing background contamination with no additional purification steps, materials, or costs, and thus pushes the limits of standard His-tag purifications.

Keywords: BL21(DE3); E. coli protein expression strain; His-tag affinity purification; LOBSTR.

Figure 1. LOBSTR and the parental BL21(DE3) strain show comparable growth

The growth (OD600) of both LOBSTR and the parental BL21(DE3) strain was measured from initial synchronization at 0 hours until the final harvest. Both strains carried the same expression plasmid and were grown at 37°C until an OD600 ~0.7, at which point protein expression was induced at 18, 25 and 37°C (black arrow). The growth curves for LOBSTR and BL21(DE3) are shown in red and black, respectively. Cell growth during log phase and final cell density is similar for both strains.

Figure 2. ArnA and SlyD are eliminated from His-tag purifications from LOBSTR

Elution samples of test purifications from BL21(DE3) and LOBSTR using common metal affinity resins are shown. A. Seven protein constructs were purified from both the parental BL21(DE3) strain and LOBSTR using Ni Sepharose 6FF resin (GE Healthcare). The constructs are numbered 1–7, and contain either a 6×His-tag (1 and 4) or a 10×His-tag (2,3,5–7). See table S2 for a list of all test constructs. The elution samples were run on an SDS-PAGE gel and stained with Coomassie Blue R250. ArnA and SlyD are indicated by arrows and target proteins indicated with a black circle (●). The double asterisk (**) indicates Hsp15, another protein showing reduced Ni-binding affinity in LOBSTR. B. Purifications of constructs 1 and 5 from BL21(DE3) and LOBSTR were also carried out on two additional commonly used resins, Ni-NTA (Qiagen) and Talon (Clontech). In each case, ArnA and SlyD are successfully eliminated in LOBSTR.