OptimizedE. coliexpression 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.

Natively Unfolded FG Repeats Stabilize the Structure of the Nuclear Pore Complex

“…The following E. coli strains were used in this study for production of recombinant proteins: BL21 (DE3) (Agilent), BL21(DE3)-RIL (Agilent) and LOBSTR(DE3)-RIL (kerafast) (Andersen et al, 2013a). Strain usage in each case is specified in “ Recombinant proteins ” section.…”

Allosteric activation of apicomplexan calcium-dependent protein kinases

“…For crystallization, the following changes were made. Proteins were expressed in BL21(DE3)-LOBSTR-RIL E. coli, which has been optimized for low background in Ni-affinity purifications (31). Cultures were shifted to 18°C before induction with 0.2 mM IPTG for 16 h. Cells were lysed using a cell disruptor (Constant Systems) in 50 mM potassium phosphate, pH 8.0, 500 mM NaCl, , the CAD is partially occupied by Ca 2+ and can bind 1B7 (blue).…”

Structural Characterization of Bardet-Biedl Syndrome 9 Protein (BBS9)

“…15, kerafast) cells transformed with the His 10 -Arg 8 -ScSUMO-3C-HsBBS9 1-407 pETDuet-1 vector were grown in LB medium at 37°C to an optical density of 0.65. Cultures were shifted to 18°C for 30 min prior to induction with 0.2 mM isopropyl-␤-D-1-thiogalactopyranoside.…”