Conjoint expression and purification strategy for acquiring proteins with ultra-low DNA N6-methyladenine backgrounds in Escherichia coli

Abstract

DNA N6-methyladenine (6mA), a kind of DNA epigenetic modification, is widespread in eukaryotes and prokaryotes. An enzyme activity study coupled with 6mA detection using ultra-high-performance liquid chromatography-quadruple mass spectrometry (UHPLC-MS/MS) is commonly applied to investigate 6mA potentially related enzymes in vitro. However, the protein expressed in a common Escherichia coli (E. coli) strain shows an extremely high 6mA background due to minute co-purified bacterial DNA, though it has been purified to remove DNA using multiple strategies. Furthermore, as occupied by DNA with abundant 6mA, the activity of 6mA-related proteins will be influenced seriously. Here, to address this issue, we for the first time construct a derivative of E. coli Rosetta (DE3) via the λRed knockout system specifically for the expression of 6mA-related enzymes. The gene dam encoding the 6mA methyltransferase (MTase) is knocked out in the newly constructed strain named LAMBS (low adenine methylation background strain). Contrasting with E. coli Rosetta (DE3), LAMBS shows an ultra-low 6mA background on the genomic DNA when analyzed by UHPLC-MS/MS. We also demonstrate an integral strategy of protein purification, coupled with the application of LAMBS. As a result, the purified protein expressed in LAMBS exhibits an ultra-low 6mA background comparing with the one expressed in E. coli Rosetta (DE3). Our integral strategy of protein expression and purification will benefit the in vitro investigation and application of 6mA-related proteins from eukaryotes, although these proteins are elusive until now.

Keywords: DNA N6-methyladenine; E. coli Rosetta (DE3); UHPLC-MS/MS; low background; λRed system.

Figure 1. UHPLC-MS/MS analysis of the 6mA background in LAMBS

(A) UHPLC-MS/MS chromatograms of 6mA (m/z 266–150) and dC (m/z 228–112). The genomic DNA of LAMBS and Rosetta (DE3) was extracted and then digested to 2′-deoxynucleosides for the detection of 6mA and dC using UHPLC-MS/MS. (B) Quantification of 6mA backgrounds in five independent clones of LAMBS with Rosetta (DE3) as a control.

Figure 2. SDS/PAGE gel analysis of the target fraction and the chromatograms of protein purification

(A) SDS/PAGE analysis of 20 μl of total protein, supernatant, resuspension after AS precipitation, HIC, and MBP target fraction with BSA as a standard. The lanes were from the same gel, and the marker lane was cut and moved to the left of the image. (B) SDS/PAGE analysis of the target protein after dialysis, digestion with thrombin, and MonoQ chromatography. (C–E) Chromatograms of the target protein purification using HIC, MBP, and MonoQ chromatography respectively. The lanes were from the same gel and were cut and moved horizontally.

Figure 3. UHPLC-MS/MS analysis of the 6mA background in the purified target protein

(A) UHPLC-MS/MS chromatograms of 6mA (m/z 266–150) and dC (m/z 228–112). The DNA co-purified with the target protein was extracted and then digested to 2′-deoxynucleosides for the detection of 6mA and dC using UHPLC-MS/MS. (B) Quantification of the 6mA background in the purified target protein after the MBP affinity chromatography and after the MonoQ chromatography.