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DNA N 6 -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 ( l ow  a denine  m ethylation  b ackground  s train). 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.

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