The E. coli lsrK gene contained the sequence of the Bam HI restriction enzyme site, and thus, the lsrK gene was amplified from the genomic DNA of the E. coli K strain using the 5′- and 3′-primers with the Bgl II and Xho I restriction enzyme sites, respectively. The polymerase chain reaction (PCR) product was cloned into the pGEX-4T-1 expression vector (GE Healthcare) using the Bam HI and Xho I sites (21 (link)). For the coexpression of the N-terminal His-tagged LsrK and the native HPr proteins simultaneously, the lsrK and ptsH genes were cloned into the pACYCDuet-1 expression vector (Novagen). The lsrK gene was inserted into the first multiple cloning site (MCS) of the vector by using the Bam HI and Not I sites, and the ptsH gene was inserted into the second MCS by using the Nde I and Xho I sites, respectively.
To express the E. coli EI and HPr proteins as an intact form, the ptsI and ptsH genes were cloned into the pET-11a vector (37 (link)). The ptsH gene was additionally cloned into the pET-24a vector using the Nde I and Xho I sites to express the C-terminal six-His–tagged (C-His) HPr protein. The mutations resulting in the mutants H15E and H15A were introduced using the QuikChange Site-Directed Mutagenesis Kit (Stratagene).
To express the E. coli EI and HPr proteins as an intact form, the ptsI and ptsH genes were cloned into the pET-11a vector (37 (link)). The ptsH gene was additionally cloned into the pET-24a vector using the Nde I and Xho I sites to express the C-terminal six-His–tagged (C-His) HPr protein. The mutations resulting in the mutants H15E and H15A were introduced using the QuikChange Site-Directed Mutagenesis Kit (Stratagene).
