Lac repressor N-terminal DNA binding domain: cloning, isolation and preliminary 15N NMR observations

E. coli Lac repressor N-terminal DNA binding domain (headpiece) has previously been isolated by cleavage with clostripain, chymotrypsin, and trypsin to yield N-terminal fragments of 51, 56, and 59 amino acids respectively. This method is both inefficient and limited by the specificity of available proteases. To circumvent these difficulties, the authors are cloning the DNA sequence encoding various lengths of the lac1 gene into a plasmid containing the P/sub L/ promoter from bacteriophage γ as well as an improved ribosome binding site. In the authors initial efforts they cloned a 56 amino acid headpiece (pAK3-4); they describe here the isolation and characterization of this headpiece. It has been shown that 15N NMR can be extremely useful in investigating the structure and dynamics of the bacteriophage γ cro repressor protein with its binding site O/sub R/3. Uniformly labelled lac headpiece (56 aa) has been prepared by growth of E. coli transformed with pAK3-4 in a minimal medium containing 15N ammonium sulfates as the sole nitrogen source. NMR experiments were used to measure 15N-{1H} NOEs which are sensitive to mobility on a nanosecond timescale. They are thus able to observe changes in dynamics of individual amino acid side chains and backbone nitrogen of the peptide upon binding operator DNA