The recombinant production of Lac repressor (Lad) in Escherichia coli is complicated by its ubiquitous use as a regulatory element in commercially-available expression vectors and host strains. While LacI-regulated expression systems are often used to produce recombinant Lad, the product can be heterogeneous and unsuitable for some studies. Alternative approaches include using unregulated vectors which typically suffer from low yield or vectors with promoters induced by metabolically active sugars which can dilute isotope labels necessary for certain biophysical studies. Here, an optimized expression system and isolation protocol for producing various constructs of Lad is introduced which eliminates these complications. The expression vector is an adaptation of the pASK backbone wherein expression of the lad gene is regulated by an anhydrotetracyline inducible tetA promoter and the host strain lacks the lad gene. Typical yields in highly deuterated minimal medium are nearly 2-fold greater than those previously reported. Notably, the new expression system is also able to produce the isolated regulatory domain of Lad without co-expression of the full-length protein and without any defects in cell viability, eliminating the inconvenient requirement for strict monitoring of cell densities during pre-culturing. Typical yields in highly deuterated minimal medium are significantly greater than those previously reported. Characterization by solution NMR shows that Lad constructs produced using this expression system are highly homogenous and functionally active. (C) 2016 Elsevier Inc. All rights reserved.