title: Optimized expression and purification of biophysical quantities of Lac repressor and Lac repressor regulatory domain.
abstract: The recombinant production of Lac repressor (LacI) 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 LacI, 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 LacI is introduced which eliminates these complications. The expression vector is an adaptation of the pASK backbone wherein expression of the lacI gene is regulated by an anhydrotetracyline inducible tetA promoter and the host strain lacks the lacI 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 LacI 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 LacI constructs produced using this expression system are highly homogenous and functionally active.
authors.affiliation: Johnson Research Foundation and Department of Biochemistry & Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Stetz
authors.affiliation: Johnson Research Foundation and Department of Biochemistry & Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: Marie V
authors.initials: MV
authors.lastname: Carter
authors.affiliation: Johnson Research Foundation and Department of Biochemistry & Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. Electronic address: wand@upenn.edu.
authors.forename: A Joshua
authors.initials: AJ
authors.lastname: Wand
mesh_terms: D017920:Antiporters / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics*; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D056968:Lac Repressors / Q000737:chemistry / Q000235:genetics*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D017920:Antiporters; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D011994:Recombinant Proteins; C079051:tetA protein, Bacteria
keywords: Lac repressor; NMR spectroscopy; Protein deuteration; tetA promoter