Overexpression and biosynthetic deuterium enrichment of TEM-1 beta-lactamase for structural characterization by magnetic resonance methods

An expression system has been developed that allows high levels of producti on of TEM-1 beta-lactamase with ease of biosynthetic incorporation of nucle ar isotopes. The gene for mature TEM-1 beta-lactamase fused to the leader s equence of the ompA protein was subcloned into the pET-24a(+) vector by int roduction of an NdeI restriction site at the first codon of the fused genes and transformed into Escherichia coli BL21 (DE3) cells. With protein induc tion at 25 degrees C supported by LB medium supplemented with osmolytes (30 0 mM sucrose and 2.5 mM betaine), the extracellular, mature form of wild-ty pe TEM-1 beta-lactamase was recovered at a level of 140 mg/L, The productio n level of E166N, E240C, E104C, and M272C mutants depended on the mutation but was invariably higher than reported by others for expression systems of the wild-type enzyme. Comparison of different carbon sources on the effici ency of biosynthetic incorporation of covalent deuterium showed maximal (90 %) incorporation with minimal medium containing 99% (H2O)-H-2 and sodium d( 3)-acetate (99 atom% H-2). The yield of deuterium-enriched wild-type enzyme was 80 mg/L with yields for mutants proportionally reduced, The high level of protein deuteration achieved with this system allowed detection of the hyperfine coupling between the paramagnetic nitroxyl group of a spin-labele d penicillin substrate and hydrogens on the penicillin moiety in a cryokine tically isolated acylenzyme reaction intermediate because of the decrease i n overlapping resonances of active site residues. The overexpression system is readily adaptable for other target proteins and facilitates studies req uiring large quantities of protein in isotopically enriched forms. (C) 2000 Academic Press.