eNOS was expressed in E. coli as a soluble, catalytically active enzyme using the pCW expression vector coexpressed with a plasmid, pGroESL, encoding the chaperonins groEL and groES. The E. coli BL21 cultures synthesized 6-8 mg of recombinant enzyme per liter of culture. The eNOS protein was purified using 2'5'-ADP Sepharose exhibiting a single band of apparent molecular mass 135 kDa on SDS/PAGE. The resting enzyme is predominantly high spin with Amax, at 406 nm. The dithionite-reduced, CObound form shows Amax, at 444 nm. The spectral properties of eNOS from E. coli are identical to those obtained from stably transfccted HEK 293 cells or those from baculovirus expression systems. Enzymatic activity of eNOS from E. coli ranges between 65-135 nmol NO or L-citrulline formed/min/mg at 25°C. The enzyme is replete with heme and flavins and, after incubation with tetrahydrobiopterin, contains -0.6 pmol of pterin/pmol of eNOS. The heterologous expression of eNOS offers many advantages over tissue sources of the protein. Strategies employed to investigate NOS structure-function relationships demand larger-scale preparations of the protein and expression of functionally active eNOS in E. coli provides this capability. (Supported by Welch Grant AQ-1192 to BSSM).
|Original language||English (US)|
|State||Published - Dec 1 1996|
ASJC Scopus subject areas
- Molecular Biology