Improved method for assembly of hemeprotein neuronal NO-synthase heterodimers

Abstract

The assembly of mutated and wild type monomers into functional heterodimeric hemeproteins has provided important mechanistic insights. As in the case of NO synthase (NOS), the existing methods to make such heterodimeric NOSs are inefficient and labor intensive with typical yields of about 5%. We have found that expression of neuronal NOS heterodimers in insect cells, where we take advantage of an exogenous heme-triggered chaperone-assisted assembly process, provides an approximately 43% yield in heterodimeric NOS. In contrast, in E. coli little heterodimerizaton occurred. Thus, insect cells are preferred and may represent a valuable method for assembly of other dimeric hemeproteins.

Dynamic cycling with a unique Hsp90/Hsp70-dependent chaperone machinery and GAPDH is needed for heme insertion and activation of neuronal NO synthase

“…Sf9 cells were grown in Sf-900 II SFM serum-free, protein-free insect cell culture medium (Thermo Fisher Scientific) supplemented with 1% (v/v) bovine calf serum in suspension cultures maintained at 27 °C with continuous shaking (160 rpm). The Sf9 cells in growing phase were infected for 72 h with 0.2% (v/v) of baculovirus containing complementary DNA encoding rat nNOS that is tagged at C terminus with FLAG peptide as previously described ( 48 ), being referred to as nNOS-FLAG. For production of holo-nNOS-FLAG, hemin (7 μM) and N -acetyl- l -cysteine (2 mM) were added 6 h prior to harvesting as an albumin conjugate, prepared as described previously ( 7 ).…”

Hsp70:CHIP Ubiquitinates Dysfunctional but Not Native Neuronal NO Synthase

“…Hsp90 was purified from rabbit reticulocyte lysate by sequential chromatography on DE52, hydroxylapatite, and ATP-agarose as previously described (Dittmar et al, 1996). Rat untagged and c-terminal FLAG-tagged apo-nNOS (Morishima et al, 2016) was expressed in Sf9 insect cells using a recombinant baculovirus and purified by 29,59-ADP-sepharose and gel-filtration chromatography as described previously (Bender et al, 1999). The same method was used for the generation of FLAG-tagged holo-nNOS, except that exogenous heme was added to Sf9 cells (Bender et al, 1999).…”

“…The FLAG-tagged and untagged nNOS was ubiquitinated to the same degree by a purified protein system, as determined by Western blot analysis (data not shown). The specific activity of the FLAG-tagged and untagged nNOS were equivalent, as determined by an oxyhemoglobin assay for the measurement of nitric oxide (Morishima et al, 2016).…”