Spinach holo-acyl carrier protein: Overproduction and phosphopantetheinylation in Escherichia coli BL21(DE3), in vitro acylation, and enzymatic desaturation of histidine-tagged isoform I

Spinach ACP isoform I was overexpressed in Escherichia coli BL21(DE3) using a gene synthesized from codons associated with high-level expression in E. coli The synthetic gene has extensive changes in codon usage (23 of 77 tot al codons) relative to that of the originally synthesized plant gene (P. D. Beremand et at, 1987, Arch. Biochem Biophys. 256, 90-100). After expressio n of the new synthetic gene, purified ACP and ACP-His(6) were obtained in y ields of up to 70 mg L-1 of culture medium, compared to similar to 1-6 mg L -1 of purified ACP obtained from the gene composed of predicted spinach cod ons. In either shaken flask or fermentation culture, similar to 15% convers ion to holo-ACP or holo-ACP-Ris, was obtained regardless of the level of pr otein expression. However, coexpression of ACP-His(6) with E. coli holo-ACP synthase in E. coli BL21(DE3) during pH- and dissolved O-2-controlled ferm entation routinely yielded greater than 95% conversion to holo-ACP-Hiss. El ectrospray ionization mass spectrometric analysis of the purified recombina nt ACPs revealed that the amino terminal. Met was efficiently removed, but only if the bacterial cell lysates were prepared in the absence of EDTA. Th is observation is consistent with the inhibition of endogenous Met-aminopep tidase by removal of catalytically essential Co(II) and introduces the impo rtance of considering the catalytic properties of host enzymes providing ad hoc posttranslational modification of recombinant proteins. Stearoyl-ACP-H is(6) was shown to be indistinguishable from stearoyl-ACP as a substrate fo r enzymatic acylation and desaturation, In combination, these studies provi de a coordinated scheme to produce and characterize quantities of acyl-ACPs sufficient to support expanded biophysical and structural studies. (C) 199 9 Academic Press.