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 total codons) relative to that of the originally synthesized plant gene (P. D. Beremand et at, 1987, Arch. Biochem Biophys. 256, 90-100). After expression of the new synthetic gene, purified ACP and ACP-His(6) were obtained in yields 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 codons. In either shaken flask or fermentation culture, similar to 15% conversion to holo-ACP or holo-ACP-Ris, was obtained regardless of the level of protein 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 fermentation routinely yielded greater than 95% conversion to holo-ACP-Hiss. Electrospray ionization mass spectrometric analysis of the purified recombinant ACPs revealed that the amino terminal. Met was efficiently removed, but only if the bacterial cell lysates were prepared in the absence of EDTA. This observation is consistent with the inhibition of endogenous Met-aminopeptidase by removal of catalytically essential Co(II) and introduces the importance of considering the catalytic properties of host enzymes providing ad hoc posttranslational modification of recombinant proteins. Stearoyl-ACP-His(6) was shown to be indistinguishable from stearoyl-ACP as a substrate for enzymatic acylation and desaturation, In combination, these studies provide a coordinated scheme to produce and characterize quantities of acyl-ACPs sufficient to support expanded biophysical and structural studies. (C) 1999 Academic Press.