title: Spinach holo-acyl carrier protein: overproduction and phosphopantetheinylation in Escherichia coli BL21(DE3), in vitro acylation, and enzymatic desaturation of histidine-tagged isoform I.
abstract: 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 al., 1987, Arch. Biochem. Biophys. 256, 90-100). After expression of the new synthetic gene, purified ACP and ACP-His6 were obtained in yields of up to 70 mg L-1 of culture medium, compared to approximately 1-6 mg L-1 of purified ACP obtained from the gene composed of predicted spinach codons. In either shaken flask or fermentation culture, approximately 15% conversion to holo-ACP or holo-ACP-His6 was obtained regardless of the level of protein expression. However, coexpression of ACP-His6 with E. coli holo-ACP synthase in E. coli BL21(DE3) during pH- and dissolved O2-controlled fermentation routinely yielded greater than 95% conversion to holo-ACP-His6. 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-His6 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.
authors: Graduate School and Department of Biochemistry, University of Wisconsin, Madison, Wisconsin, 53705, USA.
authors: J A
authors: JA
authors: Broadwater
authors: B G
authors: BG
authors: Fox
mesh_terms: D000213:Acyl Carrier Protein / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D000215:Acylation; D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli; D017343:Genes, Plant; D005813:Genes, Synthetic; D006639:Histidine; D008969:Molecular Sequence Data; D010204:Pantetheine / Q000031:analogs & derivatives* / Q000378:metabolism; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D018724:Spinacia oleracea / Q000235:genetics / Q000378:metabolism*
chemical_list: D000213:Acyl Carrier Protein; D020033:Protein Isoforms; D011994:Recombinant Proteins; D010204:Pantetheine; D006639:Histidine; C003129:4'-phosphopantetheine