Mar 25, 2018 - expressed in E. coli contains pantetheine and approx- imately 50% is present in vivo as acyl-ACP. We have purified and characterized the ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 263, No. 9, Issue of March 25, pp. 4386-4391, 1988 Printed m U.S.A.
Purification and Characterizationof Recombinant SpinachAcyl Carrier Protein I Expressed inEscherichia coZi* (Received for publication, September 8,1987)
Daniel J. Guerra$Ji,Katarzyna Dziewanowskazy, John B. Ohlroggell, and Phillip D. Beremand** From the **United States Department of Agriculture/Agricultural Research Service, Northern Regional Research Center, Peoria, Illinois 61604, SBwtechnica Canada, Znc., Calgary,Alberta, Canada, and the IIDepartment of Botany and Plant Physiology, Michigan State University, East Lansing,Michigan 48824
Expression of plant acyl carrier protein (ACP) in Escherichia coli at levels above that of constitutive E. coli ACP does not appear to substantially alter bacterial growth or fatty acid metabolism. The plant ACP expressed in E. coli contains pantetheine and approximately 50% is present in vivo as acyl-ACP. We have purified and characterized the recombinant spinach ACP-I. NHz-terminalamino acid sequencing indicated identity to authentic spinach ACP-I, and there was no evidence for terminal methionine or formylmethionine. RecombinantACP-I was found to completely cross-react immunologically with polyclonal antibody raised to spinach ACP-I. Recombinant ACP-I was a poor substrate for E. coli fatty acid synthesis. In contrast, Brassica napus fatty acid synthetase gave similar reaction rates with both recombinant and E. coli ACP. Similarly, malonyl-coenzymeA:acyl carrier protein transacylase isolated from E. coli was only poorly able to utilize the recombinant ACP-I while the same enzyme from B. napus reacted equally well witheither E. coli ACP or recombinant ACP-I. E. coli acyl-ACP synthetase showed a higher reaction rate forrecombinant ACP-I than for E. coli ACP. Expression of spinach ACP-I in E. coli provides, for the first time, plant ACP in large quantities and should aid in both structural analysis of this protein and in investigations of the many ACP-dependentreactions of plant lipid metabolism. All de nouo plant fatty acid synthesis (FAS)’ and certain reactions of end product transfer and lipid synthesis require acyl carrier protein (ACP) (1). ACP provides these reactions with thioester free energy potential via a phosphopantetheine group derived from coenzyme A (2). Previously, the purification of ACP from spinach and barley leaf tissue resulted in the separation of two isoforms (3, 4). ACP-I is the major isoform in leaf tissue whereas ACP-I1 is expressed as a minor * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should be addressed: Dept. of Botany and Plant Pathology, Michigan State University, East Lansing, MI 48824. B Present address: Dept. of Biochemistry, University of Warsaw, Warsaw, Poland. ’ The abbreviations used are: FAS, fatty acid synthesis; ACP, acyl carrier protein; IPTG, isopropyl-0-D-thiogalactopyranoside; TCA, trichloroacetic acid; MCT, malonyl-CoAACP transacylase); GLC, gasliquid chromatography; HPLC, high pressure liquid chromat~graphy; DTT, dithiothreitol; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; FAME, fatty acid methyl esters; MES, 2(N-morpho1ino)ethanesulfonicacid; PVP, polyvinylpyrrolidone;RIA, radioimmunoassay.
component (3, 5). The relationship between ACP structure and expression may be important to the molecular basis of‘ FAS tissue specificity. In this regard, analyses in uitro have indicated that leaf ACP-I and ACP-I1 react differently in the reaction of oleoyl-ACP thioesterase and glycerol 3-phosphate acyl transferase (6). These enzymes provide a branch point for oleic acid utilization and may act tocontrol the proposition of acyl groups retained in or exported from chloroplasts. In the past, careful kinetic analyses using plant ACP have been limited due to the relative difficulty in obtaining plant ACP (6, 7). Recently, Beremand et al. (8)synthesized a spinach ACP-I gene and obtained expression in a bacterial system. We report here the purification and characterization of the synthetic ACP-I gene product. Acid precipitation, ion exchange chromatography, and gel permeation high pressure liquid chromatography (HPLC) were used to purify the recombinant ACP-I proteinto homogeneity. Eucaryotic proteins synthesized from plasmids in Escherichia coli often possess the amino-terminal methionine residue. This host modification i n vivo may cause alterations in the reactivity and immunogenicity of recombinant proteins (9). Evidence suggests that the nearest neighbor residue to the amino terminusplays a role in determining if the methionine is retained (10). To address this transgenic phenomenon and to verify that the purified ACP-I was from the pIasmid-born synthetic gene, the first 30 amino acid residues were sequenced. To determine the reactivity of synthetic ACP-I, a series of in uitro FAS reactions was conducted. Immunological crossreactivity of synthetic ACP-I in two independent analyses was also performed with the homogeneous protein. ACP possesses @-alanine inthe phosphopantetheine prosthetic group. An investigation of holo-ACP synthesis and acylation was conducted in the E. coli @-alanine auxotroph, SJ16. Cells were labeled with [3H]@-alanineand radioactive proteins were analyzed by electrophoresis ~ u o r o ~ a p h(11). y Finally, gas-liquid chromatography analyses were conducted on fatty acid methyl ester profiles of transformed E. coli JMlOl cells grown to midlog phase. These results and certain aspects of recombinant ACP-I structure andfunction relationships will be discussed. EXPERIMENTAL PROCEDURES’
RESULTS AND DISCUSSION
In Vitro Characterization of Recombinant SpinachACP-IAll characterizations in vitro were conducted with homogePortions of this paper (including “Experimental Procedures,” part of “Results,” Tables 1-111, and Figs. 1-8) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.
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S p i n a c h Acyl Carrier Protein I from E. coli neous preparations of recombinant ACP-I. The HPLC-purified recombinant ACP-I UV spectra was analyzed and compared with purified E. coli ACP (Fig. 2). Only the absorbance range between 245 and 300 nm isdisplayed because lower UV wavelength absorbances are essentially identical. This typeof analysis is similar to that described by Hoj and Svendsen (22). It is clear that recombinant ACP-I does not possess the same absorption maxima as E. coli ACP and that thespectral properties of the two ACP structures are quitedissimilar. The difference in aromatic amino acid residues is probably the basis for spectral divergence in the UV absorbance between E. coli ACP and recombinant ACP-I. A radioimmunoassay (17) was used to assess the structural relationship of recombinant ACP-I and authentic spinach ACP-I. Only minor differences in cross-reactivity to polyclonal antibody were evident in the comparisons shown in Fig. 3. Spinach ACP-I (isolated from spinach leaves) and recombinant ACP-I competed with approximately equal effectiveness for binding to polyclonal antibodies raised to spinach ACP-I. Further evidence for recombinant ACP-I reactivity to ACP-I specific antibody is shown by the decrease of acyl-ACP synthetase activity by adding antibody to the reaction mixtures. Less than 20% maximal activity was observed at the highest antibody concentration (Fig. 3, inset). Therefore, expression of ACP-I inE. coli did not significantly alter its immunological properties. Using a cell-free fraction from E. coli JMlOl cells, an in vitro fatty acid synthetase assay was performed. Table I1 shows that recombinant ACP-Iwas a relatively poor substrate for the E. coli FAS. With the ratio of ACP-I/[14CJmalonylCoA kept at a constant (
