Abstract
A synthetic gene encoding spinach acyl carrier protein I (ACP-I) was fused to a gene encoding the Fc-binding portion of staphylococcal protein A. This gene fusion, under the control of the λPR promoter, was expressed at high levels in Escherichia coli producing a 42 kDa fusion protein. This fusion protein was phosphopantethenylated in E. coli. In vitro the ACP portion of the fusion protein was able to participate in acyl ACP synthetase reactions, plant malonyl-CoA:ACP transacylase (MCT) reactions, and plant fatty acid synthetase (FAS) reactions. Inhibitory effects of high ACP concentrations on in vitro plant FAS were observed with the unfused ACP-1 but not with the fusion protein. As with unfused ACP-I, the fusion protein was a poor substrate for E. coli FAS reactions. When injected into rabbits, the fusion protein was also able to generate antiserum to spinach ACP-I.
Similar content being viewed by others
References
Amann E, Bröker M, Warm F: Expression of Herpes simplex virus type 1 glycoprotein C antigens in Escherichia coli. Gene 32: 203–215 (1984).
Beremand PD, Guerra DJ, Hannapel DJ, Kuhn DN, Ohlrogge JB: Expression of a synthetic spinach acyl carrier protein-I gene in Escherichia coli. J Cellular Biochem 11B: 48 (1987).
Beremand PD, Hannapel DJ, Guerra DJ, Kuhn DN, Ohlrogge JB: Synthesis, cloning, and expression in Escherichia coli of a spinach acyl carrier protein-I gene. Arch Biochem Biophys 256: 90–100 (1987).
Davis AR, Nayak DP, Ueda M, Hiti AL, Dowbenko D, Kleid DG: Expression of antigenic determinants of the hemagglutinin gene of a human influenza virus in Escherichia coli. Proc Natl Acad Sci USA 78: 5376–5380 (1981).
Geronimo J, Gray JG, Charbonneau H, Vanaman T, Bastia D: Use of gene fusions and protein-protein interaction in the isolation of a biologically active regulatory protein: The replication initiator protein of plasmid RGK. Proc Natl Acad Sci USA 80: 6848–6852 (1983).
Goeddel DV, Kleid DG, Bolivar F, Heyneker HL, Yansura DG, Crea R, Hirose T, Kraszewski A, Itakura K, Riggs AD: Expression in Escherichia coli of chemically-synthesized genes for human insulin. Proc Natl Acad Sci USA 76: 106–110 (1979).
Gottesman ME, Adhya S, Das A: Transcription antitermination by bacteriophage lambda N gene product. J Mol Biol 140: 57–75 (1980).
Guerra DJ, Dziewanowska K, Ohlrogge JB, Beremand PD: Purification and characterization of recombinant spinach acyl carrier protein I expressed in Escherichia coli. J Biol Chem 263: 4386–4391 (1988).
Guerra DJ, Ohlrogge JB: Partial purification and characterization of two forms of malonyl coenzyme A: Acyl carrier protein transacylase from soybean leaf tissue. Arch Biochem Biophys 246: 274–285 (1986).
Hanahan D: Techniques for transformation of E. coli. In: Glover D (ed) DNA Cloning, Vol. 1, p. 109–136. IRL Press, Washington DC (1985).
Harker CL, Mallineaux PM, Bryant JA, Maule AJ: Detection of CaMV gene I and gene VI protein products in vivo using antisera raised to COOH-terminal β-galactosidase fusion proteins. Plant Mol Biol 8: 275–287 (1987).
Hoj PB, Svendsen I: Barley chloroplasts contain two acyl carrier proteins coded for by different genes. Carlsberg Res Commun 49: 483–492 (1984).
Itakura K, Hirose T, Crea R, Riggs AD, Heyneker HL, Bolivar F, Boyer HW: Expression in Escherichia coli of a chemically-synthesized gene for the hormone somatostatin. Science 198: 1056–1063 (1977).
Kleid DG, Yansura D, Small B, Dowbenko D, Moore DM, Grubman MJ, McKercher PD, Morgan D, Robertson BH, Bachrach HL: Cloned viral protein vaccine for foot-and-mouth disease: Responses in cattle and swine. Science 214: 1125–1129 (1981).
Kuo TM, Ohlrogge JB: Acylation of plant acyl carrier proteins by acyl-acyl carrier protein synthetase from Escherichia coli. Arch Biochem Biophys 230: 110–116 (1984).
Kuo TM, Ohlrogge JB: A novel, general radioimmunoassay for acyl carrier proteins. Anal Biochem 136: 479–502 (1984).
Langone JJ: Protein A of Staphylococcus aureus and related immunoglobulin receptors produced by Streptococci and Pneumococci. In: Dixon JF, Kunhel H (eds), Advances in Immunology, Vol 32, pp. 157–252. Academic Press, New York (1982).
Nilsson B, Abrahmsen L, Uhlen M: Immobilization and purification of enzymes with staphylococcal Protein A gene fusion vectors. EMBO J 4: 1075–1080 (1985).
Ohlrogge JB: Biochemistry of plant acyl carrier proteins In: Stumpf PK (ed), The Biochemistry of Plants, Vol 9, pp. 137–157. Academic Press, Orlando FL (1987).
Ohlrogge JB, Kuo TM: Plants have isoforms for acyl carrier protein that are expressed differently in different tissues. J Biol Chem 260: 8032–8037 (1985).
Reichlin M: Use of glutaraldehyde as a coupling agent for proteins and peptides. In: Vunakis HV, Langone JJ (eds), Methods in Ezymology, Vol 70, pp. 159–165. Academic Press, New York (1980).
Rock CO, Garwin JL: Preparative enzymatic synthesis and hydrophobic chromatography of acyl-acyl carrier protein. J Biol Chem 254: 7123–7128 (1979).
Rüther U, Koenen M, Sippel AE, Muller-Hill B: Exon cloning: Immunoenzymatic identification of exons of the chicken lysozyme gene. Proc Natl Acad Sci USA 79: 6852–6855 (1982).
Stumpf PK: Fatty acid biosynthesis in higher plants. In: Numa S (ed), Fatty Acid Metabolism and its Regulation, pp. 155–199. Elsevier, Amsterdam (1984).
Uhlen M, Nilsson B, Gass B, Lindberg M, Gatenbeck S, Philipson L: Gene fusion vectors based on the gene for staphylococcal protein A. Gene 23: 369–378 (1983).
Ullman A: One-step purification of hybrid proteins which have β-galactosidase activity. Gene 29: 27–31 (1984).
Zabeau M, Stanley KK: Enhanced expression of cro-β-galactosidase fusion proteins under the control of the PR promoter of bacteriophage λ. EMBO J 1: 1217–1224 (1982).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Beremand, P.D., Elmore, D.D., Dziewanowska, K. et al. Expression of an active spinach acyl carrier protein-I/protein-A gene fusion. Plant Mol Biol 12, 95–104 (1989). https://doi.org/10.1007/BF00017452
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00017452