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. 1993 Sep 1;294(Pt 2):521–527. doi: 10.1042/bj2940521

Purification and separation of holo- and apo-forms of Saccharopolyspora erythraea acyl-carrier protein released from recombinant Escherichia coli by freezing and thawing.

S A Morris 1, W P Revill 1, J Staunton 1, P F Leadlay 1
PMCID: PMC1134486  PMID: 8373367

Abstract

Saccharopolyspora erythraea acyl-carrier protein, highly expressed from a T7-based expression plasmid in Escherichia coli, can be selectively released from the cells in near-quantitative yield by a single cycle of freezing and thawing in a neutral buffer. Electrospray mass spectrometry was used to confirm that the recombinant S. erythraea acyl-carrier protein over-expressed in E. coli is present predominantly as the holo-form, with variable amounts of apo-acyl-carrier protein, holo-acyl-carrier protein dimer and holo-acyl-carrier protein glutathione adduct. The holo- and apo-acyl-carrier proteins are both readily purified on a large scale from the freeze-thaw extracts and can be separated from one another by octyl-Sepharose chromatography. The holo-acyl-carrier protein obtained in this way was fully active in supporting the synthesis of acyl-acyl-carrier protein by extracts of S. erythraea.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bayan N., Therisod H. Membrane-binding sites for acyl carrier protein in Escherichia coli. FEBS Lett. 1989 Aug 14;253(1-2):221–225. doi: 10.1016/0014-5793(89)80963-9. [DOI] [PubMed] [Google Scholar]
  2. Bevitt D. J., Cortes J., Haydock S. F., Leadlay P. F. 6-Deoxyerythronolide-B synthase 2 from Saccharopolyspora erythraea. Cloning of the structural gene, sequence analysis and inferred domain structure of the multifunctional enzyme. Eur J Biochem. 1992 Feb 15;204(1):39–49. doi: 10.1111/j.1432-1033.1992.tb16603.x. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Brody S., Mikolajczyk S. Neurospora mitochondria contain an acyl-carrier protein. Eur J Biochem. 1988 Apr 15;173(2):353–359. doi: 10.1111/j.1432-1033.1988.tb14005.x. [DOI] [PubMed] [Google Scholar]
  5. Brozek K. A., Raetz C. R. Biosynthesis of lipid A in Escherichia coli. Acyl carrier protein-dependent incorporation of laurate and myristate. J Biol Chem. 1990 Sep 15;265(26):15410–15417. [PubMed] [Google Scholar]
  6. Butt A. D., Ohlrogge J. B. Acyl carrier protein is conjugated to glutathione in spinach seed. Plant Physiol. 1991 Jul;96(3):937–942. doi: 10.1104/pp.96.3.937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Caffrey P., Green B., Packman L. C., Rawlings B. J., Staunton J., Leadlay P. F. An acyl-carrier-protein-thioesterase domain from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea. High-level production, purification and characterisation in Escherichia coli. Eur J Biochem. 1991 Feb 14;195(3):823–830. doi: 10.1111/j.1432-1033.1991.tb15771.x. [DOI] [PubMed] [Google Scholar]
  8. Cortes J., Haydock S. F., Roberts G. A., Bevitt D. J., Leadlay P. F. An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea. Nature. 1990 Nov 8;348(6297):176–178. doi: 10.1038/348176a0. [DOI] [PubMed] [Google Scholar]
  9. Cronan J. E., Jr Beta-alanine synthesis in Escherichia coli. J Bacteriol. 1980 Mar;141(3):1291–1297. doi: 10.1128/jb.141.3.1291-1297.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Donadio S., Katz L. Organization of the enzymatic domains in the multifunctional polyketide synthase involved in erythromycin formation in Saccharopolyspora erythraea. Gene. 1992 Feb 1;111(1):51–60. doi: 10.1016/0378-1119(92)90602-l. [DOI] [PubMed] [Google Scholar]
  11. Donadio S., Staver M. J., McAlpine J. B., Swanson S. J., Katz L. Modular organization of genes required for complex polyketide biosynthesis. Science. 1991 May 3;252(5006):675–679. doi: 10.1126/science.2024119. [DOI] [PubMed] [Google Scholar]
  12. Dunn J. J., Studier F. W. Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J Mol Biol. 1983 Jun 5;166(4):477–535. doi: 10.1016/s0022-2836(83)80282-4. [DOI] [PubMed] [Google Scholar]
  13. Elovson J., Vagelos P. R. Acyl carrier protein. X. Acyl carrier protein synthetase. J Biol Chem. 1968 Jul 10;243(13):3603–3611. [PubMed] [Google Scholar]
  14. Guerra D. J., Dziewanowska K., Ohlrogge J. B., Beremand P. D. Purification and characterization of recombinant spinach acyl carrier protein I expressed in Escherichia coli. J Biol Chem. 1988 Mar 25;263(9):4386–4391. [PubMed] [Google Scholar]
  15. Hale R. S., Jordan K. N., Leadlay P. F. A small, discrete acyl carrier protein is involved in de novo fatty acid biosynthesis in Streptomyces erythraeus. FEBS Lett. 1987 Nov 16;224(1):133–136. doi: 10.1016/0014-5793(87)80436-2. [DOI] [PubMed] [Google Scholar]
  16. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  17. Holak T. A., Frederick A. F., Prestegard J. H. Purification and NMR characterization of acyl carrier protein. J Biol Chem. 1987 Mar 15;262(8):3685–3689. [PubMed] [Google Scholar]
  18. Hopwood D. A., Sherman D. H. Molecular genetics of polyketides and its comparison to fatty acid biosynthesis. Annu Rev Genet. 1990;24:37–66. doi: 10.1146/annurev.ge.24.120190.000345. [DOI] [PubMed] [Google Scholar]
  19. Issartel J. P., Koronakis V., Hughes C. Activation of Escherichia coli prohaemolysin to the mature toxin by acyl carrier protein-dependent fatty acylation. Nature. 1991 Jun 27;351(6329):759–761. doi: 10.1038/351759a0. [DOI] [PubMed] [Google Scholar]
  20. Jackowski S., Rock C. O. Ratio of active to inactive forms of acyl carrier protein in Escherichia coli. J Biol Chem. 1983 Dec 25;258(24):15186–15191. [PubMed] [Google Scholar]
  21. Jackowski S., Rock C. O. Regulation of coenzyme A biosynthesis. J Bacteriol. 1981 Dec;148(3):926–932. doi: 10.1128/jb.148.3.926-932.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jacobson G. R., Rosenbusch J. P. Abundance and membrane association of elongation factor Tu in E. coli. Nature. 1976 May 6;261(5555):23–26. doi: 10.1038/261023a0. [DOI] [PubMed] [Google Scholar]
  23. Jacobson G. R., Takacs B. J., Rosenbusch J. P. Properties of a major protein released from Escherichia coli by osmotic shock. Biochemistry. 1976 Jun 1;15(11):2297–2303. doi: 10.1021/bi00656a008. [DOI] [PubMed] [Google Scholar]
  24. Joseph-Liauzun E., Leplatois P., Legoux R., Guerveno V., Marchese E., Ferrara P. Human recombinant interleukin-1 beta isolated from Escherichia coli by simple osmotic shock. Gene. 1990 Feb 14;86(2):291–295. doi: 10.1016/0378-1119(90)90293-z. [DOI] [PubMed] [Google Scholar]
  25. Kim Y., Prestegard J. H. Refinement of the NMR structures for acyl carrier protein with scalar coupling data. Proteins. 1990;8(4):377–385. doi: 10.1002/prot.340080411. [DOI] [PubMed] [Google Scholar]
  26. LaVallie E. R., DiBlasio E. A., Kovacic S., Grant K. L., Schendel P. F., McCoy J. M. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology (N Y) 1993 Feb;11(2):187–193. doi: 10.1038/nbt0293-187. [DOI] [PubMed] [Google Scholar]
  27. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  28. Loo J. A., Udseth H. R., Smith R. D. Peptide and protein analysis by electrospray ionization-mass spectrometry and capillary electrophoresis-mass spectrometry. Anal Biochem. 1989 Jun;179(2):404–412. doi: 10.1016/0003-2697(89)90153-x. [DOI] [PubMed] [Google Scholar]
  29. Lunn C. A., Pigiet V. P. Localization of thioredoxin from Escherichia coli in an osmotically sensitive compartment. J Biol Chem. 1982 Oct 10;257(19):11424–11430. [PubMed] [Google Scholar]
  30. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  31. Nossal N. G., Heppel L. A. The release of enzymes by osmotic shock from Escherichia coli in exponential phase. J Biol Chem. 1966 Jul 10;241(13):3055–3062. [PubMed] [Google Scholar]
  32. Revill W. P., Leadlay P. F. Cloning, characterization, and high-level expression in Escherichia coli of the Saccharopolyspora erythraea gene encoding an acyl carrier protein potentially involved in fatty acid biosynthesis. J Bacteriol. 1991 Jul;173(14):4379–4385. doi: 10.1128/jb.173.14.4379-4385.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Roberts G. A., Staunton J., Leadlay P. F. Heterologous expression in Escherichia coli of an intact multienzyme component of the erythromycin-producing polyketide synthase. Eur J Biochem. 1993 May 15;214(1):305–311. doi: 10.1111/j.1432-1033.1993.tb17925.x. [DOI] [PubMed] [Google Scholar]
  34. Rock C. O., Cronan J. E., Jr Acyl carrier protein from Escherichia coli. Methods Enzymol. 1981;71(Pt 100):341–351. doi: 10.1016/0076-6879(81)71043-7. [DOI] [PubMed] [Google Scholar]
  35. Rock C. O., Cronan J. E., Jr Re-evaluation of the solution structure of acyl carrier protein. J Biol Chem. 1979 Oct 10;254(19):9778–9785. [PubMed] [Google Scholar]
  36. Rock C. O., Garwin J. L. Preparative enzymatic synthesis and hydrophobic chromatography of acyl-acyl carrier protein. J Biol Chem. 1979 Aug 10;254(15):7123–7128. [PubMed] [Google Scholar]
  37. Rock C. O., Jackowski S. Regulation of phospholipid synthesis in Escherichia coli. Composition of the acyl-acyl carrier protein pool in vivo. J Biol Chem. 1982 Sep 25;257(18):10759–10765. [PubMed] [Google Scholar]
  38. Runswick M. J., Fearnley I. M., Skehel J. M., Walker J. E. Presence of an acyl carrier protein in NADH:ubiquinone oxidoreductase from bovine heart mitochondria. FEBS Lett. 1991 Jul 29;286(1-2):121–124. doi: 10.1016/0014-5793(91)80955-3. [DOI] [PubMed] [Google Scholar]
  39. Shen B., Summers R. G., Gramajo H., Bibb M. J., Hutchinson C. R. Purification and characterization of the acyl carrier protein of the Streptomyces glaucescens tetracenomycin C polyketide synthase. J Bacteriol. 1992 Jun;174(11):3818–3821. doi: 10.1128/jb.174.11.3818-3821.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Swan D. G., Cortes J., Hale R. S., Leadlay P. F. Cloning, characterization, and heterologous expression of the Saccharopolyspora erythraea (Streptomyces erythraeus) gene encoding an EF-hand calcium-binding protein. J Bacteriol. 1989 Oct;171(10):5614–5619. doi: 10.1128/jb.171.10.5614-5619.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tabor S., Huber H. E., Richardson C. C. Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7. J Biol Chem. 1987 Nov 25;262(33):16212–16223. [PubMed] [Google Scholar]
  42. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Therisod H., Kennedy E. P. The function of acyl carrier protein in the synthesis of membrane-derived oligosaccharides does not require its phosphopantetheine prosthetic group. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8235–8238. doi: 10.1073/pnas.84.23.8235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Van Den Bosch H., Williamson J. R., Vagelos P. R. Localization of acyl carrier protein in Escherichia coli. Nature. 1970 Oct 24;228(5269):338–341. doi: 10.1038/228338a0. [DOI] [PubMed] [Google Scholar]
  45. Vanden Boom T., Cronan J. E., Jr Genetics and regulation of bacterial lipid metabolism. Annu Rev Microbiol. 1989;43:317–343. doi: 10.1146/annurev.mi.43.100189.001533. [DOI] [PubMed] [Google Scholar]
  46. Zensen R., Husmann H., Schneider R., Peine T., Weiss H. De novo synthesis and desaturation of fatty acids at the mitochondrial acyl-carrier protein, a subunit of NADH:ubiquinone oxidoreductase in Neurospora crassa. FEBS Lett. 1992 Sep 28;310(2):179–181. doi: 10.1016/0014-5793(92)81324-f. [DOI] [PubMed] [Google Scholar]

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