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. 1991 Dec 11;19(23):6405–6412. doi: 10.1093/nar/19.23.6405

A highly efficient, cell-free translation/translocation system prepared from Xenopus eggs.

G Matthews 1, A Colman 1
PMCID: PMC329185  PMID: 1754376

Abstract

We describe the use of a Xenopus laevis egg extract for the in vitro translation and post translational modification of membrane and secretory proteins. This extract is capable of the translation and segregation into membranes of microgram per millilitre levels of protein from added mRNAs. Signal sequences of segregated proteins are efficiently cleaved and appropriate N-linked glycosylation patterns are produced. The extract also supports the quantitative assembly of murine immunoglobulin heavy and light chains into tetramers, and two events which take place beyond the endoplasmic reticulum, mannose 6 phosphorylation of murine cathepsin D and O-linked glycosylation of coronavirus E1 protein, also occur, but at reduced efficiency. The stability of the membranes allows protease protection studies and quantitative centrifugal fractionation of segregated and unsegregated proteins to be performed. Conditions for the use of stored extract have also been determined.

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Selected References

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

  1. Almouzni G., Mousseron-Grall S., Méchali M. Oligonucleotide site-directed mutagenesis in Xenopus egg extracts. Nucleic Acids Res. 1988 Sep 12;16(17):8525–8539. doi: 10.1093/nar/16.17.8525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Armstrong J., McCrae M., Colman A. Expression of coronavirus E1 and rotavirus VP10 membrane proteins from synthetic RNA. J Cell Biochem. 1987 Oct;35(2):129–136. doi: 10.1002/jcb.240350206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baranski T. J., Faust P. L., Kornfeld S. Generation of a lysosomal enzyme targeting signal in the secretory protein pepsinogen. Cell. 1990 Oct 19;63(2):281–291. doi: 10.1016/0092-8674(90)90161-7. [DOI] [PubMed] [Google Scholar]
  4. Bass B. L., Weintraub H. A developmentally regulated activity that unwinds RNA duplexes. Cell. 1987 Feb 27;48(4):607–613. doi: 10.1016/0092-8674(87)90239-x. [DOI] [PubMed] [Google Scholar]
  5. Blobel G., Dobberstein B. Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J Cell Biol. 1975 Dec;67(3):852–862. doi: 10.1083/jcb.67.3.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blow J. J., Laskey R. A. A role for the nuclear envelope in controlling DNA replication within the cell cycle. Nature. 1988 Apr 7;332(6164):546–548. doi: 10.1038/332546a0. [DOI] [PubMed] [Google Scholar]
  7. Blow J. J., Laskey R. A. Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. Cell. 1986 Nov 21;47(4):577–587. doi: 10.1016/0092-8674(86)90622-7. [DOI] [PubMed] [Google Scholar]
  8. Ceriotti A., Colman A. Binding to membrane proteins within the endoplasmic reticulum cannot explain the retention of the glucose-regulated protein GRP78 in Xenopus oocytes. EMBO J. 1988 Mar;7(3):633–638. doi: 10.1002/j.1460-2075.1988.tb02857.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ceriotti A., Colman A. Protein transport from endoplasmic reticulum to the Golgi complex can occur during meiotic metaphase in Xenopus oocytes. J Cell Biol. 1989 Oct;109(4 Pt 1):1439–1444. doi: 10.1083/jcb.109.4.1439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dale L., Matthews G., Tabe L., Colman A. Developmental expression of the protein product of Vg1, a localized maternal mRNA in the frog Xenopus laevis. EMBO J. 1989 Apr;8(4):1057–1065. doi: 10.1002/j.1460-2075.1989.tb03473.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Deshaies R. J., Koch B. D., Werner-Washburne M., Craig E. A., Schekman R. A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature. 1988 Apr 28;332(6167):800–805. doi: 10.1038/332800a0. [DOI] [PubMed] [Google Scholar]
  12. Devchand M., Gwynne D., Buxton F. P., Davies R. W. An efficient cell-free translation system from Aspergillus nidulans and in vitro translocation of prepro-alpha-factor across Aspergillus microsomes. Curr Genet. 1988 Dec;14(6):561–566. doi: 10.1007/BF00434081. [DOI] [PubMed] [Google Scholar]
  13. Dobberstein B., Garoff H., Warren G., Robinson P. J. Cell-free synthesis and membrane insertion of mouse H-2Dd histocompatibility antigen and beta 2-microglobulin. Cell. 1979 Aug;17(4):759–769. doi: 10.1016/0092-8674(79)90316-7. [DOI] [PubMed] [Google Scholar]
  14. Faust P. L., Wall D. A., Perara E., Lingappa V. R., Kornfeld S. Expression of human cathepsin D in Xenopus oocytes: phosphorylation and intracellular targeting. J Cell Biol. 1987 Nov;105(5):1937–1945. doi: 10.1083/jcb.105.5.1937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hansen W., Garcia P. D., Walter P. In vitro protein translocation across the yeast endoplasmic reticulum: ATP-dependent posttranslational translocation of the prepro-alpha-factor. Cell. 1986 May 9;45(3):397–406. doi: 10.1016/0092-8674(86)90325-9. [DOI] [PubMed] [Google Scholar]
  16. Hardy S., Aoufouchi S., Thiebaud P., Prigent C. DNA ligase I from Xenopus laevis eggs. Nucleic Acids Res. 1991 Feb 25;19(4):701–705. doi: 10.1093/nar/19.4.701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hutchison C. J., Cox R., Ford C. C. The control of DNA replication in a cell-free extract that recapitulates a basic cell cycle in vitro. Development. 1988 Jul;103(3):553–566. doi: 10.1242/dev.103.3.553. [DOI] [PubMed] [Google Scholar]
  18. Kornfeld R., Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. doi: 10.1146/annurev.bi.54.070185.003215. [DOI] [PubMed] [Google Scholar]
  19. Kornfeld S., Mellman I. The biogenesis of lysosomes. Annu Rev Cell Biol. 1989;5:483–525. doi: 10.1146/annurev.cb.05.110189.002411. [DOI] [PubMed] [Google Scholar]
  20. Laskey R. A., Mills A. D., Morris N. R. Assembly of SV40 chromatin in a cell-free system from Xenopus eggs. Cell. 1977 Feb;10(2):237–243. doi: 10.1016/0092-8674(77)90217-3. [DOI] [PubMed] [Google Scholar]
  21. Lau J. T., Welply J. K., Shenbagamurthi P., Naider F., Lennarz W. J. Substrate recognition by oligosaccharyl transferase. Inhibition of co-translational glycosylation by acceptor peptides. J Biol Chem. 1983 Dec 25;258(24):15255–15260. [PubMed] [Google Scholar]
  22. Leaf D. S., Roberts S. J., Gerhart J. C., Moore H. P. The secretory pathway is blocked between the trans-Golgi and the plasma membrane during meiotic maturation in Xenopus oocytes. Dev Biol. 1990 Sep;141(1):1–12. doi: 10.1016/0012-1606(90)90097-3. [DOI] [PubMed] [Google Scholar]
  23. Legerski R. J., Penkala J. E., Peterson C. A., Wright D. A. Repair of UV-induced lesions in Xenopus laevis oocytes. Mol Cell Biol. 1987 Dec;7(12):4317–4323. doi: 10.1128/mcb.7.12.4317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lohka M. J., Maller J. L. Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts. J Cell Biol. 1985 Aug;101(2):518–523. doi: 10.1083/jcb.101.2.518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lohka M. J., Masui Y. Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science. 1983 May 13;220(4598):719–721. doi: 10.1126/science.6601299. [DOI] [PubMed] [Google Scholar]
  26. Meyer D. I. Signal recognition particle (SRP) does not mediate a translational arrest of nascent secretory proteins in mammalian cell-free systems. EMBO J. 1985 Aug;4(8):2031–2033. doi: 10.1002/j.1460-2075.1985.tb03888.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Murray A. W., Kirschner M. W. Cyclin synthesis drives the early embryonic cell cycle. Nature. 1989 May 25;339(6222):275–280. doi: 10.1038/339275a0. [DOI] [PubMed] [Google Scholar]
  28. Patrick T. D., Lewer C. E., Pain V. M. Preparation and characterization of cell-free protein synthesis systems from oocytes and eggs of Xenopus laevis. Development. 1989 May;106(1):1–9. doi: 10.1242/dev.106.1.1. [DOI] [PubMed] [Google Scholar]
  29. Prehn S., Wiedmann M., Rapoport T. A., Zwieb C. Protein translocation across wheat germ microsomal membranes requires an SRP-like component. EMBO J. 1987 Jul;6(7):2093–2097. doi: 10.1002/j.1460-2075.1987.tb02475.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rose J. K., Doms R. W. Regulation of protein export from the endoplasmic reticulum. Annu Rev Cell Biol. 1988;4:257–288. doi: 10.1146/annurev.cb.04.110188.001353. [DOI] [PubMed] [Google Scholar]
  31. Rothblatt J. A., Meyer D. I. Secretion in yeast: reconstitution of the translocation and glycosylation of alpha-factor and invertase in a homologous cell-free system. Cell. 1986 Feb 28;44(4):619–628. doi: 10.1016/0092-8674(86)90271-0. [DOI] [PubMed] [Google Scholar]
  32. Ryabova L. A., Ortlepp S. A., Baranov V. I. Preparative synthesis of globin in a continuous cell-free translation system from rabbit reticulocytes. Nucleic Acids Res. 1989 Jun 12;17(11):4412–4412. doi: 10.1093/nar/17.11.4412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shields D., Blobel G. Efficient cleavage and segregation of nascent presecretory proteins in a reticulocyte lysate supplemented with microsomal membranes. J Biol Chem. 1978 Jun 10;253(11):3753–3756. [PubMed] [Google Scholar]
  34. Shuttleworth J., Godfrey R., Colman A. p40MO15, a cdc2-related protein kinase involved in negative regulation of meiotic maturation of Xenopus oocytes. EMBO J. 1990 Oct;9(10):3233–3240. doi: 10.1002/j.1460-2075.1990.tb07522.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Taylor M. A., Smith L. D. Quantitative changes in protein synthesis during oogenesis in Xenopus laevis. Dev Biol. 1985 Jul;110(1):230–237. doi: 10.1016/0012-1606(85)90079-x. [DOI] [PubMed] [Google Scholar]
  36. Tooze S. A., Tooze J., Warren G. Site of addition of N-acetyl-galactosamine to the E1 glycoprotein of mouse hepatitis virus-A59. J Cell Biol. 1988 May;106(5):1475–1487. doi: 10.1083/jcb.106.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Valle G., Besley J., Colman A. Synthesis and secretion of mouse immunoglobulin chains from Xenopus oocytes. Nature. 1981 May 28;291(5813):338–340. doi: 10.1038/291338a0. [DOI] [PubMed] [Google Scholar]
  38. Valle G., Jones E. A., Colman A. Anti-ovalbumin monoclonal antibodies interact with their antigen in internal membranes of Xenopus oocytes. Nature. 1982 Nov 4;300(5887):71–74. doi: 10.1038/300071a0. [DOI] [PubMed] [Google Scholar]
  39. Varki A., Kornfeld S. The spectrum of anionic oligosaccharides released by endo-beta-N-acetylglucosaminidase H from glycoproteins. Structural studies and interactions with the phosphomannosyl receptor. J Biol Chem. 1983 Mar 10;258(5):2808–2818. [PubMed] [Google Scholar]
  40. Walter P., Blobel G. Preparation of microsomal membranes for cotranslational protein translocation. Methods Enzymol. 1983;96:84–93. doi: 10.1016/s0076-6879(83)96010-x. [DOI] [PubMed] [Google Scholar]
  41. Walter P., Blobel G. Purification of a membrane-associated protein complex required for protein translocation across the endoplasmic reticulum. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7112–7116. doi: 10.1073/pnas.77.12.7112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Walter P., Ibrahimi I., Blobel G. Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein. J Cell Biol. 1981 Nov;91(2 Pt 1):545–550. doi: 10.1083/jcb.91.2.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Walter P., Lingappa V. R. Mechanism of protein translocation across the endoplasmic reticulum membrane. Annu Rev Cell Biol. 1986;2:499–516. doi: 10.1146/annurev.cb.02.110186.002435. [DOI] [PubMed] [Google Scholar]
  44. Wasserman W. J., Richter J. D., Smith L. D. Protein synthesis during maturation promoting factor- and progesterone-induced maturation in Xenopus oocytes. Dev Biol. 1982 Jan;89(1):152–158. doi: 10.1016/0012-1606(82)90303-7. [DOI] [PubMed] [Google Scholar]
  45. Wolin S. L., Walter P. Signal recognition particle mediates a transient elongation arrest of preprolactin in reticulocyte lysate. J Cell Biol. 1989 Dec;109(6 Pt 1):2617–2622. doi: 10.1083/jcb.109.6.2617. [DOI] [PMC free article] [PubMed] [Google Scholar]

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