Use of the Gateway System for Protein Expression in Multiple Hosts

James L. Hartley1

1 Science Applications International Corporation (SAIC)/National Cancer Institute, Frederick
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.17
DOI:  10.1002/0471140864.ps0517s30
Online Posting Date:  February, 2003
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The Gateway cloning method allows a gene to be cloned and subsequently transferred into any vector by in vitro site‐specific recombination. It does not necessarily follow, however, that a gene can be cloned once and expressed in all the available host‐vector combinations with uniformly satisfactory results. This is because different organisms have different mechanisms of translating mRNA into protein, and also because choices always have to be made when designing an expression construct, for example, the presence or absence of a stop codon. This unit reviews Gateway cloning, summarizes aspects of protein expression that limit the universality of the use of one clone in many vectors and hosts, and discusses how conflicts between the structure of a Gateway clone of a gene and the rules of protein expression can be minimized or resolved.The Gateway cloning method allows a gene to be cloned and subsequently transferred into any vector by in vitro site‐specific.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • A Brief Overview of Gateway
  • Protein Expression: A Gene in a Vector in a Host
  • Combining Gateway Cloning with Protein Expression
  • Research that Combines Gateway with Protein Expression
  • Summary of Gateway Strengths and Weaknesses
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Arking, D.E., Krebsova, A., Macek, M., Sr, Arking, A., Mian, I.S., Fried, L., Hamosh, A., Dey, S., McIntosh, I., and Dietz, H.C. 2002. Association of human aging with a functional variant of klotho. Proc. Natl. Acad. Sci. U.S.A. 99:856‐861.
   Billy, E., Brondani, V., Zhang, H., Muller, U., and Filipowicz, W. 2001. Specific interference with gene expression induced by long, double‐stranded RNA in mouse embryonal teratocarcinoma cell lines. Proc. Natl. Acad. Sci. U.S.A. 98:14428‐14433.
   Boulton, S.J., Gartner, A., Reboul, J., Vaglio, P., Dyson, N., Hill, D.E., and Vidal, M. 2002. Combined functional genomic maps of the C. elegans DNA damage response. Science 295:127‐ 131
   Braun, P., Hu, Y., Shen, B., Halleck, A., Koundinya, M., Harlow, E., and LaBaer, J. 2002. Proteome‐scale purification of human proteins from bacteria. Proc. Natl. Acad. Sci. U.S.A. 99:2654‐2659.
   Carrington, J.C., Cary, S.M., Parks, T.D., and Dougherty, W.G. 1989. A second proteinase encoded by a plant potyvirus genome. EMBO J. 8:365‐370.
   Chance, M.R., Bresnick, A.R., Burley, S.K., Jiang, J.S., Lima, C.D., Sali, A., Almo, S.C., Bonanno, J.B., Buglino, J.A., Boulton, S., Chen, H., Eswa, N., He, G., Huang, R., Ilyin, V., McMahan, L., Pieper, U., Ray, S., Vidal, M., and Wang, L.K. 2002. Structural genomics: A pipeline for providing structures for the biologist. Protein Sci. 11:723‐738.
   Chang, M.J., Kuzio, J., and Blissard, G.W. 1999. Modulation of translational efficiency by contextual nucleotides flanking abaculovirus initiator AUG codon. Virology 259:369‐383.
   Cheo, D., Brasch, M.A., Temple, G.F., Hartley, J.L., and Byrd, D. 2001. Use of multiple recombination sites with unique specificity in recombinational cloning. International Patent Application Number WO 01/42509 A1.
   Dan, I., Ong, S.E., Watanabe, N.M., Blagoev, B., Nielsen, M.M., Kajikawa, E., Kristiansen, T.Z., Mann, M., and Pandey, A. 2001. Cloning of MASK, a novel member of mammalian germinal center kinase‐III subfamily, with apoptosis‐inducing properties. J. Biol. Chem. 276:32115‐32121.
   Davy, A., Bello, P., Thierry‐Mieg, N., Vaglio, P., Hitti, J., Doucette‐Stamm, L., Thierry‐Mieg, D., Reboul, J., Boulton, S., Walhout, A.J., Coux, O., and Vidal, M. 2001. A protein‐protein interaction map of the Caenorhabditis elegans 26S proteasome. EMBO Rep. 2:821‐828.
   de Smit, M.H. and van Duin, J. 1990. Control of prokaryotic translational initiation by mRNA secondary structure. Prog. Nucleic Acid Res. Mol. Biol. 38:1‐35.
   Evdokimov, A.G., Tropea, J.E., Routzahn, K.M., Copeland, T.D., and Waugh, D.S. 2001. Structure of the N‐terminal domain of Yersinia pestis YopH at 2.0 A resolution. Acta Crystallogr. D Biol. Crystallogr. 57:793‐799.
   Finley, R.L., Zhang, H., Zhong, J., and Stanyon, C. A. 2002. Regulated expression of proteins in yeast using the MAL61–62 promoter and a mating scheme to increase dynamic range. Gene 285:49‐57.
   Funk, M., Niedenthal, R., Mumberg, D., Brunkmann, K., Ronicke, V., and Henkel, T. 2002. Vector systems for the heterologous expression of proteins in Saccharomyces cerevisiae. Methods Enzymol. 350:248‐257.
   Gavin, A.C., Bosche, M., Krause, R., Grandi, P., Marzioch, M., Bauer, A., Schultz, J., Rick, J.M., Michon, A.M., Cruciat, C.M., Remor, M., Hofert, C., Schelder, M., Brajenovic, M., Ruffner, H., Merino, A., Klein, K., Hudak, M., Dickson, D., Rudi, T., Gnau, V., Bauch, A., Bastuck, S., Huhse, B., Leutwein, C., Heurtier, M.A., Copley, R.R., Edelmann, A., Querfurth, E., Rybin, V., Drewes, G., Raida, M., Bouwmeester, T., Bork, P., Seraphin, B., Kuster, B., Neubauer, G., and Superti‐Furga, G. 2002. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415:141‐147.
   Gomes, M.D., Lecker, S.H., Jagoe, R.T., Navon, A., and Goldberg, A.L. 2001. Atrogin‐1, a muscle‐specific F‐box protein highly expressed during muscle atrophy. Proc. Natl. Acad. Sci. U.S.A. 98:14440‐14445.
   Hammarström, M., Hellgren, N., van den Berg, S., Berglund, H., and Härd, T. 2002. Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli. Prot. Sci. 11:313‐321.
   Hartley, J.L., Temple, G.F., and Brasch, M.A. 2000. DNA cloning using in vitro site‐specific recombination. Genome Res. 10:1788‐1795.
   Holmberg, S.K., Mikko, S., Boswell, T., Zoorob, R., and Larhammar, D. 2002. Pharmacological characterization of cloned chicken neuropeptide Y receptors Y1 and Y5. J. Neurochem. 81:462‐471.
   Ikeda, K. and Miyasaka, H. 1998. Compilation of mRNA sequences surrounding the AUG translation initiation codon in the green alga Chlamydomonas reinhardtii. Biosci. Biotechnol. Biochem. 62:2457‐2459.
   Iwai, T., Inaba, N., Naundorf, A., Zhang, Y., Gotoh, M., Iwasaki, H., Kudo, T., Togayachi, A., Ishizuka, Y., Nakanishi, H., and Narimatsu, H. 2002. Molecular cloning and characterization of a novel UDP‐GlcNAc:GalNAc‐peptide beta1,3‐N‐acetylglucosaminyltransferase (beta 3Gn‐T6), an enzyme synthesizing the core 3 structure of O‐glycans. J. Biol. Chem. 277:12802‐12809.
   Joshi, C.P., Zhou, H., Huang, X., and Chiang, V.L. 1997. Context sequences of translation initiation codon in plants. Plant Mol. Biol. 35:993‐1001.
   Kapust, R.B., Tozser, J., Copeland, T.D., and Waugh, D.S. 2002. The P1′ specificity of tobacco etch virus protease. Biochem. Biophys. Res. Commun. 294:949‐955.
   Karimi, M., Inze, D., and Depicker, A. 2002. GATEWAY vectors for Agrobacterium‐mediated plant transformation. Trends Plant Sci. 7:193‐195.
   Kozak, M. 1987. An analysis of 5′‐noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15:8125‐8148.
   Kozak, M. 1999. Initiation of translation in prokaryotes and eukaryotes. Gene 234:187‐208.
   Liu, Q., Li, M.Z., Leibham, D., Cortez, D., and Elledge, S.J. 1998. The univector plasmid‐fusion system, a method for rapid construction of recombinant DNA without restriction enzymes. Curr. Biol. 8:1300‐1309.
   Liu, J., Yao, F., Wu, R., Morgan, M., Thorburn, A., Finley, R.L. Jr., and Chen, Y.Q. 2002. Mediation of the DCC apoptotic signal by DIP13 alpha. J. Biol. Chem. 19:26281‐26285.
   Lao, G., Polayes, D., Xia, J.L., Bloom, F.R., Levine, F., and Mansbridge, J. 2001. Overexpression of trehalose synthase and accumulation of intracellular trehalose in 293H and 293FTetR:Hyg cells. Cryobiology 43:106‐113.
   Lucast, L.J., Batey, R.T., and Doudna, J.A. 2001. Large‐scale purification of a stable form of recombinant tobacco etch virus protease. Biotechniques 30:544‐550.
   Lukaszewicz, M., Feuermann, M., Jerouville, B., Stas, A., and Boutry, M. 2000. In vivo evaluation of the context sequence of the translation initiation codon in plants. Plant Sci. 154:89‐98.
   Mankad, R.V., Gimelbrant, A.A., and McClintock, T.S. 1998. Consensus translational initiation sites of marine invertebrate phyla. Biol Bull. 195:251‐254.
   McCarthy, J.E. 1998. Posttranscriptional control of gene expression in yeast. Microbiol Mol. Biol. Rev. 62:1492‐1553.
   Mottagui‐Tabar, S. 1998. Quantitative analysis of in vivo ribosomal events at UGA and UAG stop codons. Nucl. Acids Res. 26:2789‐2796.
   Olins, P.O. and Rangwala, S.H. 1989. A novel sequence element derived from bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coli. J. Biol. Chem. 264:16973‐16976.
   Parks, T.D., Leuther, K.K., Howard, E.D., Johnston, S.A., and Dougherty, W.G. 1994. Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase. Anal. Biochem. 216:413‐417.
   Pedelacq, J.D., Piltch, E., Liong, E.C., Berendzen, J., Kim, C.Y., Rho, B.S., Park, M.S., Terwilliger, T.C., and Waldo, G.S. 2002. Engineering soluble proteins for structural genomics. Nat. Biotechnol. 20:927‐932.
   Reboul, J., Vaglio, P., Tzellas, N., Thierry‐Mieg, N., Moore, T., Jackson, C., Shin‐i, T., Kohara, Y., Thierry‐Mieg, D., Thierry‐Mieg, J., Lee, H., Hitti, J., Doucette‐Stamm, L., Hartley, J.L., Temple, G.F., Brasch, M.A., Vandenhaute, J., Lamesch, P.E., Hill, D.E., and Vidal, M. 2001. Open‐reading‐frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans. Nat. Genet. 27:332‐336.
   Rosenberg, A.H., Lade, B.N., Chui, D.S., Lin, S.W., Dunn, J.J., and Studier, F.W. 1987. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene 56:125‐135.
   Shine, J. and Dalgarno, L. 1974. The 3′‐terminal sequence of Escherichia coli 16S ribosomal RNA: Complementarity to nonsense triplets and ribosome binding sites. Proc. Natl. Acad. Sci. U.S.A. 71:1342‐1346.
   Simpson, J.C., Wellenreuther, R., Poustka, A., Pepperkok, R., and Wiemann, S. 2000. Systematic subcellular localization of novel proteins identified by large‐scale cDNA sequencing. EMBO Rep. 1:287‐292.
   Walhout, A.J., Sordella, R., Lu, X., Hartley, J.L., Temple, G.F., Brasch, M.A., Thierry‐Mieg, N., and Vidal, M. 2000. Protein interaction mapping in C. elegans using proteins involved in vulval development. Science 287:116‐122.
   Wang, Y., Bruenn, J.A., Queener, S.F., and Cody, V. 2001. Isolation of rat dihydrofolate reductase gene and characterization of recombinant enzyme. Antimicrob. Agents Chemother. 45:2517‐2523.
   Weisberg, R.A. and Landy, A. 1983. Site‐specific recombination in phage lambda. In Lambda II (R.W. Hendrix, J.W. Roberts, F.W. Stahl, and R.A. Weisberg, eds.) pp. 211‐250. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
   Yun, D.F., Laz, T.M., Clements, J.M., and Sherman, F. 1996. mRNA sequences influencing translation and the selection of AUG initiator codons in the yeast Saccharomyces cerevisiae. Mol. Microbiol. 19:1225‐1239.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library