Expression and Purification of lacZ and trpE Fusion Proteins

Timothy Hoey1

1 University of California, Berkeley, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.5
DOI:  10.1002/0471142727.mb1605s28
Online Posting Date:  May, 2001
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Abstract

Fusion proteins are commonly used as a source of antigen for producing antibodies and in many cases can be useful for biochemical analyses. This unit describes two widely used expression systems for producing large amounts of proteins in E. coli. One system expresses lacZ fusions using the pUR series of vectors and the other expresses trpE fusions using the pATH vectors. The gene of interest is first subcloned into either a pUR or pATH vector in the correct reading frame. The correct transformant is selected, grown, and then induced with either IPTG or IAA. Sonication of cells in the presence of protease inhibitors is used to prepare extracts containing both types of fusion proteins, as well as other types of proteins overexpressed in E. coli. The extracts are checked for the presence of fusion protein on an SDS‐polyacrylamide gel.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1:

  Materials
  • pUR (unit 1.5) or pATH (GenBank file name M32985) vectors
  • E. coli C600, HB101, RR1 or equivalent (Table 97.80.4711)
  • LB plates and medium containing 50 µg/ml ampicillin (unit 1.1)
  • 100 mM IPTG (store at −20°C)
  • M9 plates and medium containing 50 µg/ml ampicillin, 0.5% Casamino acids, 10 µg/ml thiamine, and with/without 20 µg/ml tryptophan (supplemented M9; unit 1.1)
  • 2.5 mg/ml indoleacrylic acid (IAA) in 95% ethanol (store at −20°C)
  • Phosphate‐buffered saline (PBS; appendix 22), ice‐cold
  • recipeHEMGN buffer, ice‐cold
  • 50 mg/ml lysozyme in 0.25 M Tris⋅Cl, pH 8.0 (store at −20°C)
  • recipeHEMGN buffer/8 M guanidine⋅HCl (prepare 100 ml and store at 4°C)
  • recipeHEMGN buffer/1 M guanidine⋅HCl (prepare 500 ml and store at 4°C)
  • Sorvall RC‐5B centrifuge with GSA rotor (or equivalent) and 200‐ml bottles
  • Sorvall Omnispin clinical centrifuge (or equivalent) and 15‐ml conical tubes
  • Sonicator with a microtip
  • Sorvall SS‐34 rotor (or equivalent) and 50‐ml tubes
  • Ultracentrifuge with Beckman 60Ti rotor (or equivalent) and tubes
  • Dialysis tubing, MWCO 12,000 to 14,000 ( appendix 3C3)
  • Additional reagents and equipment for subcloning of DNA fragments (units 1.4 & 3.16), transformation of competent E. coli cells (unit 1.8), quantitation of proteins by the Bradford method (unit 10.110.1), and SDS‐PAGE (unit 10.210.2)
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Figures

Videos

Literature Cited

   Bolivar, F. Rodriguez, R.L., Greene, T.J., Betlach, M.C., Heyneker, H.L., Boyer, H.W. 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95‐113.
   Buell, G., Schulz, M.F., Selzer, G., Chollet, A., Movva, N.R., Semon, D., Escanez, S., and Kawishima, E. 1985. Optimizing the expression in E. coli of a synthetic gene encoding somatomedin‐C (IGF‐I). Nucl. Acids Res. 13:1923‐1938.
   Desplan, C., Theis, J., and O'Farrell, P.H. 1985. The Drosophila developmental control gene, engrailed, encodes a sequence‐specific DNA binding activity. Nature (London) 318:2513‐2522.
   Gross, C., Engbaek, F., Flammang, T., and Burgess, R. 1976. Rapid micromethod for the purification of Escherichia coli ribonucleic acid polymerase and the preparation of bacterial extracts active in ribonucleic acid synthesis. J. Bacteriol. 128:382‐389.
   Joachimiak, A., Schevitz, R.W., Kelley, R.L., Yanofsky, C., and Sigler, P.B. 1983. Functional inferences from crystals of Escherichia coli trp repressor. J. Biol. Chem. 258:12641‐12643.
   Johnson, A.D. and Herskowitz, I. 1985. A repressor (MATα2 product) and its operator control expression of a set of cell type specific genes in yeast. Cell 42:237‐247.
   Kadonaga, J.T., Carner, K.R., Masiarz, F.R., and Tjian, R. 1987. Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell 51:1079‐1090.
   Koerner, T.J., Hill, J.E., Myers, A.M., and Tzagoloff, A. 1990. High‐expression vectors with multiple cloning sites for construction of trpE‐fusion genes: pATH vectors. Methods Enzymol. In press.
   Morse, D.E., Mosteller, R.D., Baker, R.F., and Yanofsky, C. 1969. Direction of in vivo degradation of tryptophan messenger RNA—a correction. Nature (London) 223:40‐43.
   Rio, D.C., Laski, F.A., and Rubin, G.M. 1986. Identification and immunological analysis of biologically active Drosophila P element transposase. Cell 44:21‐32.
   Rüther, U. and Müller‐Hill, B. 1983. Easy identification of cDNA clones. EMBO J. 2:1791‐1794.
   Sadowski, I., Stone, J.C., and Pawson, T. 1986. A noncatalytic domain conserved among cytoplasmic protein‐tyrosine kinases modifies the kinase function and transforming activity of Fujinami Sarcoma Virus P130gag‐fps. Mol. Cell. Biol. 6:4396‐4408.
   Spindler, K.R., Rossner, D.S.E., and Berk, A.J. 1984. Analysis of Adenovirus transforming proteins from early regions 1A and 1B with antisera to inducible fusion antigens produced in Escherichia coli. J. Virol. 49:132‐141.
   Tanese, N., Roth, M,, and Goff, S.G. 1985. Expression of enzymatically active reverse transcriptase in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 82:4944‐4948.
Key Reference
   Koerner et al., 1990. See above.
  Contains a complete description of the different trpE expression vectors as well as a thorough discussion of potential problems involved in maximizing expression.
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