Expression and Purification of Maltose‐Binding Protein Fusions

Paul Riggs1

1 New England Biolabs, Beverly, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.6
DOI:  10.1002/0471142727.mb1606s28
Online Posting Date:  May, 2001
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Abstract

This unit describes the procedure for subcloning the sequence encoding the protein of interest into an maltose‐binding protein (MBP) vector, and expressing and purifying the fusion protein from the cytoplasm. MBP vectors include a sequence that encodes the four‐amino‐acid recognition site for the specific protease factor Xa. The site is placed so it can be used to separate the protein of interest from MBP after affinity purification. A support protocol provides a pilot experiment for analyzing the solubility, affinity for the amylose resin, and export of a particular fusion protein. An alternate protocol gives instructions for purifying a fusion protein from the periplasm for fusions that are made in the signal sequence vector and are exported. Additional support protocols detail two different chromatographic methods for separating the protein of interest from MBP after factor Xa cleavage.

     
 
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Table of Contents

  • Basic Protocol 1: Construction, Expression, and Purification of MBP Fusion Proteins
  • Support Protocol 1: Pilot Experiment to Characterize the Behavior of an MBP Fusion Protein
  • Alternate Protocol 1: Purification of Fusion Proteins from the Periplasm
  • Support Protocol 2: Purifying the Cleaved Protein by Ion Exchange Chromatography
  • Support Protocol 3: Purifying the Cleaved Protein by Affinity Chromatography
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Construction, Expression, and Purification of MBP Fusion Proteins

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • pMAL‐c2 and/or pMAL‐p2 (New England Biolabs; see Fig. )
  • Escherichia coli TB1 (New England Biolabs) or any transformable strain of E. coli (see Table 97.80.4711)
  • LB plates and medium containing 0.2% glucose and 100 µg/ml ampicillin (unit 1.1)
  • LB plates containing 100 µg/ml ampicillin, 0.3 mM isopropyl‐1‐thio‐β‐D‐galactoside (IPTG), and 80 µg/ml Xgal (unit 1.1)
  • 1× and 2× SDS sample buffer (unit 10.210.2)
  • 0.1 M IPTG
  • recipeColumn buffer, without and with 10 mM maltose
  • Coomassie brilliant blue solution (unit 10.110.1)
  • Amylose resin (New England Biolabs)
  • Beckman JS‐4.2 and JA‐17 rotors (or equivalents)
  • Sonicator
  • 2.5 × 10–cm column
  • Centricon or Centriprep concentrator or stirred‐cell concentrator (Amicon), or equivalent
  • Boiling water bath
  • Additional reagents and equipment for replica plating (unit 1.3), subcloning of DNA fragments (units 1.4 & 3.16), preparing miniprep plasmid DNA (unit 1.6), restriction mapping (unit 3.2), quantitation of protein by the Bradford method (unit 10.110.1), and SDS‐PAGE (unit 10.210.2)
NOTE: A kit containing the pMAL vectors, TB1, amylose resin, factor Xa, anti‐MBP serum, MBP as a gel marker, and an MBP fusion as a factor Xa control is available from New England Biolabs.

Support Protocol 1: Pilot Experiment to Characterize the Behavior of an MBP Fusion Protein

  Additional MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • protocol 1Cells containing fusion plasmid ( protocol 1basic protocol)
  • 30 mM Tris⋅Cl/20% sucrose, pH 8.0
  • 0.5 M EDTA, pH 8.0 ( appendix 22)
  • 5 mM MgSO 4, ice‐cold

Alternate Protocol 1: Purification of Fusion Proteins from the Periplasm

  Additional MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • protocol 1Cells containing fusion plasmid ( protocol 1basic protocol)
  • 30 mM Tris⋅Cl/20% (w/v) sucrose, pH 8.0
  • 0.5 M EDTA, pH 8.0 ( appendix 22)
  • 5 mM MgSO 4
  • 1 M Tris⋅Cl, pH 7.4 ( appendix 22)
  • Beckman JA‐14 rotor (or equivalent)

Support Protocol 2: Purifying the Cleaved Protein by Ion Exchange Chromatography

  Additional MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • Fusion protein cleaved with factor Xa (unit 16.4)
  • 20 mM Tris⋅Cl/25 mM NaCl, pH 8.0
  • DEAE‐Sepharose or Q‐Sepharose resin
  • 20 mM Tris⋅Cl/500 mM NaCl, pH 8.0
  • 1 × 10–cm column
  • Additional reagents and equipment for purification of proteins by conventional chromatography (unit 10.10)

Support Protocol 3: Purifying the Cleaved Protein by Affinity Chromatography

  Additional MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • Hydroxylapatite resin
  • Fusion protein cleaved with factor Xa (unit 16.4)
  • 0.5 M sodium phosphate, pH 7.2
  • 1 or 1.5 × 10–cm column
  • Additional reagents and equipment for hydroxylapatite chromatography (unit 2.10)
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Figures

Videos

Literature Cited

Literature Cited
   Amann, E. and Brosius, J. 1985. ‘ATG vectors’ for regulated high‐level expression of cloned genes in Escherichia coli. Gene 40:183‐190.
   Baker, T.A., Grossman, D., and Gross, C.A. 1984. A gene regulating the heat shock response in Escherichia coli also affects proteolysis. Proc. Natl. Acad. Sci. U.S.A. 81:6779‐678.
   Bardwell, J.C.A., McGovern, K., and Beckwith, J. 1991. Identification of a protein required for disulfide bond formation in vivo. Cell 67:581‐589.
   Bedouelle, H. and Duplay, P. 1988. Production in Escherichia coli and one‐step purification of bifunctional hybrid proteins which bind maltose. Eur. J. Biochem. 171:541‐549.
   Duplay, P., Bedouelle, H., Fowler, A., Zabin, I., Saurin, W., and Hofnung, M. 1984. Sequences of the malE gene and of its product, the maltose‐binding protein of Escherichia coli K12. J. Biol. Chem. 259:10606‐10613.
   Gentz, R., Kuys, Y., Zwieb, C., Taatjes, D., Taatjes, H., Bannwarth, W., Stueber, D., and Ibrahimi, I. 1988. Association of degradation and secretion of three chimeric polypeptides in Escherichia coli. J. Bacteriol. 170:2212‐222.
   Grodberg, J. and Dunn, J.J. 1988. ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J. Bacteriol. 170:1245‐125.
   Guan, C., Li, P., Riggs, P.D., and Inouye, H. 1987. Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose‐binding protein. Gene 67:21‐30.
   Hsiung, H.M., Mayne, N.G., and Becker, G.W. 1986. High level expression, efficient secretion, and folding of human growth hormone in Escherichia coli. Bio/Technology 4:991‐995.
   Kellerman, O.K. and Ferenci, T. 1982. Maltose binding protein from E. coli. Methods Enzymol. 90:459‐463.
   Lauritzen, C., Tüchsen, E., Hansen, P.E., and Skovgaard, O. 1991. BPTI and N‐terminal extended analogues generated by Factor Xa cleavage and cathepsin C trimming of a fusion protein expressed in Escherichia coli. Prot. Expr. Purif. 2:372‐378.
   Maina, C.V., Riggs, P.D., Grandea, A.G. III, Slatko, B.E., Moran, L.S., Tagliamonte, J.A., McReynolds, L.A., and Guan, C. 1988. A vector to express and purify foreign proteins in Escherichia coli by fusion to, and separation from, maltose binding protein. Gene 74:365‐373.
   Nagai, K. and Thøgersen, H.C. 1984. Generation of β‐globin by sequence‐specific proteolysis of a hybrid protein produced in Escherichia coli. Nature 309:810‐812.
   Nagai, K. and Thøgersen, H.C. 1987. Synthesis and sequence‐specific proteolysis of hybrid proteins produced in Escherichia coli. Methods Enzymol. 153:461‐481.
   Neu, H.C. and Heppel, L.A. 1965. The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J.Biol. Chem. 240:3685‐3692.
   Reidhaar‐Olson, J.F., Parsell, D.A., and Sauer, R.T. 1990. An essential proline in λ repressor is required for resistance to intracellular proteolysis. Biochemistry 29:7563‐7571.
   Schein, C.H. 1989. Production of soluble recombinant proteins in bacteria. Bio/Technology 7:1141‐1149.
   Silber, K.R., Keiler, K.C., and Sauer, R.T. 1992. Tsp: A tail‐specific protease that selectively degrades proteins with nonpolar C termini. Proc. Natl. Acad. Sci. U.S.A. 89:295‐299.
   Silhavy, T.J., Szmelcman, S., Boos, W., and Schwartz, M. 1975. On the significance of the retention of ligand by protein. Proc. Natl. Acad. Sci. U.S.A. 72:2120‐2124.
   Strauch, K.L. and Beckwith, J. 1988. An Escherichia coli mutation preventing degradation of abnormal periplasmic proteins. Proc. Natl. Acad. Sci. U.S.A. 85:1576‐158.
   Straus, D.B., Walter, W.A., and Gross, C.A. 1988. Escherichia coli heat shock gene mutants are defective in proteolysis. Genes Dev. 2:1851‐1858.
   Sugimura, K. and Higashi, N. 1988. A novel outer‐membrane‐associated protease in Escherichia coli. J. Bacteriol. 174:3650‐3654.
   Takagi, H., Morinaga, Y., Tsuchiya, M., Ikemura, G., and Inouye, M. 1988. Control of folding of proteins secreted by a high expression secretion vector, pIN‐III‐ompA: 16‐fold increase in production of active subtilisin E in Escherichia coli Bio/Technology 6:948‐950.
   Wearne, S.J. 1990. Factor Xa cleavage of fusion proteins: Elimination of nonspecific cleavage by reversible acylation. FEBS Lett. 263:23‐26.
   Yanisch‐Perron, C., Vieira, J., and Messing, J. 1985. Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103‐119.
   Zagursky, R.J. and Berman, M.L. 1984. Cloning vectors that yield high levels of single stranded DNA for rapid DNA sequencing. Gene 27:183‐191.
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