Enzymatic and Chemical Cleavage of Fusion Proteins

Edward R. LaVallie1, John M. McCoy1, Donald B. Smith2, Paul Riggs3

1 Genetic Institute, Cambridge, Massachusetts, 2 University of Edinburg, Edinburg, Scotland, 3 New England Biolabs, Beverly, Masssachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.4B
DOI:  10.1002/0471142727.mb1604bs28
Online Posting Date:  May, 2001
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Abstract

This unit provides protocols for some commonly used methods of site‐specific cleavage of fusion proteins. The first three protocols describe enzymatic cleavage of proteins using proteases (factor Xa, thrombin, and enterokinase) that display highly restricted specificities, which greatly decrease the likelihood that unwanted secondary cuts will occur. Three additional protocols describe specific cleavage of fusion proteins with chemical reagents (cyanogen bromide, hydroxylamine, and low pH) as an alternative to enzymatic cleavage.

     
 
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Table of Contents

  • Basic Protocol 1: Enzymatic Cleavage of Fusion Proteins with Factor Xa
  • Support Protocol 1: Denaturing a Fusion Protein for Factor Xa Cleavage
  • Alternate Protocol 1: Enzymatic Cleavage of Fusion Proteins with Thrombin
  • Alternate Protocol 2: Enzymatic Cleavage of Matrix‐Bound GST Fusion Proteins
  • Alternate Protocol 3: Enzymatic Cleavage of Fusion Proteins with Enterokinase
  • Basic Protocol 2: Chemical Cleavage of Fusion Proteins Using Cyanogen Bromide
  • Alternate Protocol 4: Chemical Cleavage of Fusion Proteins Using Hydroxlamine
  • Alternate Protocol 5: Chemical Cleavage of Fusion Proteins by Hydrolysis at Low pH
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Enzymatic Cleavage of Fusion Proteins with Factor Xa

  Materials
    For recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • 1 mg/ml fusion protein
  • 200 µg/ml factor Xa (New England Biolabs) in reaction buffer (see step )
  • 2× SDS sample buffer (unit 10.210.2)
  • Boiling water bath
  • Additional reagents and equipment for SDS‐PAGE (unit 10.210.2)

Support Protocol 1: Denaturing a Fusion Protein for Factor Xa Cleavage

  Additional MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • 20 mM Tris⋅Cl (pH 7.4)/6 M guanidine⋅HCl
  • 20 mM Tris⋅Cl (pH 8.0)/1 mM CaCl 2

Alternate Protocol 1: Enzymatic Cleavage of Fusion Proteins with Thrombin

  Additional MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • recipeThrombin cleavage buffer (see recipe)
  • Heparin, sodium salt (with ≥140 U/mg activity, Sigma; optional)
  • Thrombin (human, with ∼3000 U/mg activity; Sigma or Boehringer Mannheim)

Alternate Protocol 2: Enzymatic Cleavage of Matrix‐Bound GST Fusion Proteins

  Additional MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • GST fusion protein bound to glutathione‐agarose beads (unit 16.7)
  • 1% (v/v) Triton X‐100 in phosphate‐buffered saline (PBS; appendix 22)
  • GST wash buffer: 50 mM Tris⋅Cl (pH 7.5)/150 mM NaCl
  • GST elution buffer: 50 mM Tris⋅Cl (pH 8.0)/5 mM reduced glutathione
  • 20‐ or 50‐ ml screw‐cap tube

Alternate Protocol 3: Enzymatic Cleavage of Fusion Proteins with Enterokinase

  Additional MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • 1 mg/ml thioredoxin fusion protein (unit 16.8) in 50 mM Tris⋅Cl (pH 8.0)/1 mM CaCl 2
  • 10 µg/ml bovine enterokinase (Biozyme EK‐3 grade) in 50 mM Tris⋅Cl (pH 8.0)/1 mM CaCl 2
NOTE: Many commercial preparations of enterokinase (bovine or porcine), with the exception of the source listed, are extremely impure and tend to be contaminated with, among other things, trypsin and chymotrypsin which can extensively degrade the fusion protein. It is recommended that only commercial enterokinase of the highest quality be used.

Basic Protocol 2: Chemical Cleavage of Fusion Proteins Using Cyanogen Bromide

  Materials
    For recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • 1 mg/ml fusion protein
  • 50 mg/ml cyanogen bromide (CNBr)/70% (v/v) formic acid
  • 70% (v/v) formic acid
  • 1× SDS sample buffer (unit 10.210.2)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.210.2)

Alternate Protocol 4: Chemical Cleavage of Fusion Proteins Using Hydroxlamine

  Additional MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • 1 mg/ml fusion protein in 10 mM Tris⋅Cl (pH 8.0)/150 mM NaCl
  • recipe2× hydroxylamine cleavage solution (see recipe)
  • Guanidine⋅HCl (optional)
  • 2× SDS sample buffer (unit 10.210.2)
  • Boiling water bath

Alternate Protocol 5: Chemical Cleavage of Fusion Proteins by Hydrolysis at Low pH

  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 containing an Asp‐Pro bond between the component domains
  • 70% (v/v) formic acid
  • 13% (v/v) acetic acid
  • 0.1 M Tris base
  • Guanidine⋅HCl
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Literature Cited

Literature Cited
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   Bornstein, P. and Balian, G. 1977. Cleavage at Asn‐Gly bonds with hydroxylamine. Methods Enzymol. 47:132‐145.
   Chance, R.E., Hoffman, J.A., Kroeff, E.P., Johnson, M.G., Schirmer, E.W., Bromer, W.W., Ross, M.J., and Wetzel, R. 1981. The production of humaninsulin using recombinant DNA technology and a new chain combination procedure. In Peptides: Synthesis‐Structure‐Function (D.H. Rich and E. Gross, eds.), pp. 721‐728. Pierce Chemical, Rockford, Ill.
   Chang, J.‐Y. 1985. Thrombin specificity. Eur. J. Biochem. 151:217‐224.
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   Gross, E. 1967. The cyanogen bromide reaction. Methods Enzymol. 11:238‐255.
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   LaVallie, E.R., DiBlasio, E.A., Kovacic, S., Grant, K.L., Schendel, P.F., and McCoy, J.M. 1993a. A thioredoxin gene fusion system that circumvents inclusion body formation in the E. coli cytoplasm. Bio/Technology 11:187‐193.
   LaVallie, E.R., Rehemtulla, A., Racie, L.A., DiBlasio, E.A., Ferenz, C., Grant, K.L., Light, A., and McCoy, J.M. 1993b. Cloning and functional expression of a cDNA encoding the catalytic subunit of bovine enterokinase. J. Biol. Chem. 268:23311‐23317.
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