Determining the Identity and Structure of Recombinant Proteins

Nancy D. Denslow1, Keith Rose2, Pier Giorgio Righetti3

1 University of Florida, Gainesville, 2 University Medical Center, Geneva, 3 University of Milano, Milan
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 7.3
DOI:  10.1002/0471140864.ps0703s03
Online Posting Date:  May, 2001
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Abstract

In this unit peptide mapping protocols with separation of the constituent peptides by high‐performance liquid chromatography (HPLC) analysis and by high‐resolution SDS‐PAGE are presented. Peptide mapping is ideally suited for comparative purposes‐‐for example, combined analysis of the recombinant protein and its natural counterpart (or some other well‐characterized standard). This unit also outlines the general strategy used to determine the linkage pattern of a monomeric recombinant protein containing two intramolecular disulfide bonds. The approach is an extension of peptide mapping, where the aim is to isolate and characterize peptides containing only a single disulfide bond. A two‐dimensional electrophoretic method is also described in which the protein isoelectric point is displayed as a function of pH to yield an electrophoretic titration curve. This method is especially useful for checking for deamidation (e.g., of Asn to Asp) in which additional negative charge is introduced into the modified protein.

     
 
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Table of Contents

  • Basic Protocol 1: Peptide Mapping by HPLC
  • Alternate Protocol 1: Peptide Mapping by High‐Resolution SDS‐PAGE
  • Support Protocol 1: Digestion of Protein in an SDS‐Polyacrylamide Gel Slice
  • Basic Protocol 2: Determining the Disulfide Linkage Pattern of a Recombinant Protein
  • Basic Protocol 3: Determining Charge Heterogeneity by Two‐Dimensional Titration Curve Analysis
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Peptide Mapping by HPLC

  Materials
  • Samples of recombinant and, if possible, native protein
  • Acetone/1 M HCl, −20°C (acidified acetone; optional)
  • 8 M urea/ 50 mM Tris⋅Cl, pH 7.5
  • 45 mM dithiothreitol (DTT)
  • 100 mM iodoacetamide, prepared fresh and kept wrapped in foil
  • Sequencing‐grade protease (unit 11.1) or chemical cleavage agent (unit 11.4): e.g., endoproteinase Lys‐C (Wako Chemicals)
  • recipeDigestion buffer (see recipe)
  • recipeHPLC solvents A and B for peptide mapping (see recipe)
  • 50°C water bath or heating block
  • 15‐ to 25‐cm, 2.5‐mm‐i.d. C18 HPLC column (e.g., Vydac C18, 300 Å, 5‐µm bead)
  • Analytical high‐performance liquid chromatography system
  • Additional reagents and equipment for proteolytic digestion (unit 11.1) and/or chemical cleavage (unit 11.4) of proteins in solution and for digestion in SDS‐polyacrylamide gel (optional; see protocol 3)

Alternate Protocol 1: Peptide Mapping by High‐Resolution SDS‐PAGE

  • 2× SDS sample buffer (unit 10.1)
  • 16 × 16–cm × 1‐mm or 16 × 20–cm × 1‐mm Tris‐tricine slab gel (unit 10.1)
  • 85°C water bath or heating block
  • XAR X‐ray film or equivalent (optional; for detection of radiolabeled proteins, if used)
  • Additional reagents and equipment for radiolabeling of proteins (units 3.3; optional), proteolytic digestion (unit 11.1) and/or chemical cleavage (unit 11.4) of proteins in solution, digestion in SDS‐polyacrylamide gel (optional; see 7.3), one‐dimensional SDS‐PAGE (unit 10.2), and detection of proteins in gels (unit 10.5) or on membranes (unit 10.8)

Support Protocol 1: Digestion of Protein in an SDS‐Polyacrylamide Gel Slice

  • 10 mM Tris⋅Cl (pH 7.5)/ 0.1% (w/v) SDS
  • 1.5‐ml Kontes microcentrifuge tube and pestle, or equivalent
  • 1.5‐ml low‐protein‐binding microfilterfuge tube (0.2‐µm cellulose acetate; Rainin or Costar)
  • Dry ice/ethanol bath (optional)
  • Additional reagents and equipment for one‐ or two‐dimensional SDS‐PAGE (units 10.1 & 10.4), staining with Coomassie blue (unit 10.5), and proteolytic digestion of proteins in gels (unit 11.3)

Basic Protocol 2: Determining the Disulfide Linkage Pattern of a Recombinant Protein

  Materials
  • Sample of recombinant protein
  • recipeAlkylation buffer (see recipe)
  • Nitrogen or argon, oxygen‐free
  • Guanidine hydrochloride
  • 50 mM and 2 M ammonium bicarbonate
  • recipe2 mg/ml cyanogen bromide in 70% formic acid (see recipe)
  • recipeHPLC solvents A and B for disulfide linkage analysis (see recipe)
  • 1% formic acid
  • Proteases: pepsin, Staphylococcus aureus V8 protease (endoproteinase Glu‐C), and/or trypsin (porcine variety, Sigma)
  • Glacial acetic acid
  • 1 M dithiothreitol (DTT)
  • recipePretreated dialysis tubing with appropriate MWCO (see recipe)
  • Apparatus for reversed‐phase high‐pressure liquid chromatography (RP‐HPLC), preferably with automatic sample injector
  • Appropriate analytical HPLC column: e.g., 25 cm × 4 mm i.d. filled with Nucleosil 5 µm, 300 Å C8 particles (Macherey‐Nagel)
  • Appropriate preparative HPLC column: e.g., 25 cm × 4 cm i.d. filled with Nucleosil 5 µm (as above) for purifying up to ∼0.2 mg protein, or 25 cm × 10 mm i.d. with same packing for up to ∼1.5 mg protein (see for further guidance)
  • Vacuum centrifuge: e.g., Speedvac (Savant)
  • Equipment for mass spectrometry of peptides
  • Additional reagents and equipment for dialysis (unit 4.4; appendix 3B)

Basic Protocol 3: Determining Charge Heterogeneity by Two‐Dimensional Titration Curve Analysis

  Materials
  • recipeAcrylamide/bisacrylamide stock solution (see recipe)
  • Carrier ampholytes: e.g., Pharmalyte (Pharmacia Biotech), Bio‐Lyte (Bio‐Rad), or Servalyte (Serva)
  • recipe10% (w/v) ammonium persulfate stock solution (see recipe)
  • N,N,N′,N′‐tetramethylethylene diamine (TEMED)
  • Kerosene
  • recipeElectrode solutions (see recipe)
  • Sample of recombinant protein in aqueous solution containing ≤10 mM Tris⋅acetate (or other salt or buffer consisting of weak acids and bases)
  • Coomassie blue staining solution: 0.1% (w/v) Coomassie brilliant blue R‐250/25% (v/v) ethanol/8% (v/v) acetic acid
  • Destaining solution: 25% (v/v) ethanol/8% (v/v) acetic acid
  • Gel Bond PAG sheets
  • Electrophoresis unit for horizontal slab gel operation: e.g., Multiphor (Pharmacia Biotech)
  • Levelling table
  • IEF electrode filter paper strips (Pharmacia Biotech)
  • Constant‐power supply (up to 3000 V, 200 mA, 20 W)
  • Thermostatic water bath: e.g., MultiTemp (Pharmacia Biotech)
  • Titration curve kit (Pharmacia Biotech)
  • Additional reagents and equipment for denaturing IEF in slab gels (unit 10.2)
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Figures

Videos

Literature Cited

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Key Reference
   Allen, G. 1989. See above.
  Contains excellent information about common proteolytic agents.
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