Chemoenzymatic Site‐Specific Reversible Immobilization and Labeling of Proteins from Crude Cellular Extract Without Prior Purification Using Oxime and Hydrazine Ligation

Mohammad M. Mahmoodi1, Mohammad Rashidian1, Jonathan K. Dozier2, Mark D. Distefano2

1 These authors contributed equally to this work., 2 Department of Chemistry, University of Minnesota, Minneapolis, Minnesota
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch120247
Online Posting Date:  June, 2013
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Abstract

In a facile and potentially general method for protein modification at the C‐terminus, aldehyde‐modified proteins, obtained from enzymatic protein prenylation, react rapidly with hydrazide and aminooxy surfaces and fluorophores at neutral pH and in micromolar concentration ranges of reagents. This strategy was used for fluorescent labeling of eGFP‐CVIA, as a model protein, with aminooxy and hydrazide fluorophores or PEGs, and immobilization onto and subsequent release of the protein from hydrazide‐functionalized agarose beads using hydrazone‐oxime exchange. This method is described in detail here and provides site‐specifically PEGylated or fluorescently labeled proteins starting from crude cellular extract in three steps: prenylation, capture, and release. Curr. Protoc. Chem. Biol. 5:89‐109 © 2013 by John Wiley & Sons, Inc.

Keywords: PFTase; farnesyl diphosphate; site‐specific protein modification; protein immobilization; oxime ligation; hydrazine ligation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Farnesyl Aldehyde Diphosphate (Compound 1)
  • Basic Protocol 2: Enzymatic Incorporation of Farnesylaldehyde Diphosphate into Protein, and Subsequent Site‐Specific Labeling and PEGylation
  • Basic Protocol 3: Crude Prenylation, Immobilization, and Subsequent Labeling and Release of Proteins
  • Support Protocol 1: Expression and Purification of PFTase
  • Support Protocol 2: Expression and Purification of eGFP‐CVIA
  • Support Protocol 3: Procedure for MALDI‐MS Analysis of Protein Samples
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of Farnesyl Aldehyde Diphosphate (Compound 1)

  Materials
  • Farnesol (Aldrich)
  • Dichloromethane (DCM)
  • 3,4‐dihydropyran (Aldrich)
  • Pyridinium p‐toluenesulfonate (PPTS; Aldrich)
  • Diethyl ether (Et 2O)
  • Sodium bicarbonate (NaHCO 3)
  • Sodium sulfate (Na 2SO 4)
  • Silica gel 60
  • tert‐butyl hydroperoxide (Aldrich)
  • Selenous acid (H 2SeO 3; Aldrich)
  • Salicylic acid (Aldrich)
  • Toluene
  • Ethyl acetate (EtOAc)
  • Dimethylsulfoxide (DMSO)
  • Triethylamine (TEA; Aldrich)
  • Sulfur trioxide pyridine complex (SO 3⋅Py; Aldrich)
  • Hexanes
  • 5 M HCl (aqueous)
  • Brine (saturated aqueous NaCl)
  • Isopropyl alcohol
  • Trichloroacetonitrile (CCl 3CN)
  • Acetonitrile (ACN)
  • Bis(triethylammonium) hydrogen phosphate (Et 3NH) 2HPO 4
  • Ammonium bicarbonate (NH 4HCO 3)
  • Acetonitrile (CH 3CN)
  • Heavy water (D 2O)
  • Sodium pyrophosphate (Na 2H 2P 2O 7)
  • 200‐ml and 50‐ml round‐bottom flasks
  • Magnetic stirrer and stir bar
  • Büchi Rotavapor model R‐114 or equivalent rotary evaporator
  • 10 × 2–in. (25.4 × 5.0–cm) chromatography column(s)
  • TLC plates (silica gel 60 F‐254; Merck)
  • Separatory funnel
  • Oil bath
  • Büchner funnel
  • 0.45‐µm syringe filter
  • HPLC instrument: Beckman model 127/166 equipped with a UV detector and a Phenomenex C18 column (Luna, 10 µm, 10 × 250 mm) with a 5‐cm guard column
  • Electro spray ionization mass spectrometer (ESI‐MS; Bruker BioTOF II)
  • Lyophilizer
  • 500‐MHz 1H NMR instrument (Oxford VI‐500 MHz)

Basic Protocol 2: Enzymatic Incorporation of Farnesylaldehyde Diphosphate into Protein, and Subsequent Site‐Specific Labeling and PEGylation

  Materials
  • Tris hydrochloride
  • Tris base
  • MgCl 2
  • KCl
  • ZnCl 2
  • Dithiothreitol (DTT)
  • 6×His–tagged eGFP‐CVIA stock solution (see protocol 5)
  • Compound 1 stock solution (see protocol 1)
  • PFTase stock solution (see protocol 4)
  • Aminooxy‐AlexaFluor 488 (Anaspec)
  • Dimethylsulfoxide (DMSO)
  • Aniline or m‐phenylenediamine (Aldrich)
  • 1.0 M sodium phosphate buffer, pH 7
  • Aminooxy‐PEG (9; mol. wt. 5,000; NOF Corp., http://nofamerica.net/)
  • 30°C incubator
  • Amicon Centriprep centrifugal filter (MWCO 10,000; Millipore)
  • Centrifuge (Beckman‐Coulter)
  • UV‐Vis spectrophotometer (50 Bio; Varian)
  • 600‐µl microcentrifuge tubes
  • NAP‐5 column (Amersham)
  • LC‐MS instrument (Waters Synapt G2 Quadropole TOF mass spectrometer instrument)
  • MALDI‐MS instrument (Bruker MALDI TOF)
  • Additional reagents and equipment for analyzing the efficiency of prenylation ( protocol 6), SDS‐PAGE (e.g., Gallagher, ), and MALDI‐MS (e.g., Carr and Annan, )

Basic Protocol 3: Crude Prenylation, Immobilization, and Subsequent Labeling and Release of Proteins

  Materials
  • 6×His–tagged eGFP‐CVIA cell extracts (see protocol 5)
  • Tris base
  • Tris hydrochloride
  • MgCl 2
  • KCl
  • ZnCl 2
  • Dithiothreitol (DTT)
  • Compound 1 ( protocol 1)
  • PFTase stock solution (see protocol 4)
  • 50 mM Tris⋅Cl, pH 7.5
  • Hydrazide‐functionalized agarose beads (Thermo Scientific)
  • Purified 6×His–tagged eGFP‐CVIA solution (see protocol 5)
  • 1.0 M sodium phosphate buffer, pH 7
  • Aniline or m‐phenylenediamine (Aldrich)
  • 1 M KCl in 50 mM Tris⋅Cl, pH 7.5
  • 50 mM Tris⋅Cl, pH 7.5
  • Aminooxy‐PEG (9; mol. wt. 5,000; NOF Corp., http://nofamerica.net/) or aminooxy‐AlexaFluor 488 (Anaspec)
  • UV‐Vis spectrophotometer (50 Bio; Varian)
  • 0.45 µm syringe filter
  • Centrifuge (Beckman‐Coulter)
  • Amicon Centriprep centrifugal filter (MWCO 10,000; Millipore)
  • Additional reagents and equipment for SDS‐PAGE (Gallagher, ) and MALDI‐MS (Carr and Annan, )

Support Protocol 1: Expression and Purification of PFTase

  Materials
  • Stock of BL21(DE3)pLysS E. coli cells containing PFTase on a CDF‐Duet1 vector; available from Distefano laboratory ( )
  • LB medium (see recipe) containing 50 mg/liter streptomycin
  • IPTG
  • ZnSO 4
  • Lysis buffer A (see recipe)
  • Protease inhibitor (for use with His‐tagged proteins; Sigma‐Aldrich, cat. no. P8849)
  • Nickel affinity resin (Ni‐NTA resin; Gold Biotechnology, https://www.goldbio.com/)
  • Elution buffer A (see recipe)
  • Storage buffer A (see recipe)
  • Glycerol
  • Shaking incubator (Excella E‐24; Brunswick Scientific)
  • Spectrophotometer for determining OD 600
  • Centrifuge (Beckman Coulter)
  • Probe sonicator
  • 30‐ml chromatography column
  • Amicon Centriprep centrifugal filter (MWCO 10,000; Millipore)
  • Additional reagents and equipment for MALDI‐MS ( protocol 6)

Support Protocol 2: Expression and Purification of eGFP‐CVIA

  Materials
  • pJexpress414 plasmid containing the His‐tagged eGFP‐CVIA (eGFP with a 6xHisitidine tag at the N‐terminus and the prenylation sequence CVIA at the C‐terminus; DNA 2.0, https://www.dna20.com/)
  • One Shot BL21(DE3) chemically competent E. coli (Invitrogen)
  • LB agar plates and liquid medium (see recipe) containing 100 µg/ml ampicillin (Aldrich)
  • 1 M IPTG
  • Lysis buffer B (see recipe)
  • Protease inhibitor (for use with His‐tagged proteins; Sigma‐Aldrich, cat. no. P8849)
  • Nickel affinity resin (Ni‐NTA resin; Gold Biotechnology, https://www.goldbio.com/)
  • Elution buffer B (see recipe)
  • Storage buffer B (see recipe)
  • Glycerol
  • Shaking incubator (Excella E‐24; Brunswick Scientific)
  • UV‐Vis spectrophotometer (50 Bio; Varian)
  • Centrifuge (Beckman‐Coulter)
  • Probe sonicator
  • 25‐ml chromatography column
NOTE: BL21(DE3)pLysS E. coli cells containing 6×His–tagged eGFP‐CVIA are available from Distefano laboratory ( ). Instructions for creating this cell line are given below.

Support Protocol 3: Procedure for MALDI‐MS Analysis of Protein Samples

  Materials
  • 0.1% TFA in ACN (Solvent A)
  • 0.1 % TFA in H 2O (Solvent B)
  • 10 µl of protein sample
  • Freshly made saturated solution of sinapinic acid (Aldrich) in H 2O (MALDI matrix)
  • C4 ZipTip micro columns (Millipore)
  • MALDI plate (Bruker)
  • MALDI‐MS instrument (Bruker MALDI‐TOF)
  • Additional reagents and equipment for MALDI‐TOF (Carr and Annan, )
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Figures

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Literature Cited

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