Incorporation of Isotopically Enriched Amino Acids

Mini Samuel‐Landtiser1, Cherian Zachariah1, Chris R. Williams1, Arthur S. Edison1, Joanna R. Long1

1 University of Florida, Gainesville, Florida
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 26.3
DOI:  10.1002/0471140864.ps2603s47
Online Posting Date:  February, 2007
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Abstract

The incorporation of isotope labels into proteins is extremely useful for the application of nuclear magnetic resonance (NMR), X‐ray or neutron‐diffraction crystallography, and mass spectrometry (MS) methodologies to investigate the structure and dynamics of proteins. This unit presents methods for incorporating isotopic labels into proteins via expression in E. coli and baculovirus transfected Sf9 insect cells or through cell‐free means. The unit also presents methods for introducing isotopic labels by chemical means into synthetic peptides by solid phase peptide synthesis or into isolated proteins by chemical modification of labile protein groups.

Keywords: ??

     
 
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Table of Contents

  • Strategic Planning
  • Isotopic Labeling of Proteins Produced Recombinantly in E. coli
  • Basic Protocol 1: Uniform Isotopic Labeling of Proteins Through Expression in E. coli Using IPTG Induction
  • Alternate Protocol 1: Uniform Isotopic Labeling of Proteins Through Expression in E. coli Using Auto‐Induction
  • Alternate Protocol 2: Uniform Isotopic Labeling of Specific Carbons in Proteins Expressed in E. coli
  • Alternate Protocol 3: Uniform Deuteration with Concomitant 13C and 15N Incorporation in Proteins Expressed in E. coli
  • Alternate Protocol 4: Selective Isotopic Labeling of Proteins Through Expression in Bacteria Using the “TEASE” Labeling Scheme
  • Alternate Protocol 5: Preparation of Selectively Labeled Proteins in E. coli by Incorporation of Specifically Labeled Amino Acids
  • Alternate Protocol 6: Preparation of 19F‐Labeled Proteins in E. coli Using 19F‐Labeled Aromatic Amino Acids
  • Basic Protocol 2: Expression of Uniformly 15N or 13C/15N Labeled Recombinant Proteins Using the Gateway Technology and the Baculodirect Baculovirus Expression Systems
  • Isotopic Labeling of Proteins Produced in Cell‐Free Extracts
  • Basic Protocol 3: Cell‐Free Protein Expression Using S30 Extract and T7 RNA Polymerase
  • Support Protocol 1: Preparation of S30 Extract from E. coli
  • Support Protocol 2: Preparation of T7 RNA Polymerase
  • Basic Protocol 4: Incorporation of an Isotopically Enriched Amino Acid in a Peptide Via Solid Phase Peptide Synthesis Utilizing 9‐Fluorenylmethyloxycarbonyl (Fmoc) Amino Acids
  • Support Protocol 3: Preparation of a 9‐Fluorenylmethyloxycarbonyl (Fmoc)‐Protected Amino Acid
  • Support Protocol 4: Ninhydrin Test to Monitor Amino Acid Coupling Reaction
  • Basic Protocol 5: Isotopic Labeling of Amino Acids in a Protein Via Chemical Exchange Using Deuterated Water
  • Alternate Protocol 7: Isotopic Labeling of Amino Acids in a Protein Via Chemical Exchange Using Deuterated Guanidinium Chloride
  • Basic Protocol 6: Selective Isotopic Labeling of Proteins by Chemical Modification: Reductive Alkylation of Primary Amines
  • Basic Protocol 7: Selective Isotopic Labeling of Protein Sulfhydryl Groups by Covalent Chemical Modification
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Uniform Isotopic Labeling of Proteins Through Expression in E. coli Using IPTG Induction

  Materials
  • Lysogenic expression host, e.g., E. coli BL21 (DE3) transformed with a pET or similar vector containing a T7 lac promoter (EMD Biosciences) and the gene for the protein of interest (see Chapter 5)
  • LB agar plate containing the appropriate antibiotic ( appendix 4A)
  • Overnight medium (see recipe)
  • Antibiotic solutions (see reciperecipes) appropriate for expression host and vector
  • Glucose‐based minimal medium, isotopically enriched (see recipe)
  • 0.5 M isopropyl‐1‐thio‐β‐D‐galactopyranoside (IPTG; Research Products International): sterilize by passing through a 0.22‐µm filter and store in 5‐ml and 500‐µl aliquots up to 6 months at −20°C
  • 250‐ml and 4‐liter culture flasks, sterile and preferably baffled
  • 37°C orbital shaker incubator
  • 500‐ml centrifuge tubes
  • High speed refrigerated centrifuge with appropriate rotors (e.g., Beckman J2‐HS, with JA 10 and JA 20 rotors)
  • Additional reagents and equipment for performing SDS‐PAGE (unit 10.1) and purifying recombinant proteins (see Chapter 6)

Alternate Protocol 1: Uniform Isotopic Labeling of Proteins Through Expression in E. coli Using Auto‐Induction

  • Auto‐induction minimal medium, isotopically enriched (see recipe)

Alternate Protocol 2: Uniform Isotopic Labeling of Specific Carbons in Proteins Expressed in E. coli

  • Specific carbon source‐based minimal medium, isotopically enriched (see recipe)
  • 500× α‐ketoisovalerate and α‐ketobutyrate (see recipe), labeled

Alternate Protocol 3: Uniform Deuteration with Concomitant 13C and 15N Incorporation in Proteins Expressed in E. coli

  • Carbon source conditioning medium (see recipe)
  • Deuterated minimal medium (see recipe)
  • 500× [3,3′‐2H 2]‐13C‐α‐ketobutyrate and [3‐2H]‐13C‐α‐ketoisovalerate stock solutions (see recipe), if required
  • 250‐ml centrifuge tubes, optional
  • 30‐ml centrifuge tubes

Alternate Protocol 4: Selective Isotopic Labeling of Proteins Through Expression in Bacteria Using the “TEASE” Labeling Scheme

  • TEASE minimal medium (see recipe)
  • 250‐ml (optional) centrifuge tubes

Alternate Protocol 5: Preparation of Selectively Labeled Proteins in E. coli by Incorporation of Specifically Labeled Amino Acids

  • 50× amino acid stock solution (see recipe)
  • 30‐ml and 250‐ml (optional) ml centrifuge tubes

Alternate Protocol 6: Preparation of 19F‐Labeled Proteins in E. coli Using 19F‐Labeled Aromatic Amino Acids

  • Fluorine‐labeling medium (see recipe)
  • Fluorinated amino acid stock: 50× fluorophenylalanine or 50× fluorotryptophan or 50× fluorotyrosine (see reciperecipes)
  • 2‐liter baffled flasks, sterile
  • 30‐ml and 250‐ml (optional) centrifuge tubes

Basic Protocol 2: Expression of Uniformly 15N or 13C/15N Labeled Recombinant Proteins Using the Gateway Technology and the Baculodirect Baculovirus Expression Systems

  Materials
  • DNA template containing the gene of interest
  • Appropriate primers to produce the PCR product
  • Thermostable, proofreading polymerase, e.g., AccuPrime Taq high fidelity DNA polymerase kit (Invitrogen): includes 10× PCR buffer
  • pENTR/D‐TOPO Cloning Kit (Invitrogen): includes One Shot Top10 chemically competent E. coli, salt solution, and SOC medium
  • LB agar plates containing 50 µg/ml kanamycin (Research Products International or see appendix 4A)
  • LB medium ( appendix 4A)
  • BaculoDirect linear DNA, 300 ng/vial (Invitrogen)
  • TE buffer, pH 8.0 ( appendix 2E)
  • LR Clonase II enzyme mix (Invitrogen)
  • 2 mg/µl proteinase K (Invitrogen)
  • Sf9, Sf21, or High Five insect cells (Invitrogen)
  • Complete growth medium Sf‐900 II SFM (Invitrogen)
  • Grace's Insect Medium, unsupplemented (Invitrogen)
  • Cellfectin Reagent (Invitrogen)
  • Complete growth medium Sf‐900 II SFM with antibiotics (Invitrogen)
  • Ganciclovir (Invitrogen)
  • BioExpress‐2000 insect cell medium: unlabeled (Cambridge Isotopes Laboratories) or
  • BioExpress‐2000 insect cell medium: U‐15N, 98% (Cambridge Isotopes Laboratories) or
  • BioExpress‐2000 insect cell medium: U‐13C 92‐98%, U‐15N, 92‐95% (Cambridge Isotopes Laboratories) or
  • BioExpress‐2000 insect cell medium: 13C/15N for L‐methionine, L‐leucine, L‐isoleucine, and L‐tyrosine (Cambridge Isotopes Laboratories)
  • 25°C, 27°C, 37°C, and 42°C water baths
  • 37°C orbital shaker incubator
  • 27°C and 37°C incubators
  • 6‐well tissue culture plates
  • Microscope
  • Plastic bag and paper towels
  • 50‐ml centrifuge tubes, sterile
  • 500‐ml Erlenmeyer flasks
  • Additional reagents and equipment for PCR ( appendix 4J), agarose gel electrophoresis ( appendix 4F), restriction analysis ( appendix 4I; optional), DNA plasmid miniprep ( appendix 4C), DNA quantitation ( appendix 4K), determination of plaque forming units (unit 5.5), and cell quantitation using a hemacytometer ( appendix 3C)

Basic Protocol 3: Cell‐Free Protein Expression Using S30 Extract and T7 RNA Polymerase

  Materials
  • DEPC‐treated H 2O: 500 µl DEPC per 100 ml H 2O
  • 1.4 M CH 3COONH 4 (ammonium acetate)
  • 0.5 M (CH 3COO) 2Mg (magnesium acetate)
  • Amino acid mixture (see recipe)
  • 0.645 M creatine phosphate
  • LM mixture (see recipe)
  • 1 mg/ml high copy DNA template plasmid with gene for protein of interest and T7 promoter in DEPC‐treated H 2O
  • 11 mg/ml T7 RNA polymerase ( protocol 11 or Fermentas Life Sciences)
  • RNase inhibitor (Fermentas Life Sciences)
  • 10 mg/ml creatine kinase (Roche)
  • S30 extract ( protocol 10)
  • Small‐scale dialysis solution (see recipe)
  • Large‐scale dialysis solution (see recipe)
  • 5‐mm or 15‐mm diameter Spectra/Por CE 50,000 MWCO dialysis tubing (Spectrum Laboratories)
  • 10‐mm or 20‐mm (i.d.) test tubes
  • 37°C shaking water bath
  • Additional reagents and equipment for performing SDS‐PAGE (unit 10.1) and purifying proteins (Chapter 6)

Support Protocol 1: Preparation of S30 Extract from E. coli

  Materials
  • E. coli cells lacking RNase activity, e.g., BL21 star (DE3) or A19 (met B, RNA), Invitrogen
  • LB agar plate containing the appropriate antibiotic ( appendix 4A)
  • LB medium ()
  • S30 buffer (see recipe), chilled to 4°C
  • 1 M dithiothreitol (DTT) or solid DTT
  • Activation solution (see recipe)
  • Polyethylene glycol 8000 (PEG 8000; Sigma‐Aldrich)
  • 500‐ml flasks
  • 4‐liter baffled culture flasks, sterile
  • 37°C orbital shaker incubator
  • 250‐ and 500‐ml centrifuge tubes
  • High‐speed refrigerated centrifuge with appropriate rotors (e.g., Beckman J2‐HS, with JA 10 and JA 20 rotors)
  • French Press
  • 20.4 mm Spectra/Por dialysis tubing, 6‐8,000 MWCO (Spectrum Laboratories)
  • 2 Hi‐Prep 26/10 columns (GE Healthcare)

Support Protocol 2: Preparation of T7 RNA Polymerase

  Materials
  • Lysogenic expression host (E. coli, such as BL21 (DE3) transformed with pAR1219 (Davanloo et al., )
  • LB agar plate containing the appropriate antibiotic ( appendix 4A)
  • LB medium ( appendix 4A)
  • 500× ampicillin (see recipe for antibiotic solutions)
  • 1 M Isopropyl‐1‐thio‐β‐D‐galactopyranoside (IPTG; Research Products International)
  • Lysis buffer for T7 RNA polymerase (see recipe), chilled to 4°C
  • Sodium deoxycholate (Sigma‐Aldrich)
  • Tris buffer for T7 RNA polymerase, pH 8.1 (see recipe), chilled to 4°C
  • 2 M ammonium sulfate: prepare fresh and sterilize by passing through a 0.22‐µm filter
  • 10% (w/v) polyethyleneimine (Sigma): prepare fresh and sterilize by passing through a 0.22‐µm filter
  • Ammonium sulfate, saturated (see recipe)
  • Dialysis buffer (see recipe)
  • 5 M NaCl
  • 0.2 M PMSF (Sigma‐Aldrich Co.) in isopropanol
  • Glycerol
  • 250‐ml and 4‐liter baffled culture flasks, sterile
  • 37°C orbital shaker incubator
  • 500‐ml centrifuge tubes
  • High speed refrigerated centrifuge with appropriate rotors (Beckman J2‐HS, with JA 10 and JA‐20 rotors)
  • Sonicator with 3/6‐mm Vibracell probe tip (Sonics & Materials)
  • 20.4‐mm dialysis tubing, 6000 to 8000 MWCO
  • 20‐ml HiTrap SP FF (GE Healthcare) or Macro‐prep high S (Bio‐Rad) column
  • Additional reagents and equipment for performing SDS‐PAGE (unit 10.1) and spectrophotometrically determining protein concentration (unit 3.1)

Basic Protocol 4: Incorporation of an Isotopically Enriched Amino Acid in a Peptide Via Solid Phase Peptide Synthesis Utilizing 9‐Fluorenylmethyloxycarbonyl (Fmoc) Amino Acids

  Materials
  • Solid phase resin with carboxy terminal amino acid attached (EMD Biosciences)
  • Dichloromethane (DCM; Fisher Scientific)
  • N‐methylpyrrolidone (NMP; Sigma‐Aldrich)
  • 25% (v/v) piperidine (Sigma‐Aldrich) in NMP
  • Isotopically labeled and unlabeled Fmoc‐protected amino acids ( protocol 13 or Cambridge Isotope Laboratories)
  • N‐hydroxybenzotriazole (HOBt; EMD Biosciences)
  • Benzotriazole‐1‐yl‐oxy‐tris‐pyrrolidino‐phosphonium hexafluorophosphate (PyBOP; EMD Biosciences)
  • Diisopropylethylamine (DIPEA; Aldrich)
  • Acetic anhydride solution (see recipe)
  • King's reagent (see recipe) or
  • TFA/TIS/H 2O solution: 9 ml trifluoroacetic acid (TFA; Acros)/2.5 ml triisopropylsilane (TIS; Sigma‐Aldrich)/2.5 ml H 2O solution
  • Diethyl ether, chilled
  • Nitrogen gas (optional)
  • peptide reaction vessel (Sigma)
  • 100‐ml round‐bottom flask
  • Rotary evaporator equipped with a base trap filled with NaOH pellets
  • Additional reagents and equipment for performing the ninhydrin test ( protocol 14)

Support Protocol 3: Preparation of a 9‐Fluorenylmethyloxycarbonyl (Fmoc)‐Protected Amino Acid

  Materials
  • Isotopically labeled amino acid (Cambridge Isotope Laboratories)
  • 10% (w/v) Na 2CO 3 solution, warm
  • Fmoc‐O‐succinimide (EMD Biosciences)
  • 1,4‐dioxane
  • Toluene/acetic acid (10:1 ratio)
  • Diethyl ether
  • HCl, concentrated and 1 N
  • Ethyl acetate
  • MgSO 4, anhydrous
  • 50‐ml round‐bottom flask
  • Aluminum‐backed thin‐layer chromatography (TLC) plates (Whatman)
  • TLC tank
  • UV light source
  • 250‐ml separatory funnel
  • Rotary evaporator

Support Protocol 4: Ninhydrin Test to Monitor Amino Acid Coupling Reaction

  Materials
  • Sample (e.g., amino acid coupling reaction from protocol 12)
  • Solution 1: 5% (w/v) ninhydrin (Fisher) in ethanol
  • Solution 2: 1 mM KCN in pyridine (see recipe)
  • Solution 3: 80% (w/v) phenol in ethanol
  • 15‐ml glass test tube
  • 100°C hotplate

Basic Protocol 5: Isotopic Labeling of Amino Acids in a Protein Via Chemical Exchange Using Deuterated Water

  Materials
  • Protein or peptide of interest
  • 1 M hydrochloric acid
  • Deuterated water (Cambridge Isotope Laboratories) containing 150 mM NaCl and 5 mM sodium phosphate, 5°C
  • 100‐ml round‐bottom flask
  • Magnetic stir bar and stirrer
  • pH meter
  • Lyophilizer

Alternate Protocol 7: Isotopic Labeling of Amino Acids in a Protein Via Chemical Exchange Using Deuterated Guanidinium Chloride

  Materials
  • Protein or peptide of interest
  • 1 M HCl
  • 6 M deuterated guanidinium chloride (Cambridge Isotope Laboratories) in deuterated water (Cambridge Isotope Laboratories)
  • 150 mM NaCl/5 mM sodium phosphate solution
  • Deuterated water (Cambridge Isotope Laboratories)
  • 10‐ml round‐bottom flask
  • Magnetic stir bar and stirrer
  • pH meter
  • Lyophilizer
  • Dialysis tubing (protein‐dependent MWCO)

Basic Protocol 6: Selective Isotopic Labeling of Proteins by Chemical Modification: Reductive Alkylation of Primary Amines

  Materials
  • 2 mg/ml solution of protein of interest in 50 mM phosphate or HEPES buffer, pH 7.0
  • 0.1 M sodium cyanoborodeuteride solution (NaCNB2H 3; Cambridge Isotope Laboratories) or
  • 0.1 M sodium cyanoborohydride solution (NaCNBH 3; Aldrich)
  • 0.1 M 2H‐labeled formaldehyde solution (see recipe) or
  • 0.1 M 13C‐labeled formaldehyde solution (see recipe)
  • 0.1 M NaCl
  • 5‐ml test tube
  • Dialysis tubing with MWCO appropriate for the size of the protein of interest

Basic Protocol 7: Selective Isotopic Labeling of Protein Sulfhydryl Groups by Covalent Chemical Modification

  Materials
  • Protein sample
  • Denaturing or nondenaturing reducing buffer (see reciperecipes)
  • 3‐Bromo‐1,1,1‐trifluoroacetone (BTFA) or iodoacetamide with either 13C or 15N enrichment (Cambridge Isotope Laboratories)
  • Dialysis tubing (if denaturing) with MWCO appropriate for the size of the protein of interest
  • Dialysis buffer (see recipe)
  • 250‐ml round‐bottom flask
  • Magnetic stir bar and stirrer
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Figures

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Literature Cited

   Brauer, M. and Sykes, B.D. 1986. 19F nuclear magnetic resonance studies of selectively fluorinated derivatives of G‐ and F‐actin. Biochemistry 25:2187‐2191.
   Brüggert, M., Rehm, T., Shankar. S., Georgescu, J., and Holak, T.A. 2003. A novel medium for expression of proteins selectively labeled with 15N‐amino acids in Spodoptera frugiperda (Sf9) insect cells. J. Biomol. N.M.R. 25:335‐348.
   Chan, W.C. and White, P.D. 2000. FMOC Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press, Oxford.
   Chrunyk, B.A., Evans, J., Lillquist, J., Young, P., and Wetzel, R. 1993. Inclusion‐body formation and protein stability in sequence variants of interleukin‐β. J. Biol. Chem. 268:18053‐18061.
   Cowburn, D. and Muir, T.W. 2001. Segmental isotopic labeling using expressed protein ligation. Method Enzymol. 339:41‐54.
   Creemers, A.F.L., Klaassen, C.H.W., Bovee‐Geurts, P.H.M., Kelle, R., Kragl, U., Raap, J., de Grip, W.J., Lugtenburg, J., and de Groot, H.J.M. 1999. Solid state 15N NMR evidence for a complex Schiff base counterion in the visual G‐protein receptor rhodopsin. Biochemistry 38:7195‐7199.
   Davanloo, P., Rosenberg, A.H., Dunn, J.J., and Studier, F.W. 1984. Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U.S.A. 81:2035‐2039.
   David, R., Richter, P.O., and Beck‐Sickinger, A.G. 2004. Expressed protein ligation, methods and applications. Eur J. Biochem. 271:663‐677.
   DeLange, F., Klaassen, C.H.W., Wallace‐Williams, S.E., Bovee‐Geurts, P.H.M., Liu, X‐M., de Grip, W.J., and Rothschild, K.J. 1998. Tyrosine structural changes detected during the photoactivation of rhodopsin. J. Biol. Chem. 273:23735‐23739.
   Englander, S.W. and Poulsen, A. 1969. Hydrogen‐tritium exchange of the random chain polypeptide. Biopolymers 7:379‐393.
   Gardner, K.H. and Kay, L.E. 1997. Production and incorporation of 15N, 13C, 2H (1H‐δ1 methyl) isoleucine into proteins for multidimensional NMR studies. J. Am. Chem. Soc. 119:7599‐7600.
   Gerig, J.T. 2001. Fluorine NMR. Biophysics Textbook Online. 1‐35.
   Goff, S.A. and Goldberg, A.L. 1987. An increased content of protease LA, the Lon gene product, increases protein degradation and blocks growth in Escherichia coli. J. Biol. Chem. 262:4508‐4515.
   Goto, N.K. and Kay, L.E. 2000. New developments in isotope labeling strategies for protein solution NMR spectroscopy. Curr. Opin. Struc. Biol. 10:585‐592.
   Goto, N.K., Gardner, K.H., Mueller, G.A., Willis, R.C., and Kay, L.E. 1999. A robust and cost‐effective method for the production of Val, Leu, Ile (δ1) methyl‐protonated 15N‐, 13C‐, 2H‐labeled proteins. J. Biomol. N.M.R. 13:369‐374.
   Hajduk, P.J., Augeri, D.J., Mack, J., Mendoza, R., Yang, J., Betz, S.F. and Fesik, S.W. 2000. NMR‐based screening of proteins containing 13C‐labeled methyl groups. J. Am. Chem. Soc. 122:7898‐7904.
   Henrich, B., Lubitz, W. and Plapp, R. 1982. Lysis of Escherichia coli by induction of cloned ϕ X174 genes. Mol. Gen. Genet. 185:493‐497.
   Hong, M. and Jakes, K. 1999. Selective and extensive 13C labeling of a membrane protein for solid‐state NMR investigations. J. Biomol. N.M.R. 14:71‐74.
   Ishima, R., Louis, J.M., and Torchia, D.A. 1999. Transverse 13C relaxation of CHD2 methyl isotopmers to detect slow conformational changes of protein side chains. J. Am. Chem. Soc. 121:11589‐11590.
   Jentoft, N. and Dearborn, D.P.G., 1983. Protein labeling by reductive alkylation. Methods in Enzymolgy 19:570‐579.
   Klammt, C., Lohr, F., Schafer, B., Haase, W. Dotsch, V., Ruterjans, H., Glaubitz, C., and Bernhard, F. 2004. High level cell free expression and specific labeling of integral membrane proteins. Eur. J. Biochem. 271:568‐580.
   Kotik, M., Radford, S.E., and Dobson, C.M. 1995. Comparison of the refolding of hen lysozyme from dimethyl sulfoxide and guanidinium chloride. Biochemistry 34:1714‐1724.
   Kranz, James K., Lu, J. and Hall, K.B. 1996. Contribution of the tyrosines to the structure and Function of the human U1A N‐terminal RNA binding domain. Protein Sci. 5:1567‐1583.
   Lee, A.L., Urbauer, J.L. and Wand, J.A. 1997. Improved labeling strategy for 13C relaxation measurements of methyl groups in proteins. J. Biomol. N.M.R. 9:437‐440.
   LeMaster, D.M. and Kushlan, D.M. 1996. Dynamical Mapping of E. coli Thioredoxin via 13C NMR Relaxation Analysis. J. Am. Chem. Soc. 118:9255‐9264.
   Lian, L‐Y. and Middleton, D.A. 2001. Labelling approaches for protein structural studies for solution‐state and solid‐state NMR. Prog. Nucl. Mag. Res. Spec. 39:171‐190.
   Nirenberg, M.W. and Matthaei, J.H. 1961. The dependence of cell‐free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc. Natl. Acad. Sci. U.S.A. 47:1588‐1602.
   Ozawa, K., Wu, P.S., Dixon, N.E., and Otting, G. 2006. 15N‐labeled proteins by cell‐free protein synthesis. Strategies for high‐throughput NMR studies of proteins and protein‐ligand complexes. FEBS J. 273:4154‐4159.
   Ralf, D., Richter, M.P.O., and Beck‐Sickinger, A.G. 2004. Expressed protein ligation. Eur. J. Biochem. 271:663‐677.
   Rohl, C.A., Scholtz, J.M., York, E.J., Stewart, J.M. and Baldwin, R.L. 1992. Kinetics of amide proton exchange in helical peptides of varying chain lengths. Interpretation by the Lifson‐Roig Equation. Biochemistry 31:1263‐1269.
   Rosen, M.K., Gardner, K.H., Willis, R.C., Parris, W.E., Pawson, T., and Kay, L.E. 1996. Selective methyl group protonation of perdeuterated proteins. J. Mol. Biol. 263:627‐636.
   Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, New York.
   Spooner, P.J. and Watts, A., 1991. Reversible unfolding of cytochrome c upon interaction with cardiolipin bilayers. 1.Evidence from deuterium NMR measurements. Biochemistry 30:3871‐3879.
   Strauss, A., Bitsch, F., Fendrich, G., Cutting, B., Fendrich, G., Graff, P., Liebetanz, J., Zurini, M. and Jahnke, W. 2003. Amino‐acid‐type selective isotope labeling of protein expressed in Baculovirus‐infected cells useful for NMR studies. J. Biomol. N.M.R. 26:347‐372.
   Strauss, A., Bitsch, F., Fendrich, G., Graff, P., Knecht, R., Meyhack, B. and Jahnke, W. 2005. Efficient uniform isotope labeling of ab1 kinase expressed in Baculovirus‐infected cells. J. Biomol. N.M.R. 31:343‐349.
   Studier, F.W. 2005. Protein production by auto‐induction in high density shaking cultures. Protein Expr. Purif. 41:207‐234.
   Studier, F.W. and Moffatt, B.A. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high‐level expression of cloned genes. J. Mol. Biol. 189:113‐130.
   Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. 1990. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185:60‐89.
   Torizawa, T., Shimizu, M., Taoka, M., Miyano, H., and Kainosho, M. 2004. Efficient production of isotopically labeled proteins by cell‐free synthesis: A practical protocol. J. Biomol. N.M.R. 30:311‐325.
   Tyler, R.C., Sreenath, H.K., Singh, S., Aceti, D.J., Bingman, C.A., Markley, J.L., and Fox, B.G. 2005. Auto‐induction medium for the production of [U‐15N]‐ and [U‐13C, U‐15N]‐labeled proteins for NMR screening and structure determination. Protein Expr. Purif. 40:268‐278.
   Venters, R.A., Huang, C‐C., Farmer II, B.T., Trolard, R., Spicer, L.D., and Fierke, C.A. 1995. High‐level 2H/13C/15N labeling of proteins for NMR studies. J. Biomol. N.M.R. 5:339‐344.
   Vinarov, D.A., Lytle, B.L., Peterson, F.C., Tyler, E.M., Volkman, B.F., and Markley, J.L. 2004. Nat. Methods 1:1‐5.
Key References
   BaculoDirect Baculovirus Expression Systems Instruction Manual, Version F, 2004. Invitrogen, Carlsbad, Calif.
  This manual describes the steps involved in forming a recombinant baculovirus with the gene of interest, which can subsequently be used to transfect insect cells for protein expression, using the BaculoDirect Baculovirus Expression System.
   Chan, W.C. and White, P.D. 2000. Fmoc Solid Phase Peptide Synthesis: A Practical Approach. Oxford University Press, Oxford.
  Covers the essential procedures for the production of linear peptides as well as new methodological advances in the field.
   Englander, S.W. and Poulsen, A. 1969. Hydrogen‐tritium exchange of the random chain polypeptide. Biopolymers 7:379‐393.
  Describes the kinetics of hydrogen exchange in proteins as a functin of pH.
   Fields, G.B. and Noble, R.L. 1990. Solid phase peptide synthesis utilizing 9‐fluorenylmethoxycarbonyl amino acids, Int. J. Peptide Protein Res. 35:161‐214.
  An overview of solid phase peptide synthesis methodology.
   pENTR Directional TOPO Cloning Kit Instruction Manual, Version G, 2006. Invitrogen, Carlsbad, Calif.
  Comprehensive instruction manual for the pENTR Directional TOPO Cloning Kits for directional cloning of a blunt‐end PCR product into a vector for entry into Invitrogen's Gateway system.
   Sambrook et al., 1989. See above.
  A resource for general molecular biology cloning and expression techniques.
   Strauss et al., 2005. See above.
  Describes the use of commercially available BioExpress media to express both unlabeled and isotopically labeled recombinant proteins using insect cells.
   Studier, 2005. See above.
  This provides a detailed account of auto induction of recombinant proteins in E. coli.
   Torizawa et al., 2004. See above.
  This provides an excellent account of the protocol for the expression of labeled proteins using cell‐free systems which includes modifications such as continuous diffusion of by‐products and replenishments of precursors and energy regeneration.
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