Native Chemical Ligation of Polypeptides

Julio A. Camarero1, Tom W. Muir1

1 The Rockefeller University, New York
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 18.4
DOI:  10.1002/0471140864.ps1804s15
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The total synthesis and semisynthesis of proteins allows the site‐specific incorporation of unnatural amino acids, post‐translational modifications, and biophysical/biochemical probes into the target molecule. Among the various chemical and enzymatic approaches available for the synthesis/semisynthesis of proteins, the native chemical ligation technique has proven especially useful and is the exclusive focus of this unit. This unit first discusses how to choose the ligation site(s) in the target protein and then outlines how to obtain the necessary polypeptide building blocks using either chemical synthesis or recombinant DNA expression. Next, the synthesis of a protein by native chemical ligation of two polypeptide fragments is described. The synthesis of a protein from three polypeptide fragments using a sequential native chemical ligation strategy is also described. Support protocols describe how to obtain the necessary polypeptide fragments using either chemical synthesis or recombinant DNA expression.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Native Chemical Ligation of Two Polypeptides
  • Basic Protocol 2: Sequential Chemical Ligation of Three Polypeptides
  • Support Protocol 1: Chemical Synthesis of N‐Terminal CYS‐Polypeptides
  • Support Protocol 2: Biosynthesis of N‐Terminal CYS Polypeptides
  • Support Protocol 3: Chemical Synthesis of α‐Thioester Polypeptides
  • Support Protocol 4: Bacterial Expression of α‐Thioester Polypeptides
  • Support Protocol 5: Chemical Synthesis of Nα(Msc)‐CYS, α‐Thioester Polypeptides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Native Chemical Ligation of Two Polypeptides

  Materials
  • recipe6 M guanidine⋅HCl buffer (see recipe)
  • Nitrogen source (ultrapure)
  • Benzyl mercaptan (Aldrich)
  • Thiophenol (Aldrich)
  • Polypeptide with N‐terminal Cys residue (see Support Protocols protocol 31 and protocol 42)
  • Polypeptide with α‐thioester functionality (see Support Protocols protocol 53 and protocol 64)
  • 1 M NaOH
  • Tris(2‐carboxyethyl)phosphine hydrochloride (TCEP, 99% pure; Strem Chemicals)
  • Buffers for analytical C18 reversed‐phase HPLC
    • Buffer A: H 2O containing 0.1% trifluoroacetic acid (TFA)
    • Buffer B: 1 part H 2O/9 parts acetonitrile/0.1% TFA
  • Appropriate final buffer in which target protein will be dissolved (e.g., for long‐term storage, activity studies, or structural studies), containing recipe6 M guanidine⋅HCl and 5 mM DTT
  • Appropriate final buffer in which target protein will be dissolved, containing 4 M, 2 M, and 0 M guanidine⋅HCl ( appendix 3A) and 1 mM DTT
  • Dialysis membrane with MWCO substantially smaller than target protein
  • Centricon filter (Amicon; optional)
  • Tabletop centrifuge (Sorvall RT‐7 or equivalent)
  • Additional reagents and equipment for analytical and semipreparative or preparative C18 reversed‐phase HPLC, electrospray ionization (ESI) or matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry (MS; units 16.1 & 16.2), dialysis (unit 4.4 & 3.NaN), and spectrophotometric determination of protein concentration (unit 3.1)

Basic Protocol 2: Sequential Chemical Ligation of Three Polypeptides

  Materials
  • Three polypeptide fragments to be ligated:
    • Polypeptide with N‐terminal Cys residue (see Support Protocols protocol 31 and protocol 42)
    • Polypeptide with Nα(Msc)‐Cys, α‐thioester functionality (see protocol 7)
    • Polypeptide with α‐thioester functionality (see Support Protocols protocol 53 and protocol 64)
  • 1 M HCl
  • Additional reagents and equipment for native chemical ligation of two polypeptides (see protocol 1)

Support Protocol 1: Chemical Synthesis of N‐Terminal CYS‐Polypeptides

  Materials
  • Methylbenzhydrylamine (MBHA) resin (Peptides International)
  • Dimethylformamide (DMF, spectrophotometric grade; Fisher)
  • 5% (v/v) diisopropylethylamine (DIEA, peptide synthesis grade; Perkin‐Elmer Applied Biosystems) in DMF (store up to 1 month at room temperature)
  • 97% 3‐bromopropionic acid
  • Dichloromethane (DCM, spectrophotometric grade; Fisher)
  • 99% diisopropyl carbodiimide (DIPC, 99%; Aldrich)
  • Diisopropylethylamine (DIEA, peptide synthesis grade; Perkin‐Elmer Applied Biosystems)
  • recipeAc 2O/DIEA/DMF solution (see recipe)
  • recipeAcSH/DIEA/DMF solution (see recipe)
  • recipeBME/DIEA/DMF solution (see recipe)
  • Boc–amino acyl–N‐hydroxysuccinimide ester (Boc‐AA‐OSu; where AA is the first amino acid to be incorporated into the polypeptide; Bachem)
  • Trifluoroacetic acid (TFA, BioGrade)
  • Ninhydrin test reagents: monitor 1, monitor 2, and monitor 3 (Perkin‐Elmer Applied Biosystems)
  • recipeHF/p‐cresol solution (see recipe)
  • Diethyl ether, cold
  • 10% to 50% acetonitrile in H 2O containing 0.1% TFA
  • 15‐ml manual peptide synthesis vessel (Peptides International)
  • Black rubber tubing (1/4 in. i.d. × 5/8 in. o.d. × 3/16 in. wall thickness; Fisher), resistant to acids and organic solvents
  • 2‐liter side‐arm flasks with rubber stoppers and glass tubing to fit
  • Pasteur pipet containing glass wool for filtration
  • 2‐ml polypropylene column (Microcolumn X from Isolab) with Teflon stopcock
  • 13 × 100–mm glass test tube
  • 110°C heating block
  • 60% ethanol
  • HF cleavage apparatus (Peptides International)
  • Additional material and equipment for solid‐phase peptide synthesis (Schnölzer et al., ; unit 18.1), preparative C18 reversed‐phase HPLC (see protocol 1, step ), and ESI‐MS (Chapter 1)

Support Protocol 2: Biosynthesis of N‐Terminal CYS Polypeptides

  Materials
  • Gene construct encoding target polypeptide
  • pTYB1 vector (New England Biolabs)
  • E. coli BL21 (or any other suitable E. coli strain)
  • LB medium containing 100 µg/ml ampicillin ( appendix 4A)
  • 1 M isopropyl‐1‐thio‐β‐D‐galactopyranoside (IPTG), filter sterilized
  • Chitin beads slurry: 50% (w/v) suspension of chitin beads (New England Biolabs) in 40% ethanol
  • recipeColumn buffer (see recipe)
  • recipeLysis buffer (see recipe)
  • recipeCleavage buffer (see recipe)
  • Refrigerated centrifuge with Beckman JA‐10 rotor and centrifuge buckets to hold 1 liter of culture
  • 1.5 × 10–cm glass or polypropylene column
  • Refrigerated centrifuge with Beckman JA‐17 rotor and appropriate centrifuge tubes
  • Shaker
  • Additional reagents and equipment for introducing plasmid vectors into bacterial cells ( appendix 4D), growth of bacteria in liquid medium ( appendix 4A), lysis of bacterial cells using a French press (unit 6.3), ESI‐MS (units 16.1 & 16.2)

Support Protocol 3: Chemical Synthesis of α‐Thioester Polypeptides

  • Fully protected Boc‐polypeptide‐3‐mercaptopropionamide‐MBHA resin (see protocol 5, step ), dried
  • (Methylsulfonyl)‐ethyl 4‐nitrophenyl carbonate (Msc‐ONp; Fluka)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Ausubel, F.A., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.). 1999. Current Protocols in Molecular Biology. John Wiley & Sons, New York.
   Bodanszky, M. and Bodanszky, A. 1994. The Practice of Peptide Synthesis, 2nd ed. Springer‐Verlag, Berlin.
   Camarero, J.A., Cotton, G.J., Adeva, A., and Muir, T.W. 1998. Chemical ligation of unprotected peptides directly from a solid support. J. Pept. Res. 51:303‐316.
   Canne, L.E., Ferre‐D'Amare, A.R., Burley, S.K., and Kent, S.B.H. 1995. Total chemical synthesis of a unique transcription factor‐related protein: cMyc‐Max. J. Am. Chem. Soc. 117:2998‐3007.
   Chong, S., Mersha, F.B., Comb, D.G., Scott, M.E., Landry, D., Vence, L.M., Perler, F.B., Benner, J., Kucera, R.B., Hirvonen, C.A., Pelletier, J.J., Paulus, H., and Xu, M.Q. 1997. Single‐column purification of free recombinant proteins using a self‐cleavable affinity tag derived from a protein splicing element. Gene 192:271‐281.
   Dawson, P.E., Muir, T.W., Clark‐Lewis, I., and Kent, S.B.H. 1994. Synthesis of proteins by native chemical ligation. Science 266:776‐779.
   Dawson, P.E., Churchill, M.J., Ghadiri, M.R., and Kent, S.B.H. 1997. Modulation in native chemical ligation through the use of thiol additives. J. Am. Chem. Soc. 119:4325‐4329.
   Erlandson, D.A., Chytill, M., and Verdine, G.L. 1996. The leucine zipper domain controls the orientation of AP‐1 in the NFAT AP‐1 DNA complex. Chem. Biol. 3(12):981‐991.
   Evans, T.C., Benner, J.J., and Xu, M.‐Q. 1998. Semisynthesis of cytotoxic proteins using a modified protein splicing element. Protein Sci. 7:2256‐2264.
   Hackeng, T.M., Mounier, C.M., Bon, C., Dawson, P.E., Griffin, J.H., and Kent, S.B. 1997. Total chemical synthesis of enzymatically active human type II secretory phospholipase A2. Proc. Natl. Acad. Sci. U.S.A. 94:7845‐7850.
   LLoyd‐Williams, P., Albericio, F., and Giralt, E. 1993. Convergent solid‐phase peptide synthesis. Tetrahedron 49:11065‐11133.
   Lu, W., Qasim, M.A., and Kent, S.B.H. 1996. Comparative total synthesis of turkey ovomucoid third domain by both stepwise solid phase synthesis and native chemical ligation. J. Am. Chem. Soc. 118:8518‐8523.
   Merrifield, R.B. 1963. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149‐2154.
   Muir, T.W. 1995. A chemical approach to the construction of multimeric protein assemblies. Structure 3:649‐652.
   Muir, T.W., Dawson, P.E., and Kent, S.B.H. 1997. Protein synthesis by chemical ligation of unprotected peptides in aqueous solution. Methods Enzymol. 289:266‐298.
   Muir, T.W., Sondhi, D., and Cole, P.A. 1998. Expressed protein ligation: A general method for protein engineering. Proc. Natl. Acad. Sci. U.S.A. 95:6705‐6710.
   Schnölzer, M., Alewood, P., Jones, A., Alewood, D., and Kent, S.B.H. 1992. In situ neutralization in Boc‐chemistry solid phase peptide synthesis: Rapid, high yield assembly of difficult sequences. Int. J. Pept. Protein Res. 40:180‐193.
   Severinov, K. and Muir, T.W. 1998. Expressed protein ligation: A novel method for studying protein‐protein interactions in transcription. J. Biol. Chem. 273:16205‐16209.
   Tesser, G.I. and Balvert‐Geers, I.C. 1975. The methylsulfonylethyloxycarbonyl group: A new and versatile amino protective function. Int. J. Pept. Prot. Res. 7:295‐305.
   Wallace, C.J.A. 1995. Peptide ligation and semisynthesis. Curr. Opin. Biotechnol. 6:403‐410.
   Xu, M.‐Q. and Perler, F.B. 1996. The mechanism of protein splicing and its modulation by mutation. EMBO J. 15:5146‐5153.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library