Amino Acids

, Volume 38, Issue 5, pp 1313–1322 | Cite as

In vivo biosynthesis of an Ala-scan library based on the cyclic peptide SFTI-1

  • Jeffrey Austin
  • Richard H. Kimura
  • Youn-Hi Woo
  • Julio A. Camarero
Original Article

Abstract

We present the in vivo biosynthesis of wild-type sunflower trypsin inhibitor 1 (SFTI-1) inside E. coli cells using an intramolecular native chemical ligation in combination with a modified protein splicing unit. SFTI-1 is a small backbone cyclized polypeptide with a single disulfide bridge. A small library containing multiple Ala mutants was also biosynthesized and its activity was assayed using a trypsin-binding assay. This study clearly demonstrates the exciting possibility of generating large cyclic peptide libraries in live E. coli cells, and is a critical first step for developing in vivo screening and directed evolution technologies using the cyclic peptide SFTI-1 as a molecular scaffold.

Keywords

Bowman–Birk inhibitor Trypsin inhibitor Backbone cyclized peptides Genetically encoded libraries Protein splicing 

Abbreviations

AMC

7-Amido-4-methyl-coumarin

CBD

Chitin-binding domain

Cbz

Benzyloxycarbonyl

CCK

Cyclic cystine-knot motif

BBI

Bowman–Birk inhibitor

EDTA

Ethylenediaminetetraacetic acid

EtSH

Ethanethiol

GdmCl

Guanidinium hydrochloride

GSH

Glutathione

HPLC

High performance liquid chromatography

LB

Luria–Bertani

MAP

Methionyl aminopeptidase

MESNA

Mercaptoethanesulfonic acid

NHS

N-hydroxysuccinimide ester

PAGE

Polyacrylamide gel electrophoresis

PMSF

Phenylmethylsulfonyl fluoride

SDS

Sodium dodecyl sulfate

SFTI-1

Sunflower trypsin inhibitor 1

TFA

Trifluoroacetic acid

UV-Vis

Ultraviolet-visible

Notes

Acknowledgments

Work was supported by funding from the School of Pharmacy at the University of Southern California and Lawrence Livermore National Laboratory.

Supplementary material

726_2009_338_MOESM1_ESM.doc (587 kb)
Supplementary material 1 (DOC 587 kb)

References

  1. Baird T, Wang B, Lodder M, Hecht S, Craik CS (2000) Generation of active trypsin by chemical cleavage. Tetrahedron 56:9477–9485CrossRefGoogle Scholar
  2. Camarero JA, Muir TW (1997) Chemoselective backbone cyclization of unprotected peptides. J Chem Soc Chem Commun 1997:1369–1370Google Scholar
  3. Camarero JA, Muir TW (1999a) Biosynthesis of a head-to-tail cyclized protein with improved biological activity. J Am Chem Soc 121:5597–5598CrossRefGoogle Scholar
  4. Camarero JA, Muir TW (1999b) Native chemical ligation of polypeptides. Curr Protoc Protein Sci 18(4):1–21Google Scholar
  5. Camarero JA, Cotton GJ, Adeva A, Muir TW (1998a) Chemical ligation of unprotected peptides directly form a solid support. J Pept Res 51:303–316PubMedGoogle Scholar
  6. Camarero JA, Pavel J, Muir TW (1998b) Chemical synthesis of a circular protein domain: evidence for folding-assisted cyclization. Angew Chem Int Ed 37:347–349CrossRefGoogle Scholar
  7. Camarero JA, Fushman D, Cowburn D, Muir TW (2001) Peptide chemical ligation inside living cells: in vivo generation of a circular protein domain. Bioorg Med Chem 9:2479–2484CrossRefPubMedGoogle Scholar
  8. Camarero JA, Kimura RH, Woo YH, Shekhtman A, Cantor J (2007) Biosynthesis of a fully functional cyclotide inside living bacterial cells. Chembiochem 8:1363–1366CrossRefPubMedGoogle Scholar
  9. Cheng Y, Prusoff WH (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22:3099–3108CrossRefPubMedGoogle Scholar
  10. Craik DJ, Daly NL, Bond T, Waine C (1999) Plant cyclotides: a unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif. J Mol Biol 294:1327–1336CrossRefPubMedGoogle Scholar
  11. Craik DJ, Simonsen S, Daly NL (2002) The cyclotides: novel macrocyclic peptides as scaffolds in drug design. Curr Opin Drug Discov Dev 5:251–260Google Scholar
  12. Craik DJ, Daly NL, Saska I, Trabi M, Rosengren KJ (2003) Structures of naturally occurring circular proteins from bacteria. J Bacteriol 185:4011–4021CrossRefPubMedGoogle Scholar
  13. Craik DJ, Cemazar M, Daly NL (2006a) The cyclotides and related macrocyclic peptides as scaffolds in drug design. Curr Opin Drug Discov Dev 9:251–260Google Scholar
  14. Craik DJ, Cemazar M, Wang CK, Daly NL (2006b) The cyclotide family of circular miniproteins: nature’s combinatorial peptide template. Biopolymers 84:250–266CrossRefPubMedGoogle Scholar
  15. Crovella S, Antcheva N, Zelezetsky I, Boniotto M, Pacor S, Verga Falzacappa MV, Tossi A (2005) Primate beta-defensins—structure, function and evolution. Curr Protein Pept Sci 6:7–21CrossRefPubMedGoogle Scholar
  16. Daly NL, Chen YK, Foley FM, Bansal PS, Bharathi R, Clark RJ, Sommerhoff CP, Craik DJ (2006) The absolute structural requirement for a proline in the P3′-position of Bowman–Birk protease inhibitors is surmounted in the minimized SFTI-1 scaffold. J Biol Chem 281:23668–23675CrossRefPubMedGoogle Scholar
  17. Descours A, Moehle K, Renard A, Robinson JA (2002) A new family of beta-hairpin mimetics based on a trypsin inhibitor from sunflower seeds. Chembiochem 3:318–323CrossRefPubMedGoogle Scholar
  18. Evans TC, Benner J, Xu M-Q (1998) Semisynthesis of cytotoxic proteins using a modified protein splicing element. Protein Sci 7:2256–2264CrossRefPubMedGoogle Scholar
  19. Evans TC Jr, Martin D, Kolly R, Panne D, Sun L, Ghosh I, Chen L, Benner J, Liu XQ, Xu MQ (2000) Protein trans-splicing and cyclization by a naturally split intein from the dnaE gene of Synechocystis species PCC6803. J Biol Chem 275:9091–9094CrossRefPubMedGoogle Scholar
  20. Hilpert K, Hansen G, Wessner H, Schneider-Mergener J, Hohne W (2000) Characterizing and optimizing protease/peptide inhibitor interactions, a new application for spot synthesis. J Biochem 128:1051–1057PubMedGoogle Scholar
  21. Hruby VJ, Al-Obeidi F (1990) Emerging approaches in the molecular design of receptor-selective peptide ligands: conformational, topographical and dynamic considerations. J Biochem 268:249–262Google Scholar
  22. Iwai H, Pluckthum A (1999) Circular β-lactamase: stability enhancement by cyclizing the backbone. FEBS Lett 459:166–172Google Scholar
  23. Jaulent AM, Leatherbarrow RJ (2004) Design, synthesis and analysis of novel bicyclic and bifunctional protease inhibitors. Protein Eng Des Sel 17:681–687CrossRefPubMedGoogle Scholar
  24. Kimura RH, Tran AT, Camarero JA (2006) Biosynthesis of the cyclotide kalata B1 by using protein splicing. Angew Chem Int Ed Engl 45:973–976CrossRefPubMedGoogle Scholar
  25. Kimura RH, Steenblock ER, Camarero JA (2007) Development of a cell-based fluorescence resonance energy transfer reporter for Bacillus anthracis lethal factor protease. Anal Biochem 369:60–70CrossRefPubMedGoogle Scholar
  26. Korsinczky ML, Schirra HJ, Rosengren KJ, West J, Condie BA, Otvos L, Anderson MA, Craik DJ (2001) Solution structures by 1H NMR of the novel cyclic trypsin inhibitor SFTI-1 from sunflower seeds and an acyclic permutant. J Mol Biol 311:579–591CrossRefPubMedGoogle Scholar
  27. Korsinczky ML, Schirra HJ, Craik DJ (2004) Sunflower trypsin inhibitor-1. Curr Protein Pept Sci 5:351–364CrossRefPubMedGoogle Scholar
  28. Korsinczky ML, Clark RJ, Craik DJ (2005) Disulfide bond mutagenesis and the structure and function of the head-to-tail macrocyclic trypsin inhibitor SFTI-1. Biochemistry 44:1145–1153CrossRefPubMedGoogle Scholar
  29. Luckett S, Garcia RS, Barker JJ, Konarev AV, Shewry PR, Clarke AR, Brady RL (1999) High-resolution structure of a potent, cyclic proteinase inhibitor from sunflower seeds. J Mol Biol 290:525–533CrossRefPubMedGoogle Scholar
  30. Marx UC, Korsinczky ML, Schirra HJ, Jones A, Condie B, Otvos L Jr, Craik DJ (2003) Enzymatic cyclization of a potent Bowman–Birk protease inhibitor, sunflower trypsin inhibitor-1, and solution structure of an acyclic precursor peptide. J Biol Chem 278:21782–21789CrossRefPubMedGoogle Scholar
  31. Muir TW, Sondhi D, Cole PA (1998) Expressed protein ligation: a general method for protein engineering. Proc Natl Acad Sci USA 95:6705–6710CrossRefPubMedGoogle Scholar
  32. Rizo J, Gierasch LM (1992) Constrained peptides: models of bioactive peptides and protein substructures. Annu Rev Biochem 61:387–418CrossRefPubMedGoogle Scholar
  33. Scott CP, Abel-Santos E, Wall M, Wahnon D, Benkovic SJ (1999) Production of cyclic peptides and proteins in vivo. Proc Natl Acad Sci USA 96:13638–13643CrossRefPubMedGoogle Scholar
  34. Severinov K, Muir TW (1998) Expressed protein ligation, a novel method for studying protein–protein interactions in transcription. J Biol Chem 273:16205–16209CrossRefPubMedGoogle Scholar
  35. Shao Y, Lu WY, Kent SBH (1998) A novel method to synthesize cyclic peptides. Tetrahedron Lett 39:3911–3914CrossRefGoogle Scholar
  36. Trabi M, Craik DJ (2002) Circular proteins—no end in sight. Trends Biochem Sci 27:132–138CrossRefPubMedGoogle Scholar
  37. Wu H, Hu Z, Liu XQ (1998) Protein trans-splicing by a split intein encoded in a split DnaE gene of Synechocystis sp. PCC6803. Proc Natl Acad Sci USA 95:9226–9231CrossRefPubMedGoogle Scholar
  38. Zhang L, Tam JP (1997) Synthesis and application of unprotected cyclic peptides as building blocks for peptide dendrimers. J Am Chem Soc 119:2363–2370CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Jeffrey Austin
    • 2
  • Richard H. Kimura
    • 2
  • Youn-Hi Woo
    • 2
  • Julio A. Camarero
    • 1
    • 2
  1. 1.Department of Pharmaceutical Sciences and Pharmacology, School of PharmacyUniversity of Southern CaliforniaLos AngelesUSA
  2. 2.Lawrence Livermore National LaboratoryLivermoreUSA

Personalised recommendations