Generation of active trypsin by chemical cleavage

Teaster Baird, Bixun Wang, Michiel Lodder, Sidney Hecht, Charles S. Craik

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

A novel strategy has been developed to generate trypsin from trypsinogen without proteolytic processing. The unnatural amino acid allylglycine was incorporated into a trypsinogen variant to permit cleavage specifically at Ile-16, the prototypical activation site, and generate the active protease, trypsin. A suppressor tRNA, activated with allylglycine, was used to synthesize the allylglycine trypsinogen variant by in vitro translation of an mRNA transcript that placed the UAG stop codon immediately prior to the authentic trypsin coding region. Iodine treatment of the variant trypsinogen resulted in trypsin that was fully active and kinetically indistinguishable from wild type recombinant trypsin. Using single substrates, the iodine treated variant exhibited a catalytic profile essentially identical to that of wild type recombinant rat trypsin (k(cat)/k(M)=3.7±0.5 vs 3.7±0.8 μM-1 s-1, respectively). Furthermore, the specificity profiles generated from substrate combinatorial libraries are indistinguishable. These results illustrate that (1) iodine activated allylglycine trypsinogen is virtually identical to enteropeptidase activated recombinant trypsinogen and (2) this novel technique is a feasible alternative to achieve site specific cleavage of serine protease zymogens. (C) 2000 Elsevier Science Ltd.

Original languageEnglish (US)
Pages (from-to)9477-9485
Number of pages9
JournalTetrahedron
Volume56
Issue number48
StatePublished - Nov 24 2000
Externally publishedYes

Fingerprint

Trypsinogen
Allylglycine
Trypsin
Iodine
Enteropeptidase
Enzyme Precursors
Terminator Codon
Protein Biosynthesis
Serine Proteases
Substrates
Transfer RNA
Rats
Catalytic Domain
Peptide Hydrolases
Chemical activation
Amino Acids
Messenger RNA
Processing

Keywords

  • Hydrolysis
  • Protease
  • Unnatural amino acids
  • Zymogen activation

ASJC Scopus subject areas

  • Biochemistry
  • Organic Chemistry
  • Drug Discovery

Cite this

Baird, T., Wang, B., Lodder, M., Hecht, S., & Craik, C. S. (2000). Generation of active trypsin by chemical cleavage. Tetrahedron, 56(48), 9477-9485.

Generation of active trypsin by chemical cleavage. / Baird, Teaster; Wang, Bixun; Lodder, Michiel; Hecht, Sidney; Craik, Charles S.

In: Tetrahedron, Vol. 56, No. 48, 24.11.2000, p. 9477-9485.

Research output: Contribution to journalArticle

Baird, T, Wang, B, Lodder, M, Hecht, S & Craik, CS 2000, 'Generation of active trypsin by chemical cleavage', Tetrahedron, vol. 56, no. 48, pp. 9477-9485.
Baird T, Wang B, Lodder M, Hecht S, Craik CS. Generation of active trypsin by chemical cleavage. Tetrahedron. 2000 Nov 24;56(48):9477-9485.
Baird, Teaster ; Wang, Bixun ; Lodder, Michiel ; Hecht, Sidney ; Craik, Charles S. / Generation of active trypsin by chemical cleavage. In: Tetrahedron. 2000 ; Vol. 56, No. 48. pp. 9477-9485.
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