Identification and recombinant expression of a novel chymotrypsin from Spodoptera exigua

S. Herrero, E. Combes, M.M. van Oers, J.M. Vlak, R.A. de Maagd, M.J. Beekwilder

Research output: Contribution to journalArticleAcademicpeer-review

37 Citations (Scopus)

Abstract

A novel chymotrypsin which is expressed in the midgut of the lepidopteran insect Spodoptera exigua is described. This enzyme, referred to as SeCT34, represents a novel class of chymotrypsins. Its amino-acid sequence shares common features of gut chymotrpysins, but can be clearly distinguished from other serine proteinases that are expressed in the insect gut. Most notable, SeCT34 contains a chymotrypsin activation site and the highly conserved motive DSGGP in the catalytic domain around the active-site serine is changed to DSGSA. Recombinant expression of SeCT34 was achieved in Sf21 insect cells using a special baculovirus vector, which has been engineered for optimized protein production. This is the first example of recombinant expression of an active serine proteinase which functions in the lepidopteran digestive tract. Purified recombinant SeCT34 enzyme was characterized by its ability to hydrolyze various synthetic substrates and its susceptibility to proteinase inhibitors. It appeared to be highly selective for substrates carrying a phenylalanine residue at the cleavage site. SeCT34 showed a pH-dependence and sensitivity to inhibitors, which is characteristic for semi-purified lepidopteran gut proteinases. Expression analysis revealed that SeCT34 was only expressed in the midgut of larvae at the end of their last instar, just before the onset of pupation. This suggests a possible role of this protein in the proteolytic remodelling that occurs in the gut during the larval to pupal molt.
Original languageEnglish
Pages (from-to)1073-1082
JournalInsect Biochemistry and Molecular Biology
Volume35
Issue number10
DOIs
Publication statusPublished - 2005

Fingerprint

Spodoptera
Spodoptera exigua
Chymotrypsin
chymotrypsin
Insects
digestive system
Serine Proteases
Lepidoptera
Catalytic Domain
serine proteinases
Peptide Hydrolases
active sites
midgut
insects
Sf9 Cells
Baculoviridae
Substrates
Enzymes
Phenylalanine
Serine

Keywords

  • bacillus-thuringiensis toxins
  • helicoverpa-zea
  • proteinase-inhibitors
  • trypsin-inhibitor
  • agrotis-ipsilon
  • manduca-sexta
  • midgut
  • proteases
  • sequence
  • larvae

Cite this

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title = "Identification and recombinant expression of a novel chymotrypsin from Spodoptera exigua",
abstract = "A novel chymotrypsin which is expressed in the midgut of the lepidopteran insect Spodoptera exigua is described. This enzyme, referred to as SeCT34, represents a novel class of chymotrypsins. Its amino-acid sequence shares common features of gut chymotrpysins, but can be clearly distinguished from other serine proteinases that are expressed in the insect gut. Most notable, SeCT34 contains a chymotrypsin activation site and the highly conserved motive DSGGP in the catalytic domain around the active-site serine is changed to DSGSA. Recombinant expression of SeCT34 was achieved in Sf21 insect cells using a special baculovirus vector, which has been engineered for optimized protein production. This is the first example of recombinant expression of an active serine proteinase which functions in the lepidopteran digestive tract. Purified recombinant SeCT34 enzyme was characterized by its ability to hydrolyze various synthetic substrates and its susceptibility to proteinase inhibitors. It appeared to be highly selective for substrates carrying a phenylalanine residue at the cleavage site. SeCT34 showed a pH-dependence and sensitivity to inhibitors, which is characteristic for semi-purified lepidopteran gut proteinases. Expression analysis revealed that SeCT34 was only expressed in the midgut of larvae at the end of their last instar, just before the onset of pupation. This suggests a possible role of this protein in the proteolytic remodelling that occurs in the gut during the larval to pupal molt.",
keywords = "bacillus-thuringiensis toxins, helicoverpa-zea, proteinase-inhibitors, trypsin-inhibitor, agrotis-ipsilon, manduca-sexta, midgut, proteases, sequence, larvae",
author = "S. Herrero and E. Combes and {van Oers}, M.M. and J.M. Vlak and {de Maagd}, R.A. and M.J. Beekwilder",
year = "2005",
doi = "10.1016/j.ibmb.2005.05.006",
language = "English",
volume = "35",
pages = "1073--1082",
journal = "Insect Biochemistry and Molecular Biology",
issn = "0965-1748",
publisher = "Elsevier",
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}

Identification and recombinant expression of a novel chymotrypsin from Spodoptera exigua. / Herrero, S.; Combes, E.; van Oers, M.M.; Vlak, J.M.; de Maagd, R.A.; Beekwilder, M.J.

In: Insect Biochemistry and Molecular Biology, Vol. 35, No. 10, 2005, p. 1073-1082.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Identification and recombinant expression of a novel chymotrypsin from Spodoptera exigua

AU - Herrero, S.

AU - Combes, E.

AU - van Oers, M.M.

AU - Vlak, J.M.

AU - de Maagd, R.A.

AU - Beekwilder, M.J.

PY - 2005

Y1 - 2005

N2 - A novel chymotrypsin which is expressed in the midgut of the lepidopteran insect Spodoptera exigua is described. This enzyme, referred to as SeCT34, represents a novel class of chymotrypsins. Its amino-acid sequence shares common features of gut chymotrpysins, but can be clearly distinguished from other serine proteinases that are expressed in the insect gut. Most notable, SeCT34 contains a chymotrypsin activation site and the highly conserved motive DSGGP in the catalytic domain around the active-site serine is changed to DSGSA. Recombinant expression of SeCT34 was achieved in Sf21 insect cells using a special baculovirus vector, which has been engineered for optimized protein production. This is the first example of recombinant expression of an active serine proteinase which functions in the lepidopteran digestive tract. Purified recombinant SeCT34 enzyme was characterized by its ability to hydrolyze various synthetic substrates and its susceptibility to proteinase inhibitors. It appeared to be highly selective for substrates carrying a phenylalanine residue at the cleavage site. SeCT34 showed a pH-dependence and sensitivity to inhibitors, which is characteristic for semi-purified lepidopteran gut proteinases. Expression analysis revealed that SeCT34 was only expressed in the midgut of larvae at the end of their last instar, just before the onset of pupation. This suggests a possible role of this protein in the proteolytic remodelling that occurs in the gut during the larval to pupal molt.

AB - A novel chymotrypsin which is expressed in the midgut of the lepidopteran insect Spodoptera exigua is described. This enzyme, referred to as SeCT34, represents a novel class of chymotrypsins. Its amino-acid sequence shares common features of gut chymotrpysins, but can be clearly distinguished from other serine proteinases that are expressed in the insect gut. Most notable, SeCT34 contains a chymotrypsin activation site and the highly conserved motive DSGGP in the catalytic domain around the active-site serine is changed to DSGSA. Recombinant expression of SeCT34 was achieved in Sf21 insect cells using a special baculovirus vector, which has been engineered for optimized protein production. This is the first example of recombinant expression of an active serine proteinase which functions in the lepidopteran digestive tract. Purified recombinant SeCT34 enzyme was characterized by its ability to hydrolyze various synthetic substrates and its susceptibility to proteinase inhibitors. It appeared to be highly selective for substrates carrying a phenylalanine residue at the cleavage site. SeCT34 showed a pH-dependence and sensitivity to inhibitors, which is characteristic for semi-purified lepidopteran gut proteinases. Expression analysis revealed that SeCT34 was only expressed in the midgut of larvae at the end of their last instar, just before the onset of pupation. This suggests a possible role of this protein in the proteolytic remodelling that occurs in the gut during the larval to pupal molt.

KW - bacillus-thuringiensis toxins

KW - helicoverpa-zea

KW - proteinase-inhibitors

KW - trypsin-inhibitor

KW - agrotis-ipsilon

KW - manduca-sexta

KW - midgut

KW - proteases

KW - sequence

KW - larvae

U2 - 10.1016/j.ibmb.2005.05.006

DO - 10.1016/j.ibmb.2005.05.006

M3 - Article

VL - 35

SP - 1073

EP - 1082

JO - Insect Biochemistry and Molecular Biology

JF - Insect Biochemistry and Molecular Biology

SN - 0965-1748

IS - 10

ER -