Recombinant production and structural studies of the Aplysia water-borne protein pheromone enticin indicates it has a novel disulfide stabilized fold

Scott F. Cummins, Fang Xie, Milind Misra, Andinet Amare, Jennifer A. Jakubowski, Melissa R. de Vries, Jonathan V. Sweedler, Gregg Thomas Nagle, Catherine H. Schein

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Enticin is one of three Aplysia proteins released during egg laying that act in concert with the pheromone attractin to attract other Aplysia and stimulate mating behavior. Whereas the enticin cDNA predicts a 69-residue mature protein, enticin isolated from the albumen gland was found to be posttranslationally processed in vivo by cleavage at Arg 50 residue to generate a smaller 49-residue mature peptide. The Arg 50 cleavage site is conserved in enticin from both Aplysia californica and Aplysia brasiliana. In order to generate sufficient enticin for structural studies, recombinant full-length protein was produced in a soluble form in Escherichia coli using a cold shock promoter-based protein expression system. The enticin cDNA was cloned into the bacterial vector pCold III, and efficiently expressed, as determined by amino acid microsequence and immunoblot analyses. Recombinant enticin, which contained an additional N-terminal 13-residue translation-enhancing element, was purified by reversed-phase HPLC and compared to enticin isolated from the albumen gland. The three disulfide bonds in enticin were characterized by endoproteinase Glu-C proteolysis followed by mass spectrometric characterization of the fragments. The cysteine pairing, for both recombinant and native enticin, was I-II, III-IV, and V-VI, confirming that the protein produced in the bacterial system was correctly folded. The circular dichroism spectrum of the recombinant protein indicated it was predominantly α-helical. While this was consistent with fold recognition server results indicating a fold for enticin similar to that of attractin, the disulfide bonding pattern differs. A model for enticin was prepared based on its helical structure and these disulfide constraints.

Original languageEnglish (US)
Pages (from-to)94-102
Number of pages9
JournalPeptides
Volume28
Issue number1
DOIs
StatePublished - Jan 1 2007
Externally publishedYes

Fingerprint

Aplysia
Pheromones
Disulfides
Water
Proteins
Complementary DNA
Proteolysis
Circular Dichroism
Recombinant Proteins
Escherichia coli
Ovum
Cysteine
Shock
Servers
High Pressure Liquid Chromatography
Amino Acids
Peptides

Keywords

  • Aplysia
  • Attractin
  • Enticin
  • Pheromone
  • Temptin
  • pCold III

ASJC Scopus subject areas

  • Biochemistry
  • Physiology
  • Endocrinology
  • Cellular and Molecular Neuroscience

Cite this

Recombinant production and structural studies of the Aplysia water-borne protein pheromone enticin indicates it has a novel disulfide stabilized fold. / Cummins, Scott F.; Xie, Fang; Misra, Milind; Amare, Andinet; Jakubowski, Jennifer A.; de Vries, Melissa R.; Sweedler, Jonathan V.; Nagle, Gregg Thomas; Schein, Catherine H.

In: Peptides, Vol. 28, No. 1, 01.01.2007, p. 94-102.

Research output: Contribution to journalArticle

Cummins, Scott F. ; Xie, Fang ; Misra, Milind ; Amare, Andinet ; Jakubowski, Jennifer A. ; de Vries, Melissa R. ; Sweedler, Jonathan V. ; Nagle, Gregg Thomas ; Schein, Catherine H. / Recombinant production and structural studies of the Aplysia water-borne protein pheromone enticin indicates it has a novel disulfide stabilized fold. In: Peptides. 2007 ; Vol. 28, No. 1. pp. 94-102.
@article{e04f1986d7ef4d428e1e7347aa6af420,
title = "Recombinant production and structural studies of the Aplysia water-borne protein pheromone enticin indicates it has a novel disulfide stabilized fold",
abstract = "Enticin is one of three Aplysia proteins released during egg laying that act in concert with the pheromone attractin to attract other Aplysia and stimulate mating behavior. Whereas the enticin cDNA predicts a 69-residue mature protein, enticin isolated from the albumen gland was found to be posttranslationally processed in vivo by cleavage at Arg 50 residue to generate a smaller 49-residue mature peptide. The Arg 50 cleavage site is conserved in enticin from both Aplysia californica and Aplysia brasiliana. In order to generate sufficient enticin for structural studies, recombinant full-length protein was produced in a soluble form in Escherichia coli using a cold shock promoter-based protein expression system. The enticin cDNA was cloned into the bacterial vector pCold III, and efficiently expressed, as determined by amino acid microsequence and immunoblot analyses. Recombinant enticin, which contained an additional N-terminal 13-residue translation-enhancing element, was purified by reversed-phase HPLC and compared to enticin isolated from the albumen gland. The three disulfide bonds in enticin were characterized by endoproteinase Glu-C proteolysis followed by mass spectrometric characterization of the fragments. The cysteine pairing, for both recombinant and native enticin, was I-II, III-IV, and V-VI, confirming that the protein produced in the bacterial system was correctly folded. The circular dichroism spectrum of the recombinant protein indicated it was predominantly α-helical. While this was consistent with fold recognition server results indicating a fold for enticin similar to that of attractin, the disulfide bonding pattern differs. A model for enticin was prepared based on its helical structure and these disulfide constraints.",
keywords = "Aplysia, Attractin, Enticin, Pheromone, Temptin, pCold III",
author = "Cummins, {Scott F.} and Fang Xie and Milind Misra and Andinet Amare and Jakubowski, {Jennifer A.} and {de Vries}, {Melissa R.} and Sweedler, {Jonathan V.} and Nagle, {Gregg Thomas} and Schein, {Catherine H.}",
year = "2007",
month = "1",
day = "1",
doi = "10.1016/j.peptides.2006.08.027",
language = "English (US)",
volume = "28",
pages = "94--102",
journal = "Peptides",
issn = "0196-9781",
publisher = "Elsevier Inc.",
number = "1",

}

TY - JOUR

T1 - Recombinant production and structural studies of the Aplysia water-borne protein pheromone enticin indicates it has a novel disulfide stabilized fold

AU - Cummins, Scott F.

AU - Xie, Fang

AU - Misra, Milind

AU - Amare, Andinet

AU - Jakubowski, Jennifer A.

AU - de Vries, Melissa R.

AU - Sweedler, Jonathan V.

AU - Nagle, Gregg Thomas

AU - Schein, Catherine H.

PY - 2007/1/1

Y1 - 2007/1/1

N2 - Enticin is one of three Aplysia proteins released during egg laying that act in concert with the pheromone attractin to attract other Aplysia and stimulate mating behavior. Whereas the enticin cDNA predicts a 69-residue mature protein, enticin isolated from the albumen gland was found to be posttranslationally processed in vivo by cleavage at Arg 50 residue to generate a smaller 49-residue mature peptide. The Arg 50 cleavage site is conserved in enticin from both Aplysia californica and Aplysia brasiliana. In order to generate sufficient enticin for structural studies, recombinant full-length protein was produced in a soluble form in Escherichia coli using a cold shock promoter-based protein expression system. The enticin cDNA was cloned into the bacterial vector pCold III, and efficiently expressed, as determined by amino acid microsequence and immunoblot analyses. Recombinant enticin, which contained an additional N-terminal 13-residue translation-enhancing element, was purified by reversed-phase HPLC and compared to enticin isolated from the albumen gland. The three disulfide bonds in enticin were characterized by endoproteinase Glu-C proteolysis followed by mass spectrometric characterization of the fragments. The cysteine pairing, for both recombinant and native enticin, was I-II, III-IV, and V-VI, confirming that the protein produced in the bacterial system was correctly folded. The circular dichroism spectrum of the recombinant protein indicated it was predominantly α-helical. While this was consistent with fold recognition server results indicating a fold for enticin similar to that of attractin, the disulfide bonding pattern differs. A model for enticin was prepared based on its helical structure and these disulfide constraints.

AB - Enticin is one of three Aplysia proteins released during egg laying that act in concert with the pheromone attractin to attract other Aplysia and stimulate mating behavior. Whereas the enticin cDNA predicts a 69-residue mature protein, enticin isolated from the albumen gland was found to be posttranslationally processed in vivo by cleavage at Arg 50 residue to generate a smaller 49-residue mature peptide. The Arg 50 cleavage site is conserved in enticin from both Aplysia californica and Aplysia brasiliana. In order to generate sufficient enticin for structural studies, recombinant full-length protein was produced in a soluble form in Escherichia coli using a cold shock promoter-based protein expression system. The enticin cDNA was cloned into the bacterial vector pCold III, and efficiently expressed, as determined by amino acid microsequence and immunoblot analyses. Recombinant enticin, which contained an additional N-terminal 13-residue translation-enhancing element, was purified by reversed-phase HPLC and compared to enticin isolated from the albumen gland. The three disulfide bonds in enticin were characterized by endoproteinase Glu-C proteolysis followed by mass spectrometric characterization of the fragments. The cysteine pairing, for both recombinant and native enticin, was I-II, III-IV, and V-VI, confirming that the protein produced in the bacterial system was correctly folded. The circular dichroism spectrum of the recombinant protein indicated it was predominantly α-helical. While this was consistent with fold recognition server results indicating a fold for enticin similar to that of attractin, the disulfide bonding pattern differs. A model for enticin was prepared based on its helical structure and these disulfide constraints.

KW - Aplysia

KW - Attractin

KW - Enticin

KW - Pheromone

KW - Temptin

KW - pCold III

UR - http://www.scopus.com/inward/record.url?scp=33845600781&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33845600781&partnerID=8YFLogxK

U2 - 10.1016/j.peptides.2006.08.027

DO - 10.1016/j.peptides.2006.08.027

M3 - Article

VL - 28

SP - 94

EP - 102

JO - Peptides

JF - Peptides

SN - 0196-9781

IS - 1

ER -