Journal article
Recombinant production and structural studies of the Aplysia water-borne protein pheromone enticin indicates it has a novel disulfide stabilized fold
Peptides, Vol.28(1), pp.94-102
2007
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 Arg50 residue to generate a smaller 49-residue mature peptide. The Arg50 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.
Details
- Title
- Recombinant production and structural studies of the Aplysia water-borne protein pheromone enticin indicates it has a novel disulfide stabilized fold
- Authors
- Scott F Cummins (Author) - University of Texas, United StatesF Xie (Author) - University of Illinois, United StatesM Misra (Author) - University of Texas, United StatesA Amare (Author) - University of Illinois, United StatesJ A Jakubowski (Author) - University of Illinois, United StatesMelissa R De Vries (Author) - University of Texas, United StatesJ V Sweedler (Author) - University of Illinois, United StatesG T Nagle (Author) - University of Texas, United StatesC H Schein (Author) - University of Texas, United States
- Publication details
- Peptides, Vol.28(1), pp.94-102
- Publisher
- Elsevier Inc.
- DOI
- 10.1016/j.peptides.2006.08.027
- ISSN
- 0196-9781
- Organisation Unit
- School of Science and Engineering - Legacy; School of Science, Technology and Engineering; Centre for Bioinnovation; University of the Sunshine Coast, Queensland
- Language
- English
- Record Identifier
- 99449649502621
- Output Type
- Journal article
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