Gene characterisation, isoforms and recombinant expression of carcinin, an antibacterial protein from the shore crab, Carcinus maenas

V Brockton, JA Hammond, Valerie Jane Smith

Research output: Contribution to journalArticlepeer-review

56 Citations (Scopus)

Abstract

Carcinin is a whey acidic protein (WAP) domain-containing antimicrobial protein produced by the circulating haemocytes of the shore crab, Carcinus maenas. Cloning of its full coding cDNA reveals that it shows some similarity to invertebrate defensins, has a valine-rich signal sequence followed by a defined cleavage site but no obvious acidic anionic 'pro' sequence. The C-terminus exhibits a unique cysteine array that is predicted to form six disulphide bonds in the tertiary structure. This 12 cysteine array arrangement is conserved in expressed sequence tags (ESTs) from related genera and seems to represent a novel tertiary structure amongst antimicrobial proteins (AMPs), unique to the Crustacea. There are at least five putative isoforms that arise through the transcription of a multi-exon gene. These isoforms do not arise as a result of alternate splicing of the exons, but by either the transcription of different alleles and/or single point mutation of the transcript at up to four loci in the gene. Several of the same transcripts have been found in different animals. The most commonly expressed transcript of the protein was recombinantly expressed in bacterial fusion system to a yield of ca. 2-3 mu g ml(-1) of culture. In vitro expression with or without the leader sequence confirms the bioinformatic prediction that the stability of the mature protein is reduced when the leader sequence is removed. Carcinin is one of very few invertebrate AMPs characterised at the gene, transcript and protein level and to be recombinantly expressed in vitro in a bacterial system. (c) 2006 Elsevier Ltd. All rights reserved.

Original languageEnglish
Pages (from-to)943-949
Number of pages7
JournalMolecular Immunology
Volume44
Issue number5
Early online date18 May 2006
DOIs
Publication statusPublished - Feb 2007

Keywords

  • Carcinin
  • Crustin
  • Crustacean
  • Carcinus maenas
  • Antibacterial protein
  • 11.5 kDa protein
  • WAP domain
  • Bacterial expression
  • Shrimp penaeus-monodon
  • Whey acidic protein
  • Antimicrobial peptides
  • Litopenaeus-vannamei
  • Crustin-like
  • Hemocytes
  • Hemocyanin
  • Invertebrates
  • Purification
  • Defensins

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