This Article Full Text (PDF) References Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Ding, J.L. Articles by Ho, B. Search for Related Content PubMed Articles by Ding, J.L. Articles by Ho, B. Social Bookmarking                   What's this?

Expression of full length and deletion homologues of Carcinoscorpius rotundicauda Factor C in Saccharomyces cerevisiae: immunoreactivity and endotoxin binding

Marine Biotechnology Laboratory and BioScience Centre, sbsdjl{at}leonis.nus.sg

C. Chai

Department of Microbiology, School of Biological Sciences National University of Singapore, Singapore

A.W.M. Pui

Marine Biotechnology Laboratory and BioScience Centre

B. Ho

Department of Microbiology, School of Biological Sciences National University of Singapore, Singapore

Deletion homologues of the cloned Factor C cDNAs from the horseshoe crab Carcinoscorpius rotundicauda were engineered to express in Saccharomyces cerevisiae under the regulation of a galactose-inducible promoter. Expression cassettes were constructed in the vectors: pEMBLyex4 and YEpsec1 to direct, respectively, the intracellular expression, and the secretion of the protein into the culture medium using a heterologous signal sequence. The effect of insert size on the efficiency of expression and the functionality of the resulting recombinant Factor C (rFC) were studied by creating expression constructs bearing various deletion and/or hybrid fragments of Factor C. Removal of the long 5' UTR from the Factor C cDNA improved expression of the rFC. 3' Deletions of up to 84%, or internal deletions of 65% of the Factor C cDNA resulted in either the lack of detectable amounts of Factor C or loss of immunoreactivity. Depending on the construct, full length or partial rFC-related proteins were correspondingly expressed intracellularly, regardless of the vector. The rFC partitioned with the insoluble cell fraction, was solubilised with either SDS or Triton X-100, and found to be immunoreactive. The rFCs were functionally active, being able to bind Gram-negative bacterial endotoxin, provided critical regions of the endotoxin-binding domain were preserved.

Journal of Endotoxin Research, Vol. 4, No. 1, 33-43 (1997)
DOI: 10.1177/096805199700400105


CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				Y. H. Yau, B. Ho, N. S. Tan, M. L. Ng, and J. L. Ding High Therapeutic Index of Factor C Sushi Peptides: Potent Antimicrobials against Pseudomonas aeruginosa Antimicrob. Agents Chemother., October 1, 2001; 45(10): 2820 - 2825. [Abstract] [Full Text]