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

Abstract

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.