Expression of the Pasteurella haemolytica O-sialoglycoprotein endopeptidase as a GST fusion protein

by Beasley, Steven Ari

The Pasteurella haemolytica O-siaioglycoprotein endopeptidase specificdly cleaves cell-membrane glycoproteins that bear negatively charged clusters such as sialo- and sulfoglycans. This thesis describes the heterologous expression in coli of the gcp gene, in fusion with ihe gene for glutathione-StraflSferase (GST) of Schistosoma japotziaun. The GST fusion protein product, rGgcp, was purified by affin@ chromatography on a glutathione-Sepharose column, and thrombin cleavage of rGgcp Liberated the rGcp moiety. The purified recombinant protein was used as a substrate for in vitro refolding by the molecular chaperones PD1 and DnaWDnal/GrpE under a variety of expenrnentai conditions. Glycoprotease enzyme activity was not reproducibly generated in any of the rGgcp samples testeci. A novel shuttle vector, pNF2176, was used for the expression of rGcp and rGgcp in P. haernoIpku serotypes Al and Al 1. The glycoprotease antigen was expressed in P. haemolyfica serotypes Al and Al 1, but there was no increase in glycoprotease activity in either serotype associated with the gene expression. in contrast with previous reports, serotype AI l was found to constitutively express an enzymatically active glycoprotease.