The goal of this grant application is to isolate and clone the sulfatases of heparin-like glycosaminoglycans. More specifically, during the phase I part of the grant application, the investigators will focus on the 2-O sulfatase as a model enzyme, which will enable the isolation of other sulfatases during Phase II. The specific aims of this phase I project are: 1) to isolate and purify the 2-O sulfatase from Flavobacterium heparinum, and 2) clone and recombinantly express 2-O sulfatase in E. coli. The cloning of the sulfatases is expected to a) provide a methodology that will facilitate the isolation of the other heparin-like GAG sulfatases (i.e. 3-O, 6-) and the NS degrading enzymes), b) enable the development of an integrated enzyme based approach towards heparin-like GAG structure-function investigation and sequencing, and c) enable the commercial availability of the sulfatases for applications beyond heparin-like GAG sequencing, such as in the clinic.
