Patent ID: 8088604

Claim:
A method for enzymatically producing defined glycosaminoglycan polymers comprising the steps of: providing at least one functional acceptor, wherein the functional acceptor comprises at least two sugar units, and wherein at least one of the at least two sugar units is selected from the group consisting of uronic acid, a uronic acid analog comprising a substitution at at least one of the C2 and C3 positions thereof, a hexosamine and a hexosamine analog comprising a substitution at at least one of the C2 and C6 positions thereof; providing a recombinant heparosan synthase having an empty acceptor site and being capable of elongating the at least one functional acceptor in a controlled and/or repetitive fashion to form extended glycosaminoglycan molecules, and wherein the recombinant heparosan synthase is selected from the group consisting of: (a) a recombinant heparosan synthase having an amino acid sequence as set forth in one of SEQ ID NOS: 6, 8, 66, 70 and 71; (b) a recombinant heparosan synthase encoded by the nucleotide sequence of one of SEQ ID NOS: 5, 7 and 65; (c) a recombinant heparosan synthase encoded by a nucleotide sequence that is at least 90% identical to at least one of SEQ ID NOS: 5, 7 and 65; (d) a recombinant heparosan synthase encoded by a nucleic acid capable of hybridizing to the complement of the polynucleotide of at least one of SEQ ID NOS: 5, 7 and 65 under hybridization conditions comprising hybridization at a temperature of 68° C. in 5×SSC/5× Denhardt's solution/1.0% SDS, followed with washing in 3×SSC at 42° C.; (e) a recombinant heparosan synthase encoded by a nucleic acid capable of hybridizing to the complement of a polynucleotide encoding an amino acid sequence as set forth in at least one of SEQ ID NOS: 6, 8, 66, 70 and 71 under hybridization conditions comprising hybridization at a temperature of 68° C. in 5×SSC/5× Denhardt's solution/1.0% SDS, followed with washing in 3×SSC at 42° C.; and providing at least one of a UDP-sugar and a UDP-sugar analog in a stoichiometric ratio to the at least one functional acceptor such that the recombinant heparosan synthase elongates the at least one functional acceptor to provide glycosaminoglycan polymers wherein the glycosaminoglycan polymers have a desired size distribution such that the glycosaminoglycan polymers are substantially monodisperse in size such that the glycosaminoglycan polymers have a polydispersity value in a range of from 1.0 to 1.5, and wherein the desired size distribution is obtained by controlling the stoichiometric ratio of UDP-sugar to functional acceptor.