Hyaluronan synthases and methods of making and using same

Abstract

A functionally active hyaluronan synthase having at least one modified amino acid residue therein as compared to a corresponding functionally active native hyaluronan synthase such that the functionally active hyaluronan synthase has an altered enzymatic activity as compared to the corresponding functionally active native hyaluronan synthase is disclosed. Methods of producing hyaluronic acid utilizing a recombinant host cell having an expression construct encoding the functionally active hyaluronan synthase with altered enzymatic activity are also disclosed.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. Ser. No. 10 / 309,560, filed Dec. 3, 2002; which claims benefit under 35 U.S.C. 119(e) of U.S. Ser. No. 60 / 336,105, filed Dec. 3, 2001; the contents of which are hereby expressly incorporated herein by reference. [0002] Said U.S. Ser. No. 10 / 309,560 is also a continuation-in-part of U.S. Ser. No. 10 / 011,771, filed Dec. 11, 2001; the contents of which are hereby expressly incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0003] This application was supported in part by a grant from the National Institutes of Health (GM35978). The United States Government may have rights in and to this application by virtue of this funding.BACKGROUND OF THE INVENTION [0004] 1. Field of the Invention [0005] The present invention relates to nucleic acid segments having coding regions encoding enzymatically active hyaluronate synthase (HAS), and to the use of these nuclei...

AI Technical Summary

Problems solved by technology

The incidence of streptococcal infections is a major health and economic problem worldwide, particularly in developing countries.

The extrusion of the growing chain into the extracellular space also allows for unconstrained polymer growth, thereby achieving the exceptionally large size of HA, whereas confinement of synthesis within a Golgi or post-Golgi compartment limits the overall amount or length of the polymers formed.

High concentrations of HA within a confined lumen may also create a high viscosity environment that might be deleterious for other organelle functions.

Although the streptococcal and murine oligodendroglioma enzymes were successfully detergent-solubilized and studied, efforts to purify an active HAS for further study or molecular cloning remained unsuccessful for decades.

This led to a report claiming that the Group C streptococcal HAS had been cloned, which was unfortunately erroneous.

Despite these efforts, progress in understanding the regulation and mechanisms of HA synthesis was essentially stalled, since there were no molecular probes for HAS mRNA or HAS protein.

Unfortunately, several studies have employed antibodies to this uncharacterized 52-kDa streptococcal protein to investigate what was believed to be eukaryotic HAS.

It is generally felt that isolation of HA from rooster combs is laborious and difficult, since one starts with HA in a less pure state.

Unfortunately, very high molecular weight HA, such as that ranging up to 107, has been difficult to obtain by currently available isolation procedures.

However, to date the involvement of one or more of these conserved Cys residues in enzyme activity or disulfide bond formation has not been determined.

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