Method for providing hyaluronic acid

Abstract

Disclosed are DNA segments encoding <DEL-S DATE="20010821" ID="DEL-S-00001">hyaluronic acid synthase which are employed to construct recombinant cells useful in the production of hyaluronate synthase or hyaluronic acid (HA)<DEL-E ID="DEL-S-00001"> <INS-S DATE="20010821" ID="INS-S-00001">the recombinant DNA segment identified in FIG. 5. <INS-E ID="INS-S-00001">In preferred aspects, chromosomal DNA from Streptococcus equisimilis is partially digested with EcoRI and the resultant fragments are ligated to form recombinant vectors. These vectors are useful in the transformation of host cells such as E. coli and or Streptococcal hosts. <DEL-S DATE="20010821" ID="DEL-S-00002">Resultant transformants are screened by the novel screening assays to identify colonies which have incorporated HA synthase DNA in a form that is being actively transcribed into the corresponding HA synthase enzyme. These colonies may be selected and employed in the production of the enzyme itself or its product, HA.<DEL-E ID="DEL-S-00002"> <INS-S DATE="20010821" ID="INS-S-00002">The recombinant DNA segment identified in FIG. 5 is then inserted into a recombinant Streptococcal host for the production of hyaluronic acid (HA).<INS-E ID="INS-S-00002">

Description

BACKGROUND OF THE INVENTION1. Field of the InventionThe present invention relates to .[.DNA encoding the enzyme hyaluronate synthase, and to the use of this DNA in the preparation of recombinant cells for the production of the hyaluronate synthase enzyme and / or hyaluronic acid.]. .Iadd.a method for providing hyaluronic acid..Iaddend.2. Description of the Related ArtThe incidence of Streptococcal infections is a major health and economic problem worldwide, particularly in the developing countries (21). One reason for this is due to the ability of Streptococcal bacteria to often grow undetected by the body's phagocytic cells (i.e., macrophages and polymorphonuclear cells (PMNs). These cells are responsible for recognizing and engulfing foreign microorganisms. One effective way by which the bacteria evade surveillance is by coating themselves with polysaccharide capsules, such as hyaluronic acid (HA) capsules. Since polysaccharides are generally nonimmunogenic, the encapsulated bacteri...

AI Technical Summary

Benefits of technology

A preferred technique for isolation of HA is described in U.S. Pat. No. 4,517,295 in which the organic carboxylic acid, trichloroacetic acid, is added to the bacterial suspension at the end of the fermentation. The trichloroacetic acid causes the bacterial cell to clump and die and facilitates the ease of separating these cells and associated debris from HA, the desired product. The clarified supernatant is concentrated and dialyzed to remove low molecular weight contaminants including the organic acid. The aforementioned procedure utilizes Millipore TM filtration through filter cassettes containing 0.22 mm pore size filters. Diafiltration is continued until the conductivity of the solution decreases to approximately 0.5 mega-ohms.

Problems solved by technology

The incidence of Streptococcal infections is a major health and economic problem worldwide, particularly in the developing countries (21).

This may be because in vitro systems developed to date are inadequate in that de novo biosynthesis of HA has not been accomplished.

Thus, totally ex vivo methods of producing HA have not been forthcoming.

It is generally felt that isolation of HA from rooster comb 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 10.sup.7, has been difficult to obtain by currently available isolation procedures.

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