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

Through the application of techniques set forth herein, those of skill in the art will be enabled to obtain .Iadd.a recombinant .Iaddend.DNA .[.segments encoding all or a portion of the HA synthase gene.]. .Iadd.segment identified in FIG. 5.Iaddend.. Through isolation of.[.the HA gene.]. .Iadd.this DNA, .Iaddend.from whatever source is chosen, one will have the ability to realize significant advantages such as an ability to manipulate the host that is chosen to express the .[.HA synthase gene.]. .Iadd.recombinant DNA segment identified in FIG. 5, .Iaddend.the fermentation environment chosen for HA production, as well as genetic manipulation of the underlying gene. As those of skill in the art will recognize in light of the present disclosure, this will provide additional significant advantages both in the ability to control the expression of the gene and in the nature of the gene product that is realized.

In accordance with the present invention, .[.the HA synthase gene.]. .Iadd.recombinant DNA segment identified in FIG. 5, .Iaddend.when from a prokaryotic source such as a Streptococcal source, is obtained by the following general steps. First, genomic DNA is obtained from Streptococcal cells which are capable of producing the enzyme, such as S. equisimilis strain D181. This DNA is then partially digested with a selected restriction enzyme, such as EcoRI, to provide fragments having an average length that is compatible with the insert length allowed by the cloning vector that is subsequently employed. Due to the size of the .[.HA synthase enzyme.]. .Iadd.recombinant DNA segment identified in FIG. 5, .Iaddend.one will desire to obtain DNA that has an average length of up to 15 kb, and preferably about 5-10 kb. The use of the partial digestion technique is a significant factor in the practice of the invention in that it avoids the potential problem of an incomplete gene that will be realized if the selected enzyme gene has a restriction enzyme .[.site within the HA synthase coding region.]. .

Vectors such as these, exemplified by the pSA3 vector of Dao and Ferretti (4), allow one to perform clonal colony selection in an easily manipulated host such as E. coli, followed by subsequent transfer back into a Streptococcal strain for production of HA. This is advantageous in that one can augment the Streptococcal strain's inherent ability to synthesize HA through gene dosaging (i.e., providing extra copies of the .[.HA synthase gene.]. .Iadd.recombinant DNA segment identified in FIG. 5 .Iaddend.by amplification). The inherent ability of the streptococci to synthesize HA can also be augmented through the formation of extra copies, or amplification, of the plasmid that carries the .[.HA synthase gene.]. .Iadd.recombinant DNA segment identified in FIG. 5.Iaddend.. This amplification can account for up to a 10-fold increase in plasmid copy number and, therefore, the .[.HA synthase gene.]. .Iadd.recombinant DNA segment identified in FIG. 5 .Iaddend.copy number.

Once a positive clone is identified, and the presence of .[.putative HA synthase.]. .Iadd.the recombinant DNA segment identified in FIG. 5 .Iaddend.is confirmed, it will be desirable to subject the cloned insert to restriction enzyme mapping and DNA sequence analysis. This will both identify the amino acid sequence of the underlying .[.enzyme.]. .Iadd.DNA product.Iaddend., and provide the ability to further engineer the .[.HA synthase DNA.]. .Iadd.recombinant DNA segment identified in FIG. 5.Iaddend.. It is recognized that .[.HA synthase DNA.]. .Iadd.the recombinant DNA segment identified in FIG. 5 .Iaddend.may be engineered in a variety of manners and for many purposes. For example, it is possible to tailor the sequence by deletion of a transmembrane spanning domain that would normally insert the .[.enzyme.]. .Iadd.segment product .Iaddend.into the bacterial membrane. Removal of this region or regions from, and possibly addition of a leader signal sequence to .[.the HA synthase DNA.]. .Iadd.recombinant DNA segment identified in FIG. 5 .Iaddend.would then direct the completed enzyme to be secreted from the cell into the medium. Furthermore, manipulation and / or alteration of the underlying nucleotide sequences can be achieved to provide for a recombinant enzyme having improved kinetic attributes. Thus, in light of the present disclosure, once the mapping and sequence analysis is complete, those of skill in the art will have the ability to reengineer the gene into desired expression constructs, including positioning the gene to bring it under the control of whatever promoter system that is desired.

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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