Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1999-01-05
2001-07-03
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S069100, C435S320100, C530S300000, C530S326000, C536S023100, C536S023200
Reexamination Certificate
active
06255094
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a novel gene encoding &bgr;-galactoside-&agr;2,6-sialyltransferase.
Further, it relates to a gene encoding a novel signal peptide.
PRIOR ART
In recent years, biological activities of complex carbohydrates such as glycoptoteins and glycolipids have been successively clarified and the importance of sugar chains has come to be understood. Sialic acids are known as sugars often found at the nonreducing end of a sugar chain of complex carbohydrates. While the physiological functions and biological significance of sugar chains are important, it is considered that sialic acids have a particularly large number of functions. However, it is difficult to chemically synthesize these substances, in particular, to add a sialic acid to the chain of oligosaccharides, complex carbohydrates, etc. Accordingly, attention has been paid to enzymatic methods by which these products can be easily synthesized at a high yield without any side-reactions.
Sialyltransferases currently available on the market includes enzymes obtained from the submaxillary gland, liver, etc. of an animal such as rat, swine and human being [Poulson et al. J. Biol. Chem. 252, 2356-2362 1977), Weistein et al. J. Biol. Chem. 257, 13835-13844 (1982), Miyagi et al. Eur. J. Biochem. 126, 253-261 (1982)]. However, the enzymes from animals cannot be obtained in a large amount due to difficulties involving purification, which makes them highly expensive. Moreover, the poor stability of these enzymes is also a problem.
Under these circumstances, the present inventors conducted a search for a bacterium having sialyltransferase activity to provide a sialyltransferase which can be supplied in a large amount. As a result, they found that a marine bacterium Photobacterium damsela JT0160 (hereinafter referred to as “JT0160”) has such an activity. Further, they purified the sialyltransferase 0160 (hereinafter referred to as “ST0160”) produced by JT0160 to an electrophoretically homogeneous level. They furthermore analyzed the binding property of this enzyme and thus clarified that ST0160 is a 0-galactoside-&agr;2,6-sialyltransferase which transfers sialic acids, via an &agr;-2,6-linkage, to the 6-position of galactose at the nonreducing end of a sugar chain [JP (Kokai) Hei 8-154673]. Thus, it became possible to produce the sialyltransferase in a large amount by culturing JT0160 capable of producing ST0160. Since this enzyme is of the membrane-binding type, it is necessary in this process to add a surfactant in the purification of the enzyme, which gives rise to problems such as the possibile contamination of surfactants in the purified enzyme.
On the other hand, advances in genetic engineering techniques have made it possible to express certain proteins in large amounts with the use of recombinant Escherichia coli cells which have been transformed by an expression vector carrying the gene of a protein of interest. When this approach is applied to the production of &bgr;-galactoside-&agr;2,6-sialyltransferase, the problems described above can be solved and, moreover, it is possible to produce modified or non-native enzymes such as a soluble enzyme lacking the sequence which takes part in the membrane-binding of the protein, or an enzyme with a modified substrate specificity, etc. Furthermore, by using a highly efficient promoter such as T7 promoter, it becomes possible to construct a production system capble of delivering an extremely high productivity so that a desired protein amounts to 50% or more of the soluble proteins produced in microbial cells. However, a problem has existed that the genomic DNA of JT0160 could not be extracted from the culture in marine broth which has normally been employed as the growth medium. Thus no gene encoding the &bgr;-galactoside-&agr;2,6-sialyltransferase has been obtained hitherto. That is to say, the &bgr;-galactoside-&agr;2,6-sialyltransferase was not available for use in genetic engineering before the present invention, in spite of existence of a strong demand.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a novel gene encoding &bgr;-galactoside-&agr;2,6-sialyltransferase.
Another object of the present invention is to provide expression vectors for producing the &bgr;-galactoside-&agr;2,6-sialyltransferase protein containing the above-mentioned gene.
A further object of the present invention is to provide a process for producing recombinant &bgr;-galactoside-&agr;2,6-sialyltransferase proteins by using the above-mentioned expression vectors.
A further object of the present invention is to provide recombinant &bgr;-galactoside-&agr;2,6-sialyltransferase proteins produced by using the above-mentioned process.
A further object of the present invention is to provide a gene encoding a novel signal peptide.
REFERENCES:
patent: 8154673A (1996-06-01), None
patent: 173182 (1996-07-01), None
Paulson et al.,The Journal of Biological Chemistry, vol. 252, No. 7, Issue of Apr. 10, pp. 2356-2362 (1977).
Weinstein et al.,The Journal of Biological Chemistry, vol. 257, No. 22, Issue of Nov. 25, pp. 13835-13844 (1982).
Miyagi et al.,Eur. J. Biochem., vol. 126, pp. 253-261 (1982).
Weinstein et al. Primary Structure of b-Galactoside a2,6-Sialyltransferase, J. Biol. Chem. 262(36): 17735-17743.*
T. Yamamoto et al., Journal of Biochemistry (Tokyo), vol. 1200, (1996).
T. Yamamoto et al., Journal of Biochemistry (Tokyo), vol. 123, (1998).
Nakashizuka Motoko
Terada Ichiro
Yamamoto Takeshi
Birch & Stewart Kolasch & Birch, LLP
Hutson Richard
Japan Tobacco Inc.
Prouty Rebecca E.
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