Glycosyltransferase and DNA encoding the same

Details Glycosyltransferase and DNA encoding the same Glycosyltransferase and DNA encoding the same

2000-08-02

2002-11-05

Saidha, Tekchand (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S252300, C435S320100, C435S252330, C435S072000, C435S074000, C435S084000, C435S822000, C536S023200, C536S023700

Reexamination Certificate

active

06475761

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a protein having &bgr;1,4-galactosyltransferase activity, DNA encoding the protein, a recombinant DNA comprising the DNA, a transformant carrying the recombinant DNA, a process for producing &bgr;1,4-galactosyltransferase by using the transformant, and a process for producing a galactose-containing carbohydrate by using the transformant.
BACKGROUND ART
The &bgr;1,4-galactosyltransferase genes so far obtained are those derived from animals [J. Biol. Chem., 263, 10420 (1988); Biochem. Biophys. Res. Commun., 157, 657 (1988); Eur. J. Biochem., 183, 211 (1989)], those derived from
Neisseria gonorrhoeae
(WO 96/10086) and those derived from
Streptococcus pneumoniae
[Mol. Microbiol., 26, 197 (1997)].
O-antigens of lipopolysaccharides of
Helicobacter pylori
have the same structures as the Lewis X [Gal&bgr;1-4(Fuc&agr;1-3)GlcNAc] and Lewis Y [Fuc&agr;1-2Gal&bgr;1-4(Fuc&agr;1-3)GlcNAc] epitopes of mammals, and Helicobacter pylori is expected to possess &bgr;1,4-galactosyltransferase activity [Glycobiology, 5, 683 (1995)]. In
Helicobacter pylori
, however, a protein highly homologous to known &bgr;1,4-galactosyltransferases has not been found and a &bgr;1,4-galactosyltransferase gene has not been specified [Nature, 388, 539 (1997)].
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide a protein having &bgr;1,4-galactosyltransferase activity, DNA encoding the protein, a process for producing a protein having &bgr;1,4-galactosyltransferase activity by using the DNA, and a process for producing a galactose-containing carbohydrate by using the above protein.
The present inventors made an intensive investigation to attain the object. As a result, a &bgr;1,4-galactosyltransferase gene so far unspecified has been obtained by screening of
Helicobacter pylori
genomic DNA library for the gene encoding a protein having &bgr;1,4-galactosyltransferase activity and its sequence has been determined. The present invention has been completed on the basis of this result.
Thus, the first aspect of the present invention provides a protein which is selected from the group consisting of:
(a) a protein having the amino acid sequence shown in SEQ ID NO: 1; and
(b) a protein having &bgr;1,4-galactosyltransferase activity and having an amino acid sequence wherein one to several amino acid residues are deleted, substituted or added in the amino acid sequence of the protein of (a).
The above deletion, substitution or addition of amino acid residues can be caused by site-directed mutagenesis, which is a technique known in the art at the time of filing of the present application. The expression “one to several amino acid residues” is intended to mean amino acid residues in such numbers that can be deleted, substituted or added by site-directed mutagenesis.
Such a protein having &bgr;1,4-galactosyltransferase activity and having an amino acid sequence wherein one to several amino acid residues are deleted, substituted or added can be produced according to the methods described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) (hereinafter referred to as Molecular Cloning, Second Edition); Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) (hereinafter referred to as Current Protocols in Molecular Biology); Nucleic acids Research, 10, 6487 (1982); Proc. Natl. Acad. Sci. USA, 79, 6409 (1982); Gene, 34, 315 (1985); Nucleic Acids Research, 13, 4431 (1985); Proc. Natl. Acad. Sci. USA, 82, 488 (1985), etc.
The second aspect of the present invention provides DNA encoding the above protein, DNA having the nucleotide sequence shown in SEQ ID NO: 2, and DNA which hybridizes to the DNA having the nucleotide sequence shown in SEQ ID NO: 2 under stringent conditions and which encodes a protein having &bgr;1,4-galactosyltransferase activity.
The “DNA which hybridizes under stringent conditions” refers to DNA which is obtained by colony hybridization, plaque hybridization or Southern hybridization using the DNA having the nucleotide sequence shown in SEQ ID NO: 2 as a probe. Such DNA can be identified, for example, by performing hybridization at 65° C. in the presence of 0.7-1.0 M sodium chloride using a filter with colony- or plaque-derived DNA immobilized thereon and then washing the filter at 65° C. using 0.1 to 2-fold concentrated SSC solution (SSC solution: 150 mM sodium chloride and 15 mM sodium citrate).
Hybridization can be carried out according to the methods described in laboratory manuals such as Molecular Cloning, Second Edition; Current Protocols in Molecular Biology; and DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995). The hybridizable DNA is, for example, DNA having at least 80% homology, preferably 95% or more homology to the nucleotide sequence shown in SEQ ID NO: 2.
The third aspect of the present invention provides a recombinant DNA which is obtained by inserting the above DNA into a vector.
The fourth aspect of the present invention provides a transformant which is obtained by introducing the above recombinant DNA into a host cell.
The fifth aspect of the present invention provides a process for producing the above protein which comprises culturing the above transformant in a medium, allowing the protein to form and accumulate in the culture, and recovering the protein from the culture.
The sixth aspect of the present invention provides a process for producing a galactose-containing carbohydrate which comprises allowing a culture of the above transformant or a treated matter thereof as an enzyme source, an acceptor carbohydrate and uridine diphosphogalactose to be present in an aqueous medium, transferring galactose to the acceptor carbohydrate by &bgr;1,4 linkage to form and accumulate the galactose-containing carbohydrate in the aqueous medium, and recovering the galactose-containing carbohydrate from the aqueous medium.
The present invention is described in detail below.
1. Preparation of the DNA of the Present Invention
(1) Construction of a Genomic DNA Library
The DNA of the present invention can be prepared from a microorganism belonging to the genus Helicobacter.
Examples of suitable microorganisms belonging to the genus Helicobacter are strains of
Helicobacter pylori
, specifically,
Helicobacter pylori
NCTC 11637.
A microorganism belonging to the genus Helicobacter is cultured by a known method [e.g., Mol. Microbiol., 20, 833 (1996)].
After the culturing, the chromosomal DNA of the microorganism is isolated and purified by a known method (e.g., Current Protocols in Molecular Biology).
The obtained chromosomal DNA is cleaved with an appropriate restriction enzyme and fractionated by means such as sucrose density gradient ultracentrifugation, and DNA fragments of 2-6 kb are recovered.
According to a conventional method (e.g., Molecular Cloning, Second Edition)., the recovered DNA fragment is inserted into an expression vector for
Escherichia coli
at a site downstream of the promoter and the thus constructed recombinant DNA is introduced into
Escherichia coli
to prepare a genomic DNA library.
Suitable expression vectors include pBTrp2, pBTac1 and pBTac2 (all available from Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (QIAGEN), pKYP10 (Japanese Published Unexamined Patent Application No. 110600/83), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK+ (Stratagene), pBluescript II SK(−) (Stratagene), pTrs30 (FERM BP-5407), pTrs32. (FERM BP-5408), pGHA2 (FERM BP-400), pGKA2 (FERM BP-6798), pTerm2 (Japanese Published Unexamined Patent Application No. 22979/91, U.S. Pat. No. 4,686,191, U.S. Pat. No. 4,939,094 and U.S. Pat. No. 5,160,735), pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (Pharmacia), pET-3 (Novagen), pSupex, pUB110, pTP5, pC1

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