WO2008126993A1

WO2008126993A1 - Novel 2, 3-sialyltransferase and use thereof

Info

Publication number: WO2008126993A1
Application number: PCT/KR2008/001728
Authority: WO
Inventors: Jin Suk Woo; Jae Kyung Sohng; Dea Hee Kim; Sun Youp Kang; Ji Young Yang; Young Soo Jung; Won Min Seo; Tae Geon Gil; Sang Hee Shim
Original assignee: Gene Chem Inc
Priority date: 2007-04-13
Filing date: 2008-03-27
Publication date: 2008-10-23
Also published as: KR100886650B1; KR20080092662A

Abstract

The present invention relates to a novel 2,3-sialyltransferase and a method for producing a material having galactose at a terminal end thereof using the same, and more particularly, to a novel 2,3-sialyltransferase derived from Haemophilus ducreyi (KCTC 2745) or a variant thereof, a method for preparing 2,3- sialyltransferase or its variant, in which a recombinant microorganism transformed with a recombinant vector containing a gene encoding 2,3-sialyltransferase or its variant, is cultured, and a method for producing a material having galactose at a terminal end thereof, such as 2,3-siayllactose etc., by using 2,3-sialyltransferase or its variant.

Description

NOVEL 2,3-SIALYLTRANSFERASE AND USE THEREOF

TECHNICAL FIELD

The present invention relates to a novel 2,3 -sialy transferase and use thereof, and more particularly, to a novel 2,3-sialyltransferase derived from Haemophilus ducreyi (KCTC 2745) or a variant thereof, a method for preparing 2,3- sialyltransferase or its variant in which a recombinant microorganism, transformed with a recombinant vector containing a gene encoding 2,3-sialyltransferase or its variant, is cultured, and a method for producing a material having galactose at a terminal end thereof by using 2,3-sialyltransferase or its variant.

BACKGROUND ART

Among various components contained in human milk, many elements for inhibiting development of various diseases are well known, which include, for example, immunoglubulin A, oligosaccharides, lactoxidants, mucin, etc. Especially, sialyllactose as one of oligosaccharides known to protect against various diseases has high affinity to influenza A virus hemagglutinin (HA), so as to prevent bacteria or viruses from adhering to the endothelial surface and thus reduce or inhibit infection caused by the same.

Sialyllactose and sialyl-oligosaccharides function as an important source in supplying sialylic acid to body organs such as skin, brain, etc.

Sialic acid is found in especially high concentrations in the gangliosides of the cerebral cortex of the human brain and, from various animal experiments, it was demonstrated that sialic acid supplied to gangliosides in brain has a strong influence on brain development.

It is generally known that sialyllactose and sialyl oligosaccharides are contained in human colostrum and bovine colostrum. However, preparation of sialyllactose and sialyl oligosaccharides is possible only by isolation of sialyllactose and sialyl oligosaccharides from bovine colostrum. The sialyllactose and sialyl oligosaccharides, obtained by the above method, have limitations on general use thereof, although these are used for manufacturing powdered milk products by some manufacturers. That is, there is a strong possibility that sialyllactose and sialyl oligosaccharides are employed in development of health food supplements or a new type of anti-adhesion drugs, but supply of these materials is limited because these are mostly isolated from bovine colostrum to use.

Accordingly, there is great demand for developing mass production of sialyllactose and sialyl oligosaccharides by enzyme process and methods for synthesis of various sialyl oligosaccharides.

Whole genome sequence of Haemophilus ducreyi 3500HP has been identified (NCBI AE017143), and Haemophilus ducreyi 3500HP-derived sialyltransferase and its gene (1^st gene) have also been identified, as disclosed in Bozue, et al, J. Biol. Chem., 274(7):4106, 1999.

Therefore, the present inventors have made extensive efforts to develop a simple method for economically producing 2,3 -sialyllactose, and as a result, identified a gene encoding a novel 2,3 -sialyltransferase derived from Haemophilus ducreyi KCTC 2745, and confirmed that when 2,3-sialytransferase, obtained by culturing a transformant into which a recombinant vector containing the above gene is introduced, is used, the 2,3-sialyllactose can be economically mass-produced, thereby completing the present invention. SUMMARY OF INVENTION

The main object of the present invention is to provide a novel 2,3-sialyltransferase or its variant.

Another object of the present invention is to provide a gene encoding 2,3- sialyltransferase or its variant, a recombinant vector containing the gene, a recombinant microorganism transformed with the recombinant vector, and a method for preparing 2,3-sialyltransferase or its variant, which comprises culturing the recombinant microorganisms.

Still another object of the present invention is to provide a method for producing a material having galactose at a terminal end thereof, which comprises using 2,3- sialyltransferase or its variant.

In order to accomplish the above objects, the present invention provides a 2,3- sialyltransferase having an amino acid sequence of SEQ ID NO: 1 or 2,3- sialyltransferase variant in which an amino acid at position 21 or 112 of the above amino acid sequence is substituted with alternative amino acid.

According to the present invention, the 2,3-sialyltransferase variant is preferably obtained by substituting cysteine (Cys) for tyrosine (Tyr) at position 21 or substituting glutamine (GIn) for proline (Pro) at position 112 of the amino acid sequence.

The present invention provides a gene encoding 2,3-sialyltransferase or a variant thereof, a recombinant vector containing the gene, and a recombinant microorganism transformed with the recombinant vector.

The present invention also provides a method for preparing 2,3-sialyltransferase or its variant, which comprises culturing the recombinant microorganism described above.

The present invention further provides a method for producing a compound having galactose at a terminal end thereof, which comprises using 2,3-sialyltransferase or its variant.

In the present invention, the compound having galactose at a terminal end thereof is preferably 2,3-sialyllactose.

Other features and embodiments as well as the above objects of the present invention will become more apparent to those skilled in the related art by the following detailed description and appended claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a recombinant vector containing α-2,3-sialyltransferase-encoding sialT gene.

FIG. 2 shows whether there was the expression of enzymes and the results of examining purified enzymes by SDS-PAGE gel (Lane S: marker, Lane 1 : expressed 2,3-sialyltransferase, Lane 2: supernatant after being passed affinity chromatography column, Lane 3: fraction eluted using 2OmM imidazole, Lane 4: fraction eluted using 5OmM imidazole, Lane 5: fraction eluted using 25OmM imidazole, Lane 6: 2,3-sialyltransferase after desalting).

FIG. 3 is a schematic view illustrating a synthetic process of 2,3-sialyllactose.

FIG. 4 shows a result of monitoring the extent of reaction by TLC when 2,3- sialyltransferase of the present invention is added to a mixture containing 5mM MgCl₂.6H₂O, 2OmM CMP-N-acetyl-neuraminic acid and 1OmM lactose.

FIG. 5 shows a result of monitoring 2,3-sialyllactose by Bio-LC.

FIG. 6 shows a result of analyzing 2,3-sialyllactose by LC-Mass.

DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED EMBODIMENTS

In one aspect, the present invention relates to a novel 2,3-sialyltransferase or its variant, and a method for preparing 2,3-sialyltransferase or its variant, which comprises culturing a transformant into which a recombinant vector, containing a gene encoding the 2,3-sialyltransferase or its variant, is introduced.

The sequence of the 1^st gene derived from Haemophilus ducreyi HP35000 encoding sialyltransferase was already known in the art. However, this sequence is different from that of Haemophilus ducreyi KCTC 2745 according to the present invention. In addition, a gene encoding sialytransferase derived from Haemophilus ducreyi KCTC 2745 is still unknown in the art.

According to the present invention, in order to clone sialT gene encoding α-2,3- sialyltransferase, sialT gene was amplified by PCR using chromosomal DNA of Haemophilus ducreyi KCTC 2745 strain as a template. Thereafter, the amplified gene was ligated to plasmid pET32a and introduced into E. coli XLl -Blue to form a transformant, from which plasmid pSIALT is isolated and introduced into E. coli BL21 (DE3), thus constructing E. cø/ϊ/pSIALT.

Although the present invention described only E. coli BL21 (DE3) as an illustrative example of microorganisms used for the transformation, it is obvious to those skilled in the art that a transformant can also be constructed by introducing the inventive recombinant vector into bacteria, fungi, yeast and the like without particular limitation thereto.

After culturing the above transformant E. coli/pSIALT, the cultured cells were collected to examine whether the α-2,3-sialyltransferase was expressed. As a result, it was demonstrated that α-2,3-sialyltransferase was over-expressed in the transformant.

Alternatively, α-2,3-sialyltransferase was prepared by culturing the transformant and purifying supernatant obtained after cell lysis, cells using affinity chromatography resin.

2,3-sialyltransferase variant according to the present invention means a variant in which Tyr at position 21 or Pro at position 112 of the amino acid sequence (SEQ ID NO:1) is substituted with alternative amino acid. The variant can be prepared using commonly known chemical synthesis or site-directed mutagenesis. An example of the method for preparing 2,3-sialyltransferase variant may include mutation induction by substituting Tyr at position 21 with Cys, or Pro at position 112 with GIn.

As used herein, the term "vector" refers to a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of the DNA in a suitable host. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. As used herein, the term "plasmid" and "vector" are sometimes used interchangeably, because the plasmid is the most commonly used form of vector at present. For the purpose of the present invention, the plasmid vector is preferably used. A typical plasmid vector which can be used for this purpose contains the followings: (a) a replication origin by which replication occurs efficiently such that several hundred plasmid vectors per host cell are created; (b) an antibiotic-resistant gene by which host cells transformed with the plasmid vector can be selected; and (c) restriction enzyme digestion sites into which foreign DNA fragments can be inserted. Even if suitable restriction enzyme digestion sites are not present in the vector, the use of a conventional synthetic oligonucleotide adaptor or linker enables easy ligation between the vector and the foreign DNA fragments.

After ligation, the vector should be transformed into suitable host cells. The transformation can be easily achieved using the well-known calcium chloride method. Selectively, electroporation (Neumann et al, EMBO J., 1 :841, 1982) can also be used in the transformation of such host cells.

For the overexpression of a gene, an expression vector known in the art may be used in the present invention.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. By "operably linked" is meant that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s). For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and present in open reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

As well-known to the art, in order to increase expression level of a transfected gene in a host cell, the gene should be operably linked to transcriptional and translational control sequences which can work in a selected expression host. Preferably, the corresponding gene and expression control sequence are contained in an expression vector comprising a selection marker and replication origin. When an expression host is a eukaryotic cell, the expression vector should further comprise an expression marker useful in the eukaryotic expression host cell.

The host cell transformed with said recombinant vector constitutes another aspect of the present invention. As used herein, the term "transformation" refers to introducing DNA into a host cell so that the DNA is replicable, either as a chromosomal integrant or as an extrachromosomal element.

Of course, it should be understood that all the vectors do not function equally to express the DNA sequences according to the present invention. Likewise, all the host cells do not function equally for the same expression system. However, those skilled in the art may properly select a vector, expression control sequence and host cell without departing from the scope of the present invention and without undue experimentation. For example, in selection of a vector, a host cell must be considered. This is because the vector should be replicated therein. Also, the replication number and ability to control the replication number of a vector and expression of other proteins encoded by the vector, for example, antibiotic marker, should be considered.

In another aspect, the present invention relates to a method for producing a compound having galactose at a terminal thereof by using 2,3-sialyltransferase or its variant. According to the present invention, 2,3-sialyllactose was prepared by allowing a reaction mixture containing CMP-N-acetyl-neuraminic acid and lactose to react with 2,3-sialyltransferase or its variant of the present invention, as shown in FIG. 3.

Although the present invention described only 2,3-sialyllactose as a material having galactose at a terminal end thereof, other materials can also be prepared using 2,3-sialyltransferase without limitation as long as they are materials containing galactose at a terminal thereof, which include N-acetyl lactosamine, galactinol, raffinose, lactulose, melibiose, planteobiose, stachyose, lactosyl-O- hexanoic acid and N-acetyl lactosaminyl-1-O-hexanoic acid, etc.

Examples

Hereinafter, the present invention will be described in detail in the following examples with reference to the accompanying drawing. However, it is obvious to those skilled in the art that these examples are intended to illustrate the invention as preferred embodiments of the present invention and do not limit the scope of the present invention.

Example 1 : Cloning and transformation of sialT gene encoding α-2,3- sialyltransferase

In order to clone sialT gene (SEQ ID NO:2) encoding α-2,3-sialyltransferase, sialT gene was amplified by PCR using chromosomal DNA of Haemophilus ducreyi KCTC 2745 strain as a template with primers having SEQ ID NOs: 3 and 4. SEQ ID NO: 3: 5'-TTGGATCCATGCTGATTCAACAAAATC SEQ ID NO: 4: 5'-GCGTCGACATTTAATTATGTATTGTAC

PCR was performed using a reaction mixture containing 1 μi of template DNA (lOpmol), lμ& of each of the primers, 4μi of Premix (Genotech) and 14μC of distilled water under the following conditions; 30 cycles of denaturation at 96 ^"C for 1 min, annealing at 52.5⁰C for 1 min, elongation at 72 ^°C for 2 min in Mastercycler gradient PCR (Eppendorf).

The amplified PCR product was purified using plasmid mini-prep kit (Solgent) and added with 70% ethanol to precipitate DNA. The precipitated DNA was recovered and detected using agarose gel electrophoresis to separate 1.2kb DNA fragment, which was then purified. The purified DNA fragment was digested with restriction enzymes BatnHl and Sail to ligate into plasmid pET32a (Novagen) digested with the same restriction enzymes BamHl and Sail using T4 DNA ligase (Takara), and then introduced into E. coli XLl -Blue to obtain an ampicillin resistant transformant, from which plasmid pSIALT was isolated (FIG. 1). The plasmid pSIALT has a DNA fragment containing 2,3-sialyltransferase gene derived from Haemophilus ducreyi KCTC 2745, inserted into BamHl and Sail restriction sites downstream of T7 promoter in pET32a.

The obtained recombinant gene was treated with calcium chloride to introduce into an over expressing E. coli BL21 (DE3) strain (Invitrogen) by a method for introducing plasmid into a microbial strain (J. MoI. Biol, 53: 159, 1970), thus obtaining a transformant E, coli/pSlALT.

Example 2: Examination of α-2,3-sialyltransferase expression

After the transformant E. cσ///pSIALT obtained in Example 1 was incubated in LB medium containing ampicillin, 1 mi of the incubated seed culture was inoculated into 50ml of LB medium to culture at 37 "C to a cell density (OD₆₀₀) of 0.6-0.8, and then added with IPTG to a concentration of 0.4mM to culture the cells until cell density (OD₆₀₀) reaches 3-5, thus harvesting cells.

The obtained cells were disrupted using ultrasonic disruptor or French press, the expression level of enzymes was examined using SDS-PAGE gel (see FIG. 2, Lane 1).

As a result, it was demonstrated that α-2,3-sialyltransferase encoded by sialT gene, which was inserted into the transformant, was overexpressed.

Example 3: Purification of α-2,3-sialyltransferase

Since α-2,3-sialyltransferase expressed in Example 2 contains his-tag fusion protein, it was purified using affinity chromatography resin (Ni²⁺ column, Bioprogen).

Specifically, supernatant obtained by disrupting cells was passed through a column charged with affinity chromatography resin, and the column was washed with 2OmM imidazole/0.5M NaCl (5OmM Tris HCl, pH 7.5) and 5OM imidazole/0.5M NaCl (5OmM Tris HCl, pH 7.5), respectively, thus obtaining a target protein using 250M imidazole (5OmM Tris HCl, pH 7.5) in elution buffer.

The obtained target protein was desalted in a buffer solution of 5OmM Tris HCl (pH 7.5) using a dialysis membrane to examine through SDS-PAGE gel (FIG. 2, Lanes 2 to 6). As a result, it was confirmed that α-2,3-sialyltransferase was purified.

Example 4: Synthesis and analysis of α-2,3-sialyltransferase

5mM MgCl₂.6H₂O, 2OmM CMP-N-acetyl-neuraminic acid (Sigma) and 1OmM lactose in a buffer solution of 5OmM Tris HCl (pH 7.5) was allowed to react with the α-2,3-sialyltransferase purified in Example 3. The extent of the reaction was examined by TLC (a development solvent, isopropyl alcohol:NH₄OH:H₂O=7:5: l) and Bio-LC (Metrohm Ltd).

As a result, synthesis of α-2,3-sialyllactose was confirmed as shown in FIG. 4 and FIG. 5. Furthermore, NMR and LC-Mass analyses also showed that the synthesized material was α-2,3- sialyllactose (see FIG. 6).

INDUSTRIAL APPLICABILITY

As described in detail above, the present invention has an effect to provide a novel 2,3-sialyltransferase and a method for producing a material having galactose at a terminal thereof using the same. Materials having galactose at the terminal end thereof, such as 2,3 -sialyllactose can be mass-produced economically using 2,3- sialyltransferase obtained by culturing a transformant into which a gene for encoding a novel 2,3-sialyltransferase derived from Haemophilus ducreyi KCTC 2745 according to the present invention, is introduced.

While the present invention has been described with reference to the above preferred embodiments, it will be understood by those skilled in the art that various modifications and variations may be made therein without departing from the scope of the present invention as defined by the appended claims.

Claims

THE CLAIMS

What is Claimed is:

L A 2,3-sialyltransferase having an amino acid sequence of SEQ ID NO: 1 or a 2,3- sialyltransferase variant in which an amino acid at position 21 or 112 of the amino acid sequence of SEQ ID NO: 1 is substituted with alternative amino acid.

2. The 2,3-sialyltransferase variant according to claim 1, wherein tyrosine (Tyr) at position 21 of the amino acid sequence is substituted with cysteine (Cys).

3. The 2,3-sialyltransferase variant according to claim 1, wherein proline (Pro) at position 112 of the amino acid sequence is substituted with glutamine (GIn).

4. A gene encoding the 2,3-sialyltransferase or its variant of claim 1.

5. A recombinant vector containing the gene of claim 4.

6. A recombinant microorganism transformed with the recombinant vector of claim 5.

7. A method for preparing 2,3-sialyltransferase or its variant, which comprises culturing the recombinant microorganism of claim 6.

8. A method for producing a compound having galactose at a terminal end thereof, which comprises using 2,3-sialyltransferase or its variant of any one claim among claims 1 to 3.

9. The method according to claim 8, wherein the compound having galactose at a terminal end thereof is 2,3-sialyllactose.