β-galactoside-α2,3-sialyltransferase, a gene encoding thereof, and a method for producing thereof
The present invention provides a novel β-galactoside-α2,3-sialyltransferase and a nucleic acid encoding the sialyltransferase. The present invention also provides a microorganism producing the sialyltransferase, as well as a method for producing the sialyltransferase using such a microorganism. The present invention further provides a vector carrying a nucleic acid encoding the sialyltransferase, and a host cell transformed with the vector, as well as a method for producing a recombinant β-galactoside-α2,3-sialyltransferase. The present invention further provides a method for preparing a sialylsugar chain, which uses the sialyltransferase of the present invention.
1. An isolated protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acid residues 22-409 of SEQ ID NO: 2, SEQ ID NO: 29, and amino acid residues 25-409 of SEQ ID NO: 29.
2. An isolated protein having β-galactoside-α2,3-sialyltransferase activity, which comprises an amino acid sequence comprising conservative substitution of one or more amino acids in, and sharing an amino acid homology of at least 90% or more with, an amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acid residues 22-409 of SEQ ID NO: 2, SEQ ID NO: 29, and amino acid residues 25-409 of SEQ ID NO: 29.
3. An isolated protein having β-galactoside-α2,3-sialyltransferase activity, which comprises an amino acid sequence sharing an amino acid homology of at least 90% or more with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acid residues 22-409 of SEQ ID NO: 2, SEQ ID NO: 29, and amino acid residues 25-409 of SEQ ID NO: 29.
4. An isolated protein having β-galactoside-α2,3-sialyltransferase activity, which is encoded by a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 64-1230 of SEQ ID NO: 1, SEQ ID NO: 28, and nucleotides 73-1230 of SEQ ID NO: 28.
5. An isolated protein having β-galactoside-α2,3-sialyltransferase activity, which is encoded by a nucleic acid comprising a nucleotide sequence hybridizable under conditions including hybridization at 65° C. in 0.5M sodium phosphate pH 7.2, 1 mM EDTA, 7% SDS, 1% BSA, and washing at 65° C. in 40 mM sodium phosphate buffer pH 7.2, 1 mM EDTA, 5% SDS, 0.5% BSA and at 65° C. in 40 mM sodium phosphate buffer pH 7.2, 1 mM EDTA, 1% SDS with the full length complementary strand of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 28.
6. The isolated protein according to claim 3 , which is obtained from a microorganism which belongs to Vibrionaceae.
7. An isolated nucleic acid encoding a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acid residues 22-409 of SEQ ID NO: 2, SEQ ID NO: 29, and amino acid residues 25-409 of SEQ ID NO: 29.
8. An isolated nucleic acid encoding a protein having β-galactoside-α2,3-sialyltransferase activity, wherein the protein comprises an amino acid sequence comprising conservative substitution of one or more amino acids in, and sharing an amino acid homology of at least 90% or more with, an amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acid residues 22-409 of SEQ ID NO: 2, SEQ ID NO: 29, and amino acid residues 25-409 of SEQ ID NO: 29.
9. An isolated nucleic acid encoding a protein having β-galactoside-α2,3-sialyltransferase activity, wherein the protein comprises an amino acid sequence sharing a homology of at least 90% with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, amino acid residues 22-409 of SEQ ID NO: 2, SEQ ID NO: 29, and amino acid residues 25-409 of SEQ ID NO: 29.
10. An isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, nucleotides 64-1230 of SEQ ID NO: 1, SEQ ID NO: 28, and nucleotides 73-1230 of SEQ ID NO: 28.
11. An isolated nucleic acid encoding a protein having β-galactoside-α2,3-sialyltransferase activity, wherein the nucleic acid comprises a nucleotide sequence hybridizable under conditions including hybridization at 65° C. in 0.5M sodium phosphate pH 7.2, 1 mM EDTA, 7% SDS, 1% BSA, and washing at 65° C. in 40 mM sodium phosphate buffer pH 7.2, 1 mM EDTA, 5% SDS, 0.5% BSA and at 65° C. in 40 mM sodium phosphate buffer pH 7.2, 1 mM EDTA, 1% SDS, with the full length complementary strand of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 28.
12. A recombinant vector comprising the nucleic acid according to claim 9 .
13. An isolated host cell transformed with the recombinant vector according to claim 12 .
14. An isolated microorganism of the Vibrionaceae, which expresses the protein according to claim 1 .
15. The microorganism according to claim 14 , which is Photobacterium phosphoreum strain JT-ISH-467 (Accession No. NITE BP-88), or Photobacterium sp. strain JT-ISH-224 (Accession No. NITE BP-87).
16. A method for producing a recombinant protein having β-galactoside-α2,3-sialyltransferase activity, which comprises the following steps:
1) transforming and isolating a host cell with a recombinant vector comprising the nucleic acid according to claim 7 ;
2) culturing the transformed host cell; and
3) isolating the protein having β-galactoside-α2,3-sialyltransferase activity from the cultured host cell or the culture supernatant.
17. A method for preparing a sialylated sugar chain, which comprises:
(i) preparing a solution containing the protein according to claim 1 , a glycosyl donor substrate and a glycosyl acceptor substrate;
(ii) causing sialic acid transfer reaction in the solution; and
(iii) obtaining the generated sialylated sugar chain from the reaction solution.
18. The isolated protein of claim 1 , which comprises SEQ ID NO: 2.
19. The method of claim 16 , wherein said nucleic acid encodes a protein comprising SEQ ID NO: 2.