Fucose-binding protein, method for producing same, and use of same

A technology of binding and fucose, applied in the field of fucose-binding proteins, can solve problems such as non-existence

Pending Publication Date: 2021-03-26
TOSOH CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there have been no reports of BC2LCN that has improved thermal stability and binding affinity to sugar chains through amino acid substitutions at specific positions.
In addition, in order to realize the industrial application of BC2LCN, from the viewpoint of stable supply and large-scale supply, a high-productivity method such as production using microorganisms such as Escherichia coli is preferable, but there is no BC2LCN with improved productivity so far. Related reports

Method used

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  • Fucose-binding protein, method for producing same, and use of same
  • Fucose-binding protein, method for producing same, and use of same
  • Fucose-binding protein, method for producing same, and use of same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0226] Example 1 Production of fucose-binding protein 129 and evaluation of productivity

[0227] Example 1 involves adding an oligopeptide containing a polyhistidine sequence to the N-terminus of the amino acid sequence of the fucose-binding protein shown in SEQ ID NO: 2 consisting of 129 amino acid residues, and adding an oligopeptide containing cysteine ​​to the C-terminus. Production and productivity evaluation of fucose-binding protein (hereinafter referred to as fucose-binding protein 129) of oligopeptide residues.

[0228] (1) Production of expression vector pET-BC2LCN(129)cys and recombinant Escherichia coli BL21(DE3) / pET-BC2LCN(129)cys

[0229] The expression vector pET-BC2LCN(129)cys is an expression vector for expressing fucose-binding protein 129. The amino acid sequence of fucose-binding protein 129 is SEQ ID NO: 33, the 5th to 10th positions are polyhistidine sequences, the 15th to 143rd positions are the amino acid sequence of SEQ ID NO: 2, and the 144th to 14t...

Embodiment 2

[0236] Example 2 Production of fucose-binding protein 127 and evaluation of productivity

[0237] Example 2 involves adding an oligopeptide containing a polyhistidine sequence to the N-terminus of the amino acid sequence of the fucose-binding protein shown in SEQ ID NO: 3 consisting of 127 amino acid residues, and adding an oligopeptide containing cysteine ​​to the C-terminus Production and productivity evaluation of fucose-binding protein (hereinafter referred to as fucose-binding protein 127) of oligopeptide residues.

[0238] (1) Production of expression vector pET-BC2LCN(127)cys and recombinant Escherichia coli BL21(DE3) / pET-BC2LCN(127)cys

[0239] The expression vector pET-BC2LCN(127)cys is an expression vector for expressing fucose-binding protein 127. The amino acid sequence of fucose-binding protein 127 is SEQ ID NO: 34, the 5th to 10th positions are polyhistidine sequences, the 15th to 141st positions are the amino acid sequence of SEQ ID NO: 3, and the 142nd to 14th...

Embodiment 3

[0246] Example 3 Production of fucose-binding protein 129G36C and evaluation of productivity

[0247] Example 3 relates to the amino acid sequence of the fucose-binding protein shown in SEQ ID NO: 4 consisting of 129 amino acid residues (where the glycine residue at position 36 in SEQ ID NO: 2 is replaced with a cysteine ​​residue Amino acid sequence) to which an oligopeptide containing a polyhistidine sequence is added to the N-terminus, and an oligopeptide containing a cysteine ​​residue is added to the C-terminus of a fucose-binding protein (hereinafter referred to as fucose-binding protein 129G36C.) Evaluation of manufacturing and productivity.

[0248] (1) Production of expression vector pET-BC2LCN(129G36C)cys and recombinant Escherichia coli BL21(DE3) / pET-BC2LCN(129G36C)cys

[0249] The expression vector pET-BC2LCN(129G36C)cys is an expression vector for expressing fucose-binding protein 129G36C. The amino acid sequence of the fucose-binding protein 129G36C is SEQ ID N...

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Abstract

The present invention addresses the problem of providing a fucose-binding protein which is improved in the productivity when expressed in a host such as Escherichia coli, the binding affinity for a fucose-containing sugar chain such as a sugar chain containing a structure composed of Fuc[alpha]1-2Gal[beta]1-3GlcNAc and / or Fuc[alpha]1-2Gal[beta]1-3GalNAc, and / or the stability to heat. The problem can be solved by, in the amino acid sequence for a fucose-binding protein BC2LCN which is represented by SEQ ID NO: 1, deleting multiple amino acid residues located on the C-terminal side and, if necessary, substituting a glycine residue located at position-36 in SEQ ID NO: 1 by a cysteine residue, substituting a glutamine residue located at position-39 in SEQ ID NO: 1 by a leucine residue or a methionine residue, substituting a glutamine residue located at position-65 in SEQ ID NO: 1 by a leucine residue, substituting a cysteine residue located at position-72 in SEQ ID NO: 1 by a glycine residue or an alanine residue, substituting a glutamic acid residue located at position-81 in SEQ ID NO: 1 by a cysteine residue, a glutamine residue, a histidine residue or a methionine residue and substituting a glycine residue that is specified as a residue located at position-36 in SEQ ID NO: 1 by a cysteine residue.

Description

technical field [0001] The present invention relates to a fucose-binding protein, its production method, and its use. In particular, the present invention may relate to productivity when expressed in hosts such as Escherichia coli , and fucose-containing products such as sugar chains containing a structure composed of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc. A fucose-binding protein having improved sugar chain binding affinity and / or thermal stability. Background technique [0002] BC2LCN derived from the N-terminal domain of the BC2L-C lectin produced by Gram-negative bacteria (Burkholderia cenocepacia) has binding affinity for sugar chains containing fucose residues. Proteins, for example, are known to be excluding H-type 1-type sugar chains (Fucα1-2Galβ1-3GlcNAc) and H-type In addition to high binding affinity to type 3 sugar chains (Fucα1-2Galβ1-3GalNAc), it also has a high binding affinity for Lewis (Lewis) Y-type sugar chains (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc),...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31B01D15/38B01J20/24B01J20/30C07K14/195C12N1/21C12N15/70C12P21/02C07K17/08
CPCC07K14/195C12N15/70C07K17/08B01D15/3823B01J20/3274B01J2220/58B01J20/286B01J20/3219B01J20/24B01J20/30C07H1/06C12N5/0693
Inventor 林政浩丸山高广伊藤博之浅越绫畑山耕太穗谷惠
Owner TOSOH CORP
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