JP2020058343A - Method for producing fucose-binding protein - Google Patents

Abstract

To provide methods for efficiently producing the protein by culturing Escherichia coli capable of expressing a fucose-binding protein.SOLUTION: When culturing E. coli obtained by transforming with an expression vector containing a DNA encoding a fucose-binding protein, the growth state of E. coli is appropriately measured, then a carbon source and a nitrogen source are supplied little by little at a specific rate according to its growth rate, thereby E. coli can be cultivated at high density, and as a result it becomes possible to express the protein in a large amount.SELECTED DRAWING: None

Description

  The present invention relates to an efficient method for producing a fucose-binding protein by efficiently culturing Escherichia coli capable of expressing the protein.

  With the recent progress in research on functional analysis of sugar chains, it has become clear that sugar chains play an important role in the functions of biomolecules such as glycoproteins and in the differentiation and recognition of cells. Of the sugar chains, fucose-containing sugar chains are often associated with physiological functions. For example, in Patent Document 1, an H type 1 type sugar chain (Fucα1-2Galβ1-, which is an undifferentiated sugar chain marker on the surface of human iPS cells, is described. 3GlcNAc) and an H-type 3 type sugar chain (Fucα1-2Galβ1-3GalNAc) are used to detect undifferentiated cells. The lectin capable of binding to the H-type 1 type sugar chain and the H-type 3 type sugar chain is a recombinant BC2LCN derived from the N-terminal domain of BC2L-C lectin produced by Gram-negative bacterium (Burkholderia cenocepacia). In addition to the H type 1 type sugar chain and the H type 3 type sugar chain, BC2LCN contains Lewis Y type sugar chain (Fucα1-2Galβ1-4 (Fucα1-3) GlcNAc) and Lewis X type sugar chain (Galβ1-4 (Fucα1). -3) It is known to have a high binding property to plural types of fucose-containing sugar chains such as GlcNAc) (Non-patent Document 1).

  On the other hand, for the purpose of inexpensively producing a protein having a specific function, a production method using a gene recombinant microorganism capable of expressing the protein has been studied so far (Patent Document 2). A method for producing an Fc-binding protein using the modified Escherichia coli has been reported (Patent Document 3). In the industrial culture of microorganisms such as Escherichia coli and yeast, compared to the batch culture method in which necessary nutrients are added at one time and then the culture is performed, fed-batch culture in which the medium components are continuously supplied during the culture It is known that the method gives a higher yield of the microorganism or its product (hereinafter, continuous feeding of medium components during culture is called fed-batch). The fed-batch method has the advantage that the concentration of the nutrient source supplied can be controlled arbitrarily.Therefore, when a high-concentration substrate inhibits the growth of microorganisms or the production of the target substance, or by-products such as alcohol and organic acids are generated. It is particularly effective when the product is produced. The production of by-products is not preferable because it not only inhibits the growth of microorganisms but also reduces the quality of the target product and makes purification difficult.

  Examples of medium components to be fed-batch include carbon sources such as sugars, which are consumed in large amounts. However, since the consumption rate of carbon source is not constant depending on the growth condition of microorganisms, in order to keep the concentration of carbon source constant during culture, the carbon source consumption rate by microorganisms should be monitored by some method while feeding. You need to control the quantity. As a method for monitoring the consumption rate of a carbon source, for example, a method of measuring and controlling the glucose concentration by an online glucose analyzer, a method of feeding a carbon source using oxygen consumption as an index, a method of analyzing exhaust gas composition The method of fed-batch using the respiratory quotient as an index, the method of monitoring the amount of cells by an online laser turbidimeter, the method of using pH change, and the detection of carbon source depletion by the change of dissolved oxygen concentration in the culture solution Then, a method of supplying a carbon source is disclosed. Among these, the method of supplying a carbon source by detecting a change in the dissolved oxygen concentration in the culture medium has an advantage that the carbon source in the medium can be controlled to an extremely low concentration.

  In order to efficiently culture Escherichia coli, it is necessary that the medium contains a nitrogen source in addition to the carbon source, and it is important that the ratio of the two is appropriate. When culturing Escherichia coli by the fed-batch culture method, it is necessary to feed both the carbon source and the nitrogen source. Regarding the effect of feeding a plurality of medium components for culturing the recombinant Escherichia coli to produce a target product, and the method of controlling the feeding, Escherichia coli capable of expressing human proinsulin can be used. ) A human proinsulin production system using BL21 (DE3) strain is disclosed (Non-patent document 2). In Non-Patent Document 2, human proinsulin is obtained by feeding a solution obtained by mixing a carbon source and a nitrogen source (yeast extract) to a medium to culture Escherichia coli capable of expressing human proinsulin (molecular weight about 10,000). The preferable concentrations of the carbon source and the nitrogen source (yeast extract) to be fed to the medium during the production of are investigated.

  On the other hand, the recombinant BC2LCN is a protein having a molecular weight of about 17,000 and is considered to associate with each other to form a trimer, which is greatly different from human proinsulin in terms of molecular weight and three-dimensional structure. Therefore, even if Non-Patent Document 2 discloses the preferable concentrations of the carbon source and the nitrogen source fed to the medium, it is difficult to apply this condition to the recombinant BC2LCN. Furthermore, there has been no report on a highly efficient expression system of recombinant BC2LCN using a genetically modified microorganism, and a problem remains in productivity from the viewpoint of stable supply and large-scale supply.

WO 2013/065302 Special table 2000-501936 gazette JP, 2008-245580, A

Sulak, O et al., Structure, 18 (1), 59-72, 2010. Chul Soo Shin et al., Biotechnol. Prog. 13, 249-257, 1997.

  An object of the present invention is to provide a method for producing a protein having excellent productivity by efficiently culturing Escherichia coli capable of expressing a fucose-binding protein. Specifically, when culturing Escherichia coli obtained by transforming with an expression vector containing a DNA encoding a fucose-binding protein, the growth of E. coli is monitored and the carbon source and nitrogen source are adjusted according to the growth rate. The purpose of the present invention is to provide a method for culturing Escherichia coli at a high density and supplying a large amount of the protein by supplying a small amount at a specific ratio.

  With respect to the above problems, the present inventors have conducted intensive studies on stirring conditions for controlling the dissolved oxygen concentration in a fed-batch culture method, and a feeding method of a carbon source and a nitrogen source, and as a result, the present invention has been completed. .

That is, the present invention includes the following first to seventh inventions:
A first invention is a method for producing a fucose-binding protein using Escherichia coli obtained by transforming an expression vector containing a DNA encoding a fucose-binding protein, comprising the steps (1) to ( A method for producing a fucose-binding protein, which comprises the step 5).
(1) A medium containing a carbon source and a nitrogen source is added to a culture tank, Escherichia coli obtained by transforming with an expression vector containing a DNA encoding a fucose-binding protein is inoculated, and a carbon source in a culture solution is added. A step of culturing with stirring while maintaining the dissolved oxygen concentration in the culture solution at 1.0 mg / L or more until the concentration becomes 5.0 g / L or less.
(2) At any time when the concentration of carbon source in the culture solution becomes 5.0 g / L or less, the weight of the nitrogen source supplied is 0.15 times or more and 2.00 times the weight of the carbon source supplied. Supplying (feeding) carbon and nitrogen sources to the culture broth was started while maintaining the range to less than double, and the density of E. coli in the culture broth (OD ₆₀₀ , turbidity of the culture at ₆₀₀ nm) was 60 or more. The process of stirring culture until it becomes.
(3) At any time when the density of E. coli (OD _{600 nm} , turbidity of the culture medium at 600 _nm ) in the culture medium of step (2) reached 60 or more, the concentration after the addition of the inducer was 0.005 mM. The step of adding to the culture solution so as to be 5.0 mM or less.
(4) After the inducer is added to the culture medium in the step (3), the carbon source and the nitrogen source are supplied (fed-batch) to the culture medium and the stirring culture is continued, and the density of E. coli in the culture medium is 90 or more. A step of stopping the culture at any time when it becomes 180 or less.
(5) A step of recovering a fucose-binding protein from the culture obtained by culturing Escherichia coli in steps (1) to (4).
A second aspect of the invention is to supply the carbon source and the nitrogen source while maintaining the dissolved oxygen concentration in the culture solution in the range of 0.01 mg / L to 5.0 mg / L in the steps (2) to (4). (Fed-batch) is the production method according to the first invention.
A third aspect of the invention is to supply (fed-batch) a carbon source and a nitrogen source while maintaining the carbon source concentration in the culture solution at 5.0 g / L or less in the steps (2) to (4). It is the manufacturing method according to the first or second invention.
A fourth invention is the production method according to any one of the first to third aspects, wherein the carbon source is glucose and the nitrogen source is yeast extract.
5th invention is a manufacturing method in any one of 1st-4th whose inducer is isopropyl- (beta) -thiogalactopyranoside.
A sixth invention is the production method according to any one of the first to fifth aspects, in which the fucose-binding protein is the following (a) to (d).
(A) A fucose-binding protein comprising an amino acid sequence from the 1st proline residue to the Xth amino acid residue of the amino acid sequence represented by SEQ ID NO: 1, wherein X is an integer of 120 or more. Sex protein.
(B) Amino acid in which one or more amino acid residues are deleted, substituted or added in the amino acid sequence from the 1st proline residue to the Xth amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 A fucose-binding protein containing a sequence, and having a binding affinity for a sugar chain containing a structure composed of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc, and X is an integer of 120 or more. , Fucose-binding protein.
(C) In the amino acid sequence from the 1st proline residue to the Xth amino acid residue of the amino acid sequence represented by SEQ ID NO: 1, any one of the following amino acid substitutions (1) to (3) A fucose binding protein (1) in which the amino acid sequence including the above is contained and X is an integer of 120 or more, and the 39th glutamine residue of the amino acid sequence represented by SEQ ID NO: 1 is replaced with a leucine residue (2). Substitution of the 65th glutamine residue of the amino acid sequence represented by SEQ ID NO: 1 with a leucine residue (3) Glycine residue and alanine residue of the 72nd cysteine residue of the amino acid sequence represented by SEQ ID NO: 1 Substitution with one type of amino acid residue selected from (d) In the amino acid sequence of the fucose-binding protein of (c) above, the 39th, 65th and A structure containing an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted or added in a region other than the second region, and comprising Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc A fucose-binding protein having binding affinity for a sugar chain containing the protein.
The seventh invention is the production method according to any one of the first to fifth aspects, in which the fucose-binding protein is the following (e) to (h).
(E) To the amino acid sequence from the 1st proline residue to the Xth amino acid residue of the amino acid sequence represented by SEQ ID NO: 1, an oligopeptide containing a polyhistidine sequence at the N-terminus is further added, and the C-terminus A fucose-binding protein consisting of an amino acid sequence to which an oligopeptide containing a cysteine residue is added, and X is an integer of 120 or more.
(F) An amino acid in which one or more amino acid residues are deleted, substituted or added in the amino acid sequence from the 1st proline residue to the Xth amino acid residue of the amino acid sequence represented by SEQ ID NO: 1. The sequence consists of an amino acid sequence in which an oligopeptide containing a polyhistidine sequence is further added to the N-terminus, and an oligopeptide containing a cysteine residue is added to the C-terminus. A fucose-binding protein having binding affinity for a sugar chain containing a structure consisting of -3GalNAc and X is an integer of 120 or more.
(G) To the amino acid sequence from the 1st proline residue to the Xth amino acid residue of the amino acid sequence represented by SEQ ID NO: 1, an oligopeptide containing a polyhistidine sequence at the N-terminus is further added, and the C-terminus In the amino acid sequence to which an oligopeptide containing a cysteine residue is added, an amino acid sequence containing any one or more of the following amino acid substitutions (4) to (6) is included, and X is an integer of 120 or more. Fucose binding protein (4) Substitution of 39th glutamine residue of amino acid sequence represented by SEQ ID NO: 1 with leucine residue (5) 65th glutamine residue of amino acid sequence represented by SEQ ID NO: 1 Of the amino acid sequence shown in SEQ ID NO: 1 with a glycine residue and an alanine residue (6) Substitution with one kind of amino acid residue selected from (h) in the amino acid sequence of the fucose-binding protein of (d) and / or (e), a region other than the 39th position, the 65th position and the 72nd position of SEQ ID NO: 1 In addition to the amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted or added, an oligopeptide containing a polyhistidine sequence is added to the N-terminal and a cysteine residue is included in the C-terminal. A fucose-binding protein having an amino acid sequence to which an oligopeptide is added and having a binding affinity to a sugar chain containing a structure composed of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc.

  Hereinafter, the present invention will be described in detail.

  The method for producing a fucose-binding protein of the present invention (hereinafter referred to as the production method of the present invention) is a production method including the following steps (1) to (5).

  In the step (1) of the production method of the present invention, after adding a medium to a culture tank, Escherichia coli obtained by transforming with an expression vector containing a DNA encoding a fucose-binding protein is inoculated into a culture medium. Is a step of culturing with stirring while maintaining the dissolved oxygen concentration in the culture solution at 2.0 mg / L or more until the concentration of carbon source becomes 5.0 g / L or less.

  The size and shape of the culture tank used in the production method of the present invention are not particularly limited and may be appropriately selected depending on the production amount of the fucose-binding protein. Further, the method of stirring culture is not particularly limited, and for example, a method of stirring the culture solution by rotating or shaking the entire culture tank, a method of stirring the culture solution using a culture tank having a rotating blade at the bottom, stirring A method of stirring the culture solution using a blade can be mentioned. Among these, the method of stirring the culture solution using a stirring blade is preferable because the culture apparatus as a whole can be downsized, the stirring efficiency is high, and the physical damage to the E. coli to be cultured can be reduced. . When the culture solution is stirred using the stirring blade, the diameter and shape of the stirring blade are not particularly limited, and may be appropriately selected in consideration of the size of the culture tank and the stirring efficiency of the culture solution.

  The medium that can be used in the production method of the present invention is not particularly limited, and examples thereof include Terrific Broth (TB) medium and Lysogeny Broth (LB) medium supplemented with necessary nutrient sources. In addition to the carbon source, the nitrogen source and the inorganic salt, a nutrient source commonly used in the art may be added to the medium. Examples of carbon sources supplied (fed-batch) during culture include glucose, fructose, maltose, sucrose, crude sugar, molasses, glycerin and the like. Examples of the nitrogen source supplied (fed-batch) together with the carbon source include yeast extract, polypeptone, casein and their enzymatic digests, corn steep liquor, soy protein, meat extract, fish meat extract and the like. Among these carbon sources and nitrogen sources, the growth of the transformant is good, and further, in that the fucose-binding protein can be efficiently produced, the carbon source is preferably glucose or glycerin, and the nitrogen source is yeast extract. preferable.

  The culture temperature in the production method of the present invention is not particularly limited as long as it is a temperature generally used in the art, and is 10 ° C. to 40 ° C., preferably 20 ° C. to 37 ° C., which improves the productivity of fucose-binding protein. It may be selected as appropriate while taking into consideration. The pH of the medium may be appropriately selected from the range generally used in the art. For example, when the host is Escherichia coli, the pH range is preferably 6.8 to 7.4, and more preferably pH 7.0. Before and after. In addition, the culture time in the production method of the present invention can be set arbitrarily, but is usually set between several hours and 100 hours. When the culture time is long, the activity of the host cells is weakened, which may lead to lysis due to cell death, and the structure of the target fucose-binding protein may change. Therefore, the culture time from step (1) to step (4) Is preferably 72 hours or less, more preferably 60 hours or less.

  In the production method of the present invention, in order to selectively grow Escherichia coli obtained by the above transformation depending on whether or not an expression vector containing a DNA encoding a fucose-binding protein is introduced, the expression vector is contained in the medium. It is preferable to add a drug corresponding to the drug resistance gene. For example, when the vector contains a kanamycin resistance gene, kanamycin may be added to the medium. Further, a reagent such as glycine may be added to the medium in order to promote the secretion of the protein from the Escherichia coli obtained by the transformation into the culture solution. Specifically, when the host is Escherichia coli, it may be added to the medium. Glycine is preferably added at 2% (w / v) or less. In addition to these, as components that can be added to the medium components, inorganic salts such as sodium dihydrogen phosphate, disodium hydrogen phosphate, trisodium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate Metal ions such as salt, potassium chloride, and sodium chloride are magnesium chloride, magnesium sulfate, iron sulfate (II), iron sulfate (III), iron chloride (II), iron chloride (III), iron citrate, and ammonium sulfate. Iron, calcium chloride dihydrate, calcium sulfate, zinc sulfate, zinc chloride, copper sulfate, copper chloride, manganese sulfate, manganese chloride, etc., as vitamins, biotin, nicotinic acid, thiamine, riboflavin, inositol, pyridoxine And so on.

  The concentration of the carbon source in the step (1) may be appropriately set according to the growth rate of Escherichia coli and the type of the carbon source, but when the carbon source is glucose, it is preferably 10 g / L to 50 g / L. Further, the concentration of the nitrogen source in the step (1) may be appropriately set according to the growth rate of E. coli and the type of the nitrogen source. When the carbon source is yeast extract, it may be 10 g / L to 50 g / L. preferable.

  In the step (1) of the production method of the present invention, the concentration of dissolved oxygen (hereinafter, may be abbreviated as DO) in the culture solution is measured to make the carbon source concentration in the culture solution 5.0 g / L or less. The culture is agitated while maintaining the DO concentration in the culture solution at 1.0 mg / L or more. In the step (1), the carbon source concentration decreases due to the growth of Escherichia coli, but the DO concentration decreases due to the respiration of the grown E. coli, so that the DO concentration in the culture solution is 1. Escherichia coli is cultivated by continuously controlling the stirring speed of the culture solution so as to be 0 mg / L or more. When the DO concentration in the culture solution is 1.0 mg / L or less, the proliferated E. coli may be killed. In the production method of the present invention, there is no particular limitation on the method for measuring the DO concentration, and for example, the DO electrode can be used for measurement. Further, in the production method of the present invention, the method of measuring the concentration of the carbon source in the culture solution is not particularly limited. For example, when the carbon source is glucose, the glucose concentration can be measured using a glucose analyzer.

In the step (2) of the production method of the present invention, the weight of the substance serving as the nitrogen source to be supplied is adjusted at any time when the carbon source concentration in the culture solution in the step (1) becomes 5.0 g / L or less. The carbon source and the nitrogen source are continuously supplied to the culture solution while maintaining the range of 0.15 times or more and less than 2.00 times the weight of the substance serving as the carbon source to be supplied (hereinafter, the medium during the culture. Continuous feeding of components is sometimes called fed-batch.), And stirring culture until the cell density of E. coli in the culture solution (OD _{600 nm} , turbidity of the culture solution at 600 nm) reaches 60 or more. It is a process to do. In step (2), if the culture is carried out in a state where the carbon source concentration exceeds 5.0 g / L, a large amount of organic acid is by-produced and accumulated, which may inhibit the growth of Escherichia coli and protein production. . Therefore, when the carbon source concentration becomes 5.0 g / L or less, the feeding of the carbon source and the nitrogen source to the culture solution is started, and after the feeding of the carbon source and the nitrogen source is started, the carbon source concentration is 5 g. / L or less, preferably 1 g / L or less, more preferably 0.5 g / L or less, and most preferably 0.1 g / L or less, it is preferable to control the supply amount of the carbon source. Further, in order to prevent depletion of the carbon source in the culture medium and to grow Escherichia coli at a high density, the DO concentration is in the range of 0.01 mg / L to 5.0 mg / L, preferably 0.05 mg / L to 4. It is preferable to control the feed rates of the carbon source and the nitrogen source so as to maintain the range of 0 mg / L. The fed amount of the carbon source can be grasped from, for example, the amount of decrease in the raw material tank for supplying the carbon source or the driving time of the supply pump.

  The method for measuring the depletion of the carbon source in the culture solution is not particularly limited, and can be measured, for example, by the decrease in the respiratory activity of E. coli. The decrease in respiratory activity appears, for example, as an increase in DO concentration in the culture solution, an increase in oxygen concentration in exhaust gas, a decrease in carbon dioxide concentration, and an increase in pH. In particular, an increase in DO concentration has a fast response, and when the concentration of the carbon source in the culture solution is sufficiently maintained, oxygen is consumed by the respiration of microorganisms, so that the DO concentration becomes lower than the oxygen saturation concentration. Although maintained, when the carbon source is depleted, the respiratory activity of the microorganism is reduced and the DO concentration is rapidly increased, which is a preferable index for monitoring the depletion of the carbon source in the culture solution. The method of adding a carbon source to the culture solution in conjunction with the increase in DO concentration is called the DO stat method. When monitored by an index other than the DO concentration (exhaust gas composition, carbon dioxide concentration, pH increase), there is a drawback that the response is slower than when the DO concentration is used as an index.

  The fed amount of the nitrogen source in the production method of the present invention is preferably fed while maintaining a constant ratio with the fed amount of the carbon source. When the supply of nitrogen source is low, the growth of E. coli is slow and the production of the expressed fucose-binding protein is reduced. On the other hand, if too much nitrogen source is supplied, the growth of Escherichia coli is inhibited. Therefore, the nitrogen source supply amount (weight of nitrogen source substance) at the time of fed-batch is the carbon source supply amount (weight of carbon source substance). ) Times 0.15 times or more and less than 2.00 times, preferably 0.20 times or more and less than 1.50 times, and more preferably 0.25 times or more and less than 1.00 times. When the carbon source of the fed-batch component is glucose or glycerin and the nitrogen source is yeast extract, the prepared carbon source solution and nitrogen source solution are sterilized by autoclaving (for example, 121 ° C, 20 minutes) or filter filtration. By feeding the mixed medium in a clean bench, the ratio of carbon source and nitrogen source at the time of supplying the medium can be kept constant. Further, from the viewpoint that the increase in the volume of the culture solution is suppressed, it is preferable that the carbon source and the nitrogen source to be fed are high-concentration solutions. When the carbon source is glucose and the nitrogen source is yeast extract, the fed solution is fed. The glucose solution concentration is preferably set to 300 g / L to 900 g / L, and the yeast extract solution concentration is preferably set to 100 g / L to 500 g / L.

In the step (3) of the production method of the present invention, the DO concentration in the culture solution is measured and the carbon source and the nitrogen source in the culture solution are measured even after the feeding of the carbon source and the nitrogen source to the culture solution in the step (2) is started. At any time when the density of E. coli in the culture solution (OD _{600 nm} , turbidity of the culture solution at 600 nm) reached 60 or more, the concentration after the addition of the inducer was 0.005 mM or more. This is a step of adding to the culture solution so that the concentration becomes 5.0 mM or less.

Since the expression vector used for producing Escherichia coli capable of expressing the fucose-binding protein produced by the production method of the present invention contains an inducible promoter, in step (3), the protein is efficiently expressed. An inducer is added under such conditions to induce the expression of the fucose-binding protein. The inducer that can be used in the production method of the present invention is not particularly limited, but isopropyl-β-thiogalactopyranoside (hereinafter, referred to as IPTG) in that the fucose-binding protein can be efficiently expressed. Is preferred. The concentration of IPTG added may be appropriately selected from the range of 0.005 mM to 5.0 mM after the addition, and is preferably 0.01 mM to 2.0 mM. Various conditions for inducing the expression of the fucose-binding protein by the addition of IPTG may be carried out under the conditions well known in the art. For example, at any time when the OD _{600nm of} the culture reaches 60 or more, the IPTG The fucose-binding protein expression can be induced by adding to the culture medium so that the concentration after the addition becomes 2.0 mM or less, and then culturing. The temperature of the culture medium after the induction by adding IPTG is 10 ° C. to 30 ° C., preferably 15 ° C. to 25 ° C., more preferably around 20 ° C. to suppress the growth of E. coli, The protein expression is promoted more efficiently.

In the step (4) of the production method of the present invention, after the inducer is added to the culture medium in the step (3), the carbon source and the nitrogen source are supplied (fed-batch) to the culture medium under the condition of the step (2). And agitation culture are continued, and the culture is stopped at any time when the cell density of E. coli in the culture solution becomes 90 or more and 180 or less. In step (4), the completion of E. coli culture may be judged by the OD _{600 nm} of the culture solution, and specifically, the culture may be completed at any time in the range where the OD _{600 nm} is 90 or more and 180 or less. When the OD _{600 nm of the} culture solution is 90 or less, the growth of Escherichia coli is insufficient and the yield of the target fucose-binding protein may be reduced. Further, when the OD _{600 nm of} the culture solution is 180 or more, the growth of E. coli becomes excessive, so that the lysis of E. coli may hydrolyze the target fucose-binding protein or the sugar chain containing fucose due to a change in the three-dimensional structure. There is a risk that the binding property to

  Step (5) of the production method of the present invention is a step of recovering a fucose-binding protein from the culture obtained by culturing Escherichia coli in the above steps (1) to (4). In the present specification, the culture includes not only the transformed transformant Escherichia coli itself and E. coli secretion but also the medium used for the culture and the like. In the step (5), in order to recover the fucose-binding protein from the obtained culture, a recovery method usually used by those skilled in the art may be appropriately selected depending on the expression form of the fucose-binding protein. That is, when the fucose-binding protein is expressed in Escherichia coli, the cells are collected by a centrifugation operation, and then the cells are crushed by adding an enzyme treatment agent or a surfactant to the fucose-binding protein. May be recovered as a soluble fraction. On the other hand, when the fucose-binding protein is secreted into the culture medium, the cells may be separated by centrifugation and the fucose-binding protein may be recovered from the obtained culture supernatant.

  When it is desired to improve the purity of the fucose-binding protein recovered by the above method, a method known in the art may be used, and an example thereof is a separation and purification method using liquid chromatography. As the liquid chromatography, it is preferable to use ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography and the like, and it is more preferable to perform a combination of these chromatography. Further, the purity and the molecular weight of the improved fucose-binding protein of the present invention purified by the above-mentioned chromatography may be examined using a method known in the art, and as an example, Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Examples thereof include (SDS-PAGE) method and gel filtration chromatography method.

  The fucose-binding protein in the production method of the present invention refers to an H type 1 type sugar chain (Fucα1-2Galβ1-3GlcNAc), an H type 3 type sugar chain (Fucα1-2Galβ1-3GalNAc), a Lewis Y type sugar chain (Fucα1-2Galβ1). -4 (Fucα1-3) GlcNAc), a Lewis b-type sugar chain (Fucα1-2Galβ1-3 (Fucα1-4) GlcNAc), and other fucose-containing sugar chains. Specifically, (a): the amino acid sequence of the recombinant BC2LCN represented by SEQ ID NO: 1 (which matches the amino acid sequence from the 2nd to the 156th amino acid sequence registered in GenPept as accession number WP_006490828). A protein containing an amino acid sequence from the 1st proline to the Xth amino acid, wherein X is an integer of 120 or more, or (b): from the 1st proline of the amino acid sequence shown in SEQ ID NO: 1. The amino acid sequence up to the Xth amino acid consists of an amino acid sequence in which one or more amino acids have been deleted, substituted or added, and has an H type 1 type sugar chain and / or an H type 3 type sugar chain. A protein having a binding affinity and X being an integer of 120 or more is recombined with a transformant of E. coli. In which was expressed as a Park protein. As long as the fucose-binding protein in the production method of the present invention has a binding affinity to the fucose-containing sugar chain, particularly to a sugar chain containing a structure composed of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc In the amino acid sequence from the 1st proline to the Xth amino acid of the amino acid sequence represented by SEQ ID NO: 1, one or more amino acids may be deleted, substituted or added, for example, 15 or less, preferably May have 10 or less amino acids deleted, substituted or added. Further, X may be 120 or more and 155 or less, and 125 or more and 155 or less.

  Further, the fucose-binding protein in the production method of the present invention is (1) substituted with a leucine residue at the 39th glutamine residue of the amino acid sequence represented by SEQ ID NO: 1 in order to improve stability against heat, (2) Substitution of the 72nd cysteine residue of the amino acid sequence represented by SEQ ID NO: 1 with one kind of amino acid residue selected from glycine residue and alanine residue, (3) amino acid sequence represented by SEQ ID NO: 1 Substitution of the 65th glutamine residue of the above with a leucine residue may be included. By performing the amino acid substitution described in (1) to (3) above, the stability against heat can be improved. The amino acid substitutions described in (1) to (3) above are effective in improving the stability to heat, either alone or in combination, but in that they can further improve the stability to heat. It is more preferable to combine a plurality of amino acid substitutions described in (1) to (3) above. Furthermore, the fucose-binding protein in the production method of the present invention is represented by SEQ ID NO: 1 as long as it has the ability to bind to a sugar chain containing a structure composed of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc. In the amino acid sequence from the 1st proline to the Xth amino acid of the amino acid sequence described above, one or more amino acid residues are deleted in a region other than the position substituted by the substitutions of (1) to (3) above. , May be substituted or inserted, for example, 15 or less, preferably 10 or less amino acid residues may be deleted, substituted or inserted.

  As long as the fucose-binding protein in the production method of the present invention has a binding affinity to a sugar chain containing a structure composed of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc, the fucose-binding protein has N-terminal side and / or Alternatively, the C-terminal side may have an additional amino acid sequence useful for separation from the solution in the presence of contaminants. Examples of the additional amino acid sequence include oligopeptides containing a polyhistidine sequence, glutathione S-transferase, maltose binding protein, cellulose binding domain, myc tag, FLAG tag and the like. Among these additional amino acid sequences, oligopeptides containing a polyhistidine sequence are preferable because they can be easily purified by nickel chelate affinity chromatography. The number of repeating histidine sequences in an oligopeptide containing a polyhistidine sequence is not particularly limited, but if the repeating sequences of histidine are short, purification by nickel chelate affinity chromatography becomes difficult, and if long, the sugar chain of the fucose-binding protein is The binding affinity for can be compromised. Therefore, the length of the number of repeated histidine sequences in the oligopeptide containing the polyhistidine sequence is preferably a repeated sequence consisting of 5 to 15 histidines, and more preferably a repeating sequence consisting of 5 to 10 histidines. preferable. The position where the oligopeptide containing the polyhistidine sequence is added to the fucose-binding protein is not particularly limited, and may be on both the N-terminal side and the C-terminal side, or on either the N-terminal side or the C-terminal side. However, from the viewpoint of efficient purification by nickel chelate affinity chromatography, the oligopeptide containing the polyhistidine sequence is preferably added to the N-terminal side of the fucose-binding protein.

  Furthermore, the fucose-binding protein in the production method of the present invention has a cysteine that is useful for immobilizing the fucose-binding protein on a carrier such as a support for chromatography on the N-terminal side and / or the C-terminal side. It may have an additional amino acid sequence consisting of an oligopeptide containing a residue or a lysine residue (hereinafter referred to as a carrier immobilization tag). The length of the carrier-immobilizing tag is particularly limited as long as the fucose-binding protein has a binding affinity to a sugar chain containing a structure composed of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc. There is no. As the carrier immobilization tag, an oligopeptide consisting of 2 to 10 amino acid residues containing at least one cysteine residue is preferable, since immobilization on an insoluble carrier can be performed with high selectivity and high efficiency. Is an oligopeptide consisting of 3 amino acid residues of "Gly-Gly-Cys", an oligopeptide consisting of 5 amino acid residues of "Ala-Ser-Gly-Gly-Cys" and "Gly-Gly-Gly-Ser-Gly". An oligopeptide consisting of 7 amino acid residues of "-Gly-Cys" can be exemplified. The position at which the oligopeptide containing one or more cysteines is added to the fucose-binding protein is not particularly limited, and it may be on both the N-terminal side and the C-terminal side, either the N-terminal side or the C-terminal side. From the viewpoint of efficiently immobilizing the fucose-binding protein on the carrier, the oligopeptide containing one or more cysteine residues is preferably added to the C-terminal side of the fucose-binding protein.

  Specific examples of the fucose-binding protein in the production method of the present invention include SEQ ID NO: 1, SEQ ID NO: 2 (amino acid sequence from 1 to 127 of the amino acid sequence shown in SEQ ID NO: 1), SEQ ID NO: 3 (sequence Amino acid sequence in which the 72nd cysteine residue of No. 2 is replaced with a glycine residue), SEQ ID NO: 4 (39th glutamine residue of SEQ ID NO: 2 is a leucine residue, and 72nd cysteine residue is a glycine residue) The amino acid sequence of SEQ ID NO: 5 (the glutamine residue at position 39 in SEQ ID NO: 3 is replaced with a leucine residue, the glutamine residue at position 65 is replaced with a leucine residue, and the cysteine residue at position 72 is replaced with a glycine residue) Amino acid sequence) SEQ ID NO: 6 (an oligopeptide containing a polyhistidine sequence at the N-terminal of the amino acid sequence shown by SEQ ID NO: 1 Amino acid sequence to which an oligopeptide containing an amino acid residue is added), SEQ ID NO: 7 (an amino acid sequence represented by SEQ ID NO: 2 is an oligopeptide containing a polyhistidine sequence at the N-terminal, and an oligopeptide having a cysteine residue at the C-terminal is SEQ ID NO: 8 (added amino acid sequence), SEQ ID NO: 8 (amino acid sequence represented by SEQ ID NO: 3 with an oligopeptide containing a polyhistidine sequence at the N-terminus and an oligopeptide containing a cysteine residue at the C-terminus), SEQ ID NO: 9 (an amino acid sequence obtained by adding an oligopeptide containing a polyhistidine sequence to the N-terminus of the amino acid sequence represented by SEQ ID NO: 4 and an oligopeptide containing a cysteine residue at the C-terminus) and SEQ ID NO: 10 (represented by SEQ ID NO: 5) An oligopeptide containing a polyhistidine sequence at the N-terminus of the amino acid sequence and a cysteine residue at the C-terminus It can be exemplified fucose binding protein represented by any one of the amino acid distribution) obtained by adding the oligopeptide. A signal peptide for promoting efficient expression in the host may be added to the N-terminal side of the fucose-binding protein in the production method of the present invention. When the host is Escherichia coli, examples of the signal peptide include PelB, DsbA, MalE, TorT, and other signal peptides that secrete proteins into the periplasm.

  The DNA encoding the fucose-binding protein in the production method of the present invention can be prepared by a known method. As the method for preparing the DNA, a method of converting the amino acid sequence of the fucose-binding protein into a base sequence in the production method of the present invention and artificially synthesizing a DNA containing the base sequence, or a fucose bond in the production method of the present invention Examples of the method include a method for directly artificially preparing a DNA encoding a sex protein, or a method for preparing a DNA from Burkholderia cenocepacia using a DNA amplification method such as a PCR method. In the preparation method, when designing the nucleotide sequence, it is preferable to consider the frequency of codon usage in transformed E. coli. For example, in arginine (Arg), AGA, AGG, CGG or CGA is isoleucine ( Ile) is ATA, leucine (Leu) is CTA, glycine (Gly) is GGA, and proline (Pro) is CCC, which are rare codons (rare codons). It is preferable to select and convert. Analysis of codon usage frequency is available in public databases (for example, Codon Usage Database on the website of Kazusa DNA Research Institute, http://www.kazusa.or.jp/codon/, access date: May 7, 2018). It is also possible to use.

  In order to transform Escherichia coli with the DNA encoding the fucose-binding protein prepared by the above method, the DNA itself may be used for transformation. For example, for transforming prokaryotic cells or eukaryotic cells, It is preferable to insert the DNA at an appropriate position in a vector based on a commonly used bacteriophage, cosmid, plasmid or the like to give an expression vector, and to transform the expression vector, because stable transformation can be carried out. . Here, the appropriate position means a position that does not destroy the region involved in the replication function of the expression vector, the desired antibiotic marker, and transmissibility. When inserting the DNA into the vector, it is preferable to insert the DNA in a state in which it is linked to a functional DNA such as a promoter required for expression. The vector used as the expression vector is not particularly limited as long as it can stably exist and replicate in the host, and examples thereof include pET vector, pUC vector, pTrc vector, pCDF vector, pBBR vector and the like. Examples of the promoter include trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, lpp promoter, and λ phage λPL promoter and λPR promoter. To transform Escherichia coli as a host using the expression vector, a method commonly used by those skilled in the art may be used. For example, as a host, Escherichia coli JM109 strain, Escherichia coli BL21 (DE3) strain, Escherichia coli NiCo21 (DE3) strain, When selecting Escherichia coli W3110 strain etc., the method etc. described in a well-known literature (for example, Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992) can be used.

  In addition, the binding affinity of the fucose-binding protein obtained by the production method of the present invention to a sugar chain can be evaluated by the Enzyme-linked immunosorbent assay method, the surface plasmon resonance method, or the like. The surface plasmon resonance method will be described as an example. For the binding affinity evaluation by the surface plasmon resonance method, for example, a Biacore T200 device (manufactured by GE Healthcare) can be used to measure the analyte as a recombinant protein and the solid phase as a sugar chain. The production of a sensor chip with a sugar chain immobilized was carried out by using a biotin-labeled sugar chain, a sensor chip coated with streptavidin (Sensor Chip SA, manufactured by GE Healthcare), or a sensor chip coated with dextran (Sensor Chip CM5). , GE Healthcare) to which streptavidin is immobilized in advance. In addition, the binding affinity evaluation can be performed using a kinetics analysis program attached to the device.

  According to the production method of the present invention, it is possible to suppress growth inhibition of Escherichia coli and expression inhibition of fucose-binding protein due to organic acids and the like that are by-produced by supplying an excessive carbon source. As a result, not only was it possible to efficiently produce a large amount of fucose-binding protein encoded by the expression vector introduced into E. coli, but also the effect of suppressing an increase in production cost due to excess raw materials, and after product recovery. It is also possible to obtain the effects of reducing environmental pollution and suppressing the cost of waste liquid treatment due to the reduction of organic substances remaining in the culture waste liquid.

7 is a transition of each measurement parameter in the production method of the present invention. 3 shows changes in dissolved oxygen (DO) concentration and glucose concentration during culture in Example 1. The horizontal axis represents time (unit is time), and the vertical axis represents dissolved oxygen concentration (unit is mg / L) and glucose concentration (unit is g / L).

  Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

Example 1
In Example 1, an oligopeptide containing a polyhistidine sequence at the N-terminal of the amino acid sequence of the fucose-binding protein represented by SEQ ID NO: 2 (1 to 127 of SEQ ID NO: 1) consisting of 127 amino acids and cysteine at the C-terminal The present invention relates to the production of a fucose-binding protein (hereinafter referred to as fucose-binding protein 127) to which an oligopeptide containing a residue is added.
(1) Preparation of expression vector pET-BC2LCN (127) cys and recombinant E. coli BL21 (DE3) / pET-BC2LCN (127) cys The expression vector pET-BC2LCN (127) cys expresses the fucose-binding protein 127. Is an expression vector. The amino acid sequence of the fucose-binding protein 127 is SEQ ID NO: 7, and the 5th to 10th amino acid sequences are polyhistidine sequences, the 15th to 141st amino acid sequences of SEQ ID NO: 2, and the 142nd to 148th amino acids are cysteines. Corresponds to the containing oligopeptide sequence.

The expression vector pET-BC2LCN (127) cys was prepared by the following procedure. Using the plasmid pET-BC2LCNcys disclosed in JP-A-2018-003838 as a template, each oligonucleotide comprising the sequences of SEQ ID NO: 16 and SEQ ID NO: 17 as a PCR primer, and disclosed in JP-A-2018-000038. PCR was performed according to the method described. Next, using this PCR product as a template, each oligonucleotide consisting of the sequences of SEQ ID NO: 16 and SEQ ID NO: 17 was used as a PCR primer, and PCR was carried out in the same manner. The obtained PCR product was digested with restriction enzymes NcoI and XhoI, and ligated with the expression vector pET28a (+) (manufactured by Merck Millipore) similarly treated with restriction enzymes. Escherichia coli BL21 (DE3) was transformed with this ligation product to prepare recombinant Escherichia coli BL21 (DE3) / pET-BC2LCN (127) cys. The recombinant Escherichia coli BL21 (DE3) / pET-BC2LCN (127) cys obtained by the method disclosed in Japanese Unexamined Patent Publication No. 2018-000038 is cultured, and the expression vector pET-BC2LCN ( 127) cys was obtained. As a result of confirming the base sequence by sequence analysis, it was confirmed that the expression vector pET-BC2LCN (127) cys contained the base sequence of SEQ ID NO: 12 encoding the amino acid sequence of SEQ ID NO: 2.
(2) Production of fucose-binding protein 127 using recombinant Escherichia coli The recombinant Escherichia coli BL21 (DE3) / pET-BC2LCN (127) cys produced in (1) of Example 1 above was used as a culture vessel in 500 mL of baffle. Using an Erlenmeyer flask equipped with a flask, inoculate a pre-culture medium (Table 1, sterilized) containing kanamycin sulfate at a concentration of 50 mg / L in 100 mL of 2 × YT medium and incubate at 30 ° C. for 20 hours at 130 times / min. The preculture was carried out at the shaking speed (amplitude 25 mm).

  In the preparation of the main culture medium, first, all components shown in Table 2 were added to a 3 L fermentor to prepare a solution, which was then sterilized by autoclave (121 ° C., 20 minutes) and cooled to room temperature.

  Next, an aqueous solution of each component shown in Table 3 was prepared separately, and aqueous solutions other than the kanamycin sulfate aqueous solution were sterilized by autoclaving (121 ° C, 20 minutes) and then cooled to room temperature. The kanamycin sulfate aqueous solution was sterilized with a 0.22 μm sterilizing filter (manufactured by Advantech). The sterilized aqueous solution of each component was added to the 3 L-volume fermenter containing the sterilized aqueous solution containing the components shown in Table 2 above (the amount shown in Table 3 was added) to prepare the main culture medium. did.

  Next, a feeding medium (components shown in Table 4) was prepared. Specifically, the glucose aqueous solution, the yeast extract aqueous solution, and the magnesium sulfate heptahydrate aqueous solution, which were prepared at appropriate concentrations, were autoclaved separately (121 ° C, 20 minutes), cooled to room temperature, and then autoclaved sterilized. The filter-sterilized aqueous solution of kanamycin sulfate and sterilized water were mixed in a sterilized container so as to have the composition shown in Table 4.

  To the fermenter containing the prepared main culture medium, 90 mL of the above preculture liquid was added to start main culture. In the main culture, Able's BMS-03PI type was used as the culture device, Able's φ70 mm four-blade impeller two-stage impeller combined with a cage net was used as the stirring blade, and the Able DO electrode was used as the DO electrode. It was carried out by adjusting the pH of the culture solution in the range of 6.9 to 7.1 with an air flow rate of 1.8 L / min, a culture temperature of 30 ° C. In addition, an aqueous ammonia solution or an aqueous phosphoric acid solution having an appropriate concentration was used to adjust the pH of the culture solution. The stirring speed at the start of main culture is 400 rpm, and the stirring speed is increased in steps when the dissolved oxygen concentration falls below 2.2 mg / L. Continued. In the culture device used for the main culture, the DO concentration in the culture medium, that is, the consumption state of the carbon source introduced in the initial stage of the main culture, is detected by detecting the signal from the DO electrode using a computer installed with the culture control program manufactured by Able. It was confirmed. The DO electrode was prepared by adding the above-mentioned main culture medium to a fermenter and bringing the temperature to 30 ° C., and then dissolved oxygen concentration in a state of stirring at an aeration rate of 1.8 L / min and a stirring blade speed of 900 rpm for 10 minutes. Was used as 7.5 mg / L (saturation concentration) after calibration.

The sampling solution periodically collected during the culture was analyzed by a spectrophotometer (U-2900 manufactured by Hitachi High-Tech Science) for OD ₆₀₀ (turbidity at ₆₀₀ nm), and a glucose analyzer (2700 manufactured by YSI). Was used to measure glucose concentration.

Five hours after the start of the main culture, the DO concentration did not decrease (the stirring rotation number did not increase), and the analysis values of the sample solution at this time were OD ₆₀₀ = 24 and glucose concentration = 0.1 g / L. In other words, it was suggested that glucose added to the culture medium at the start of the culture was metabolized and depleted by Escherichia coli, and the growth of E. coli was suppressed, resulting in a decrease in oxygen consumption and an increase in the DO concentration indicated by the DO sensor. ing.

  After 6 hours from the start of the main culture, the stirring rotation speed cascade control was terminated, the stirring rotation speed was fixed at 800 rpm, and at the time when the DO concentration exceeded 3.0 mg / L, the fed-batch medium (glucose, yeast, shown in Table 3 was used. The extract and magnesium sulfate) were set to be supplied dropwise by 0.2 mL each. A high-speed Watson Marlow metering pump 101U was used for supply, and the flow rate was set to 0.3 mL / min.

10 hours after the start of the main culture, OD ₆₀₀ reached 70, and the preset temperature of the culture solution was changed to 20 ° C. After confirming that the culture solution reached 20 ° C., the stirring rotation speed was changed to 600 rpm, and 1.8 mL of a 0.5 M IPTG aqueous solution prepared using IPTG (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added, Induction was initiated (final IPTG concentration = 0.5 mM). OD ₆₀₀ reached 100 after 27 hours from the start of the main culture, and the rotation speed of the stirring blade was lowered to 400 rpm. 48 hours after the start of the main culture, the OD ₆₀₀ reached 120, and the culture was completed.

  As a result of dropping the fed-batch medium by the method of adding a carbon source to the culture solution (DO stat method) in synchronism with the increase in DO concentration, as a result of the start of feeding (6 hours after the start of main culture), the end of culture (after 50 hours) ), The glucose concentration in the culture solution was maintained at 0.1 g / L or less, and the dissolved oxygen concentration during the same period fluctuated within the range of 0.1 mg / L to 3.7 mg / L (FIG. 2). ). This indicates that the carbon source and the nitrogen source were intermittently supplied by using the DO concentration that increased in response to the lack of glucose during the culture as an index. The amount of fed-batch medium used at the end of the culture was 401 g, the total carbon source (glucose) amount fed through the main culture was 156.6 g (including the initial input amount), and the total nitrogen source (yeast extract) amount was It was 75.1 g / L (including the initial input amount of 29.3 g / L).

  After completion of the culturing, 1650 mL of the obtained culture solution was dispensed into a 500 mL centrifuge container, and centrifuged using a centrifuge (main body: CR22H manufactured by Koki Holdings, rotor: R10A3 manufactured by Koki Holdings) (4 320 ° C, 3,000 xg, 15 minutes) to recover 320 g of bacterial cells. The extract having the composition shown in Table 5 was added to the collected cells and shaken and stirred at room temperature for 30 minutes, and then the additives shown in Table 6 were added and shaken at room temperature for 30 minutes and then at 4 ° C. overnight. After stirring, the soluble protein extract containing the fucose-binding protein 127 is recovered by collecting the supernatant by centrifugation (4 ° C., 12,000 × g, 30 minutes) using the aforementioned centrifuge. did. The recovered soluble protein extract containing the fucose-binding protein 127 was filtered through a 0.22 μm filter (manufactured by Advantech), and then the fucose-binding property was determined by nickel chelate affinity chromatography using His / Bind Resin (manufactured by Merck Millipore). The protein 127 was purified, and the absorbance at 280 nm of an aqueous solution containing the fucose-binding protein 127 purified using a quartz cell having an optical path length of 1 cm was measured. As a result of calculating the protein concentration in the aqueous solution by setting the molar extinction coefficient of the fucose-binding protein 127 to 1.0, and then calculating the productivity of the fucose-binding protein 127 per liter of the culture solution based on the obtained protein concentration. The productivity was 1240 mg / L-culture liquid.

Example 2
Example 2 was a fucose-binding protein in which an oligopeptide containing an N-terminal polyhistidine sequence of the amino acid sequence of recombinant BC2LCN represented by SEQ ID NO: 1 consisting of 155 amino acids was added to an oligopeptide containing a cysteine residue at the C-terminus. (Hereinafter, referred to as fucose-binding protein 155).
(1) Preparation of expression vector pET-BC2LCN (155) cys and recombinant E. coli BL21 (DE3) / pET-BC2LCN (155) cys The expression vector pET-BC2LCN (155) cys expresses fucose-binding protein 155. Is an expression vector. The amino acid sequence of the fucose-binding protein 155 is SEQ ID NO: 6, the 5th to 10th amino acid sequences are polyhistidine sequences, and the 15th to 169th amino acid sequences are SEQ ID NO: 1 (GenPept accession number: WP_006490828 (It matches the amino acid sequence of the 156th region), and the 170th to 174th positions correspond to oligopeptide sequences containing cysteine residues.

The expression vector pET-BC2LCN (155) cys has the same nucleotide sequence as the plasmid pET-BC2LCNcys disclosed in Japanese Unexamined Patent Publication No. 2018-000038, and was produced by the method disclosed in that publication. Next, E. coli BL21 (DE3) was transformed with the expression vector pET-BC2LCN (155) cys to prepare recombinant E. coli BL21 (DE3) / pET-BC2LCN (155) cys. The recombinant E. coli BL21 (DE3) / pET-BC2LCN (155) cys obtained by the method disclosed in Japanese Unexamined Patent Publication No. 2018-000038 is cultured, and the expression vector pET-BC2LCN ( 155) cys was obtained. As a result of confirming the base sequence by sequence analysis, it was confirmed that the expression vector pET-BC2LCN (155) cys contained the base sequence of SEQ ID NO: 11 encoding the amino acid sequence of SEQ ID NO: 1.
(2) Production of fucose-binding protein 155 using recombinant Escherichia coli Examples except that the recombinant Escherichia coli BL21 (DE3) / pET-BC2LCN (155) cys prepared in (1) of Example 2 was used. The fucose-binding protein 155 was produced under the culture conditions described in 1.

Glucose (19.5 g / L) contained in the initial medium was completely consumed 5 hours after the start of the main culture, and dropping of the fed-batch medium was started 6 hours after the start, and OD ₆₀₀ reached 140 after the same 50 hours. The culture was completed.

  As a result of dropping the fed-batch medium by the DO-stat method as in Example 1, the glucose concentration in the culture medium was 0 during the period from the start of feeding (6 hours after the start of main culture) to the end of the culture (after 50 hours). It was maintained below 0.1 g / L, and the dissolved oxygen concentration during the same period varied from 0.1 mg / L to 3.8 mg / L. The amount of fed-batch medium used at the end of the culture was 380 g, the amount of total carbon source (glucose) input through the main culture was 148.0 g (including the initial input amount), and the amount of total nitrogen source (yeast extract) was It was 72.3 g / L (including the initial input amount of 29.3 g / L).

  After the completion of the culture, by the method described in Example 1, 1600 mL of the obtained culture broth was centrifuged to collect 330 g of bacterial cells. Similarly, the fucose-binding protein 155 was purified from the cells recovered by the method described in Example 1 and the productivity of the fucose-binding protein 155 per liter of the culture solution was calculated. As a result, the productivity was 910 mg / L- It was a culture solution.

Example 3
Example 3 was prepared by adding a polyacetic acid at the N-terminal of the amino acid sequence of the fucose-binding protein consisting of 127 amino acid residues represented by SEQ ID NO: 3 (the amino acid sequence in which the 72nd cysteine residue of SEQ ID NO: 2 is replaced with a glycine residue). The present invention relates to the production of a fucose-binding protein (hereinafter referred to as fucose-binding protein 127C72G) in which an oligopeptide containing a histidine sequence is added to the C-terminal oligopeptide containing a cysteine residue.
(1) Preparation of expression vector pET-BC2LCN (127C72G) cys and recombinant E. coli BL21 (DE3) / pET-BC2LCN (127C72G) cys The expression vector pET-BC2LCN (127C72G) cys expresses the fucose-binding protein 127C72G. Is an expression vector. The amino acid sequence of the fucose-binding protein 127C72G is SEQ ID NO: 8, and the 5th to 10th amino acid sequences are polyhistidine sequences, the 15th to 141st amino acid sequences of SEQ ID NO: 3, and the 142nd to 148th amino acid residues are cysteine residues. Corresponds to oligopeptide sequences containing groups.

Using the expression vector pET-BC2LCN (127) cys of Example 1 (1) as a template, each oligonucleotide consisting of the sequences of SEQ ID NO: 16 and SEQ ID NO: 18 as a PCR primer, JP-A-2018-000038. PCR was performed according to the disclosed method. The obtained PCR product was digested with restriction enzymes NcoI and KpnI, and similarly ligated with the expression vector pET-BC2LCN (127) cys described in (1) of Example 1 which was treated with the restriction enzymes NcoI and KpnI. . E. coli BL21 (DE3) was transformed with this ligation product to obtain recombinant E. coli BL21 (DE3) / pET-BC2LCN (127C72G) cys. The recombinant Escherichia coli BL21 (DE3) / pET-BC2LCN (127C72G) cys obtained by the method disclosed in JP-A-2018-000038 is cultured, and the expression vector pET-BC2LCN ( 127C72G) cys was obtained. As a result of confirming the nucleotide sequence by sequence analysis, it was confirmed that the expression vector pET-BC2LCN (127C72G) cys contained the nucleotide sequence of SEQ ID NO: 13 encoding the amino acid sequence of SEQ ID NO: 3.
(2) Production of fucose-binding protein 127C72G using recombinant Escherichia coli Examples except that the recombinant Escherichia coli BL21 (DE3) / pET-BC2LCN (127C72G) cys produced in (1) of Example 3 was used. The fucose-binding protein 127C72G was produced under the culture conditions described in 1.

Glucose (19.5 g / L) contained in the initial medium was completely consumed 5 hours after the start of the main culture, dripping of the fed-batch medium was started ₆ hours later, and OD ₆₀₀ reached 110 after 52 hours later. The culture was completed.

  As a result of dropping the fed-batch medium by the DO-stat method as in Example 1, the glucose concentration in the culture medium was 0 during the period from the start of feeding (6 hours after the start of main culture) to the end of the culture (after 52 hours). It was maintained below 0.1 g / L, and the dissolved oxygen concentration during the same period varied from 0.1 mg / L to 3.8 mg / L. The amount of fed-batch medium used at the end of the culture was 450 mL, the amount of total carbon source (glucose) input through the main culture was 225.6 g (including the initial input amount), and the amount of total nitrogen source (yeast extract) was It was 1033.5 g / L (including the initial input amount).

  After the completion of the culture, by the method described in Example 1, 1570 mL of the obtained culture solution was centrifuged to recover 313 g of the bacterial cells. Similarly, the fucose-binding protein 127C72G was purified from the cells recovered by the method described in Example 1 and the productivity of the fucose-binding protein 127C72G per 1 L of the culture solution was calculated. As a result, the productivity was 920 mg / L- It was a culture solution.

Example 4
Example 4 is an amino acid sequence of the fucose-binding protein represented by SEQ ID NO: 4 consisting of 127 amino acid residues (the glutamine residue at position 39 of SEQ ID NO: 2 is a leucine residue, and the cysteine residue at position 72 is a glycine residue). An oligopeptide containing a polyhistidine sequence at the N-terminus of the amino acid sequence substituted with the group) is a fucose-binding protein (hereinafter, fucose-binding protein 127Q39L / C72G) to which an oligopeptide containing a cysteine residue is added at the C-terminus. ) Related to the production of.
(1) Preparation of expression vector pET-BC2LCN (127Q39L / C72G) cys and recombinant E. coli BL21 (DE3) / pET-BC2LCN (127Q39L / C72G) cys Expression vector pET-BC2LCN (127Q39L / C72G) cys was fucose-binding. It is an expression vector for expressing the protein 127Q39L / C72G. The amino acid sequence of the fucose-binding protein 127Q39L / C72G is SEQ ID NO: 9, the 5th to 10th amino acid sequences of which are polyhistidine sequences, the 15th to 141st amino acid sequences of SEQ ID NO: 4 and the 142nd to 148th amino acids. Corresponds to oligopeptide sequences containing cysteine residues.

A nucleotide sequence encoding the fucose-binding protein 127Q39L / C72G (a nucleotide sequence having a restriction enzyme site of XbaI and XhoI shown in SEQ ID NO: 19, GenScript) was synthesized and digested with the restriction enzymes XbaI and XhoI, and then the restriction enzyme XbaI. And the expression vector pET28a (+) (manufactured by Merck Millipore) treated with XhoI were ligated. In addition, 54th to 71st of the base sequence shown in SEQ ID NO: 19 is an oligopeptide containing a polyhistidine sequence, 84th to 464th is a polypeptide corresponding to the amino acid sequence of SEQ ID NO: 4, 465th to 485th Correspond to polynucleotides encoding respective oligopeptides containing cysteine residues. Next, by the method described in Example 1, recombinant E. coli BL21 (DE3) / pET-BC2LCN (127Q39L / C72G) cys and expression vector pET-BC2LCN (127Q39L / C72G) cys were obtained, and then the base was analyzed by sequence analysis. As a result of confirming the sequence, it was confirmed that the expression vector pET-BC2LCN (127Q39L / C72G) cys contained the base sequence of SEQ ID NO: 14 encoding the amino acid sequence of SEQ ID NO: 4.
(2) Production of fucose-binding protein 127Q39L / C72G using recombinant Escherichia coli except that the recombinant Escherichia coli BL21 (DE3) / pET-BC2LCN (127Q39L / C72G) cys produced in (1) of Example 4 was used. Produced the fucose-binding protein 127Q39L / C72G under the culture conditions described in Example 1.

Glucose (19.5 g / L) contained in the initial medium was completely consumed 5 hours after the start of the main culture, and dropping of the fed-batch medium was started ₆ hours later, and OD ₆₀₀ reached 115 after 48 hours later. The culture was completed.

  As a result of dropping the fed-batch medium by the DO-stat method as in Example 1, the glucose concentration in the culture medium was 0 during the period from the start of feeding (6 hours after the start of main culture) to the end of the culture (after 50 hours). It was maintained below 0.1 g / L, and the dissolved oxygen concentration during the same period varied from 0.1 mg / L to 3.8 mg / L. The amount of fed-batch medium used at the end of the culture was 483 g, the total carbon source (glucose) amount input through the main culture was 240.4 g (including the initial input amount), and the total nitrogen source (yeast extract) amount was It was 108.5 g / L (including the initial input amount).

  After the completion of the culture, the culture solution (1676 mL) obtained by the method described in Example 1 was centrifuged to recover 327 g of bacterial cells. Similarly, the fucose-binding protein 127Q39L / C72G was purified from the cells recovered by the method described in Example 1 and the productivity of the fucose-binding protein 127Q39L / C72G per 1 L of the culture solution was calculated. It was 883 mg / L-culture liquid.

Example 5
Example 5 is an amino acid sequence of the fucose-binding protein represented by SEQ ID NO: 5 consisting of 127 amino acid residues (the glutamine residue at position 39 of SEQ ID NO: 2 is a leucine residue, and the glutamine residue at position 65 is a leucine residue. (Amino acid sequence in which the cysteine residue at the 72nd position is replaced with a glycine residue), and a fucose-binding protein obtained by adding an oligopeptide containing a polyhistidine sequence to the N-terminus and an oligopeptide containing a cysteine residue to the C-terminus ( Hereinafter, it will be referred to as fucose-binding protein 127Q39L / Q65L / C72G.).
(1) Preparation of expression vector pET-BC2LCN (127Q39L / Q65L / C72G) cys and recombinant E. coli BL21 (DE3) / pET-BC2LCN (127Q39L / Q65L / C72G) cys Expression vector pET-BC2LCN (127Q39L / Q65L / C72G) cys is an expression vector for expressing the fucose-binding protein 127Q39L / Q65L / C72G. The amino acid sequence of the fucose-binding protein 127Q39L / Q65L / C72G is SEQ ID NO: 10, the 5th to 10th amino acid sequences of which are polyhistidine sequences, the 15th to 141st amino acid sequences of SEQ ID NO: 5 and the 142nd to 148th amino acids. Up to correspond to oligopeptide sequences containing cysteine residues.

A nucleotide sequence encoding the fucose-binding protein 127Q39L / Q65L / C72G (a nucleotide sequence having a restriction enzyme site for XbaI and XhoI shown in SEQ ID NO: 20, GenScript) was synthesized, digested with restriction enzymes XbaI and XhoI, and then restricted. A ligation reaction was performed with the expression vector pET28a (+) (manufactured by Merck Millipore) treated with the enzymes XbaI and XhoI. In addition, 54th to 71st of the base sequence shown in SEQ ID NO: 20 is an oligopeptide containing a polyhistidine sequence, 84th to 464th is a polypeptide corresponding to the amino acid sequence of SEQ ID NO: 5, 465th to 485th Correspond to polynucleotides encoding respective oligopeptides containing cysteine residues. Next, by the method described in Example 1, recombinant E. coli BL21 (DE3) / pET-BC2LCN (127Q39L / Q65L / C72G) cys and expression vector pET-BC2LCN (127Q39L / Q65L / C72G) cys were obtained, As a result of confirming the base sequence by sequence analysis, it was confirmed that the expression vector pET-BC2LCN (127Q39L / Q65L / C72G) cys contained the base sequence of SEQ ID NO: 15 encoding the amino acid sequence of SEQ ID NO: 5.
(2) Production of fucose-binding protein 127Q39L / Q65L / C72G using recombinant Escherichia coli Recombinant Escherichia coli BL21 (DE3) / pET-BC2LCN (127Q39L / Q65L / C72G) cys prepared in (1) of Example 5 above The fucose-binding protein 127Q39L / Q65L / C72G was produced under the culture conditions described in Example 1 except that the above was used.

Glucose (19.5 g / L) contained in the initial medium was completely consumed 5 hours after the start of the main culture, and dropping of the fed-batch medium was started 6 hours after the start, and OD ₆₀₀ reached 120 after 48 hours. The culture was completed.

  As a result of dropping the fed-batch medium by the DO-stat method as in Example 1, the glucose concentration in the culture medium was 0 during the period from the start of feeding (6 hours after the start of main culture) to the end of the culture (after 50 hours). It was maintained below 0.1 g / L, and the dissolved oxygen concentration during the same period varied from 0.1 mg / L to 3.8 mg / L. In addition, the amount of fed-batch medium used at the end of the culture was 470 g, the total carbon source (glucose) amount input through the main culture was 234.6 g (including the initial input amount), and the total nitrogen source (yeast extract) amount was It was 106.5 g / L (including the initial input amount).

  After the culturing was completed, according to the method described in Example 1, 1580 mL of the obtained culture solution was centrifuged to recover 310 g of bacterial cells. Similarly, the fucose-binding protein 127Q39L / Q65L / C72G was purified from the bacterial cells collected by the method described in Example 1 and the productivity of the fucose-binding protein 127Q39L / Q65L / C72G per 1 L of culture solution was calculated. The productivity was 900 mg / L-culture liquid.

Example 6
Example 6 relates to the production of fucose-binding protein 127Q39L / C72G using a host Escherichia coli different from that of Example 4.
(1) Preparation of expression vector pTrc-BC2LCN (127Q39L / C72G) cys and recombinant Escherichia coli W3110 / pTrc-BC2LCN (127Q39L / C72G) cys Expression vector pTrc-BC2LCN (127Q39L / C72G) cys is fucose binding protein 127Q39 / protein 127Q39. It is an expression vector for expressing C72G. Using the expression vector pET-BC2LCN (127Q39L / C72G) cys of Example 1 (1) as a template, each oligonucleotide consisting of the sequences of SEQ ID NO: 21 and SEQ ID NO: 22 as a PCR primer, JP-A-2018-000038. PCR was performed by the method disclosed in the publication. The obtained PCR product was digested with restriction enzymes NcoI and HindIII, and similarly digested with restriction enzymes NcoI and HindIII. Expression vector pTrc-PelBV3Km (carbenicillin in expression vector pTrc-PelBV3 prepared by the method described in WO2015 / 199154) Ligation reaction was performed with an expression vector into which a kanamycin resistance gene was introduced instead of the resistance gene. Escherichia coli W3110 was transformed with this ligation product to obtain recombinant E. coli W3110 / pTrc-BC2LCN (127Q39L / C72G) cys. The recombinant E. coli W3110 / pTrc-BC2LCN (127Q39L / C72G) cys obtained by the method disclosed in Japanese Patent Laid-Open No. 2018-000038 was cultured, and the expression vector pTrc-BC2LCN (127Q39L) was extracted by culturing the cells. / C72G) cys was obtained. As a result of confirming the base sequence by sequence analysis, it was confirmed that the expression vector pTrc-BC2LCN (127Q39L / C72G) cys contained the base sequence of SEQ ID NO: 14 encoding the amino acid sequence of SEQ ID NO: 4.
(2) Production of fucose-binding protein 127Q39L / C72G using recombinant Escherichia coli Performed except that the recombinant Escherichia coli W3110 / pTrc-BC2LCN (127Q39L / C72G) cys produced in (1) of Example 6 was used. The fucose-binding protein 127Q39L / C72G was produced under the culture conditions described in Example 1.

Glucose (19.5 g / L) contained in the initial medium was completely consumed 12 hours after the start of the main culture, and dropping of the fed-batch medium was started 13 hours after the start, and OD ₆₀₀ reached 130 after 72 hours. The culture was completed.

  As a result of dropping the fed-batch medium by the DO-stat method as in Example 1, the glucose concentration in the culture medium was 0 during the period from the start of feeding (6 hours after the start of main culture) to the end of the culture (after 50 hours). It was maintained below 0.1 g / L, and the dissolved oxygen concentration during the same period varied from 0.1 mg / L to 3.8 mg / L. The amount of fed-batch medium used at the end of the culture was 468 g, the amount of total carbon source (glucose) input through the main culture was 233.7 g (including the initial input amount), and the amount of total nitrogen source (yeast extract) was It was 106.2 g / L (including the initial input amount).

  After the completion of the culture, the obtained 1650 mL of the culture solution was centrifuged by the method described in Example 1 to recover 340 g of bacterial cells. Similarly, the fucose-binding protein 127Q39L / C72G was purified from the cells recovered by the method described in Example 1 and the productivity of the fucose-binding protein 127Q39L / C72G per 1 L of the culture solution was calculated. It was 950 mg / L-culture liquid.

Comparative Example 1
Described in Example 1 except that the composition of the fed-batch medium used in the main culture was changed to the composition shown in Table 7 and the weight ratio of the nitrogen source (yeast extract) to the carbon source (glucose) was 0.10 times. The fucose-binding protein 127 was produced by the recombinant E. coli and the culture conditions.

Glucose (19.5 g / L) contained in the initial medium was completely consumed 5 hours after the start of the main culture, the stirring rotation speed cascade mode was terminated 6 hours after the same, and the flow shown in Table 6 was fixed at a rotation speed of 800 rpm. The dropping of the supplemented medium was started. The OD ₆₀₀ was 30 after 10 hours from the start of the main culture, suggesting that the growth of E. coli was remarkably poor. 15 hours after the start of the main culture, OD ₆₀₀ was 33, but induction was initiated by adding IPTG (final IPTG concentration = 0.5 mM, culture temperature = 20 ° C.). The OD ₆₀₀ after 24 hours from the start of the main culture was 35, and the OD ₆₀₀ after 48 hours was 36. The culture was completed 56 hours after the start of the culture.

  During the period from the start of fed-batch (6 hours after the start of main culture) to the end of the culture (after 56 hours), the glucose concentration in the culture solution was maintained at 0.1 g / L or less, and the dissolved oxygen concentration during the same period was The range was from 0.7 mg / L to 3.9 mg / L. The amount of the fed-batch medium used was 120 mL, the total carbon source (glucose) amount input through the main culture was 70.9 g (including the initial input amount), and the total nitrogen source (yeast extract) amount was 34.4 g. / L (including the initial input amount of 29.3 g / L).

  After the completion of the culture, the obtained 1300 mL of culture solution was centrifuged by the method described in Example 1 to recover 100 g of bacterial cells. Similarly, the fucose-binding protein 127 was purified from the cells recovered by the method described in Example 1 and the productivity of the fucose-binding protein 127 per liter of the culture solution was calculated. As a result, the productivity was 130 mg / L- It was a culture solution.

Comparative Example 2
The fucose-binding protein 127 was produced using the recombinant E. coli and the culture conditions described in Example 1 except that induction with IPTG was not performed.

Glucose (19.5 g / L) contained in the initial medium was completely consumed 5 hours after the start of the main culture, the stirring rotation speed cascade mode was terminated 6 hours after the same, and the flow shown in Table 3 was fixed at a rotation speed of 800 rpm. The dropping of the supplemented medium was started. The OD ₆₀₀ after 10 hours from the start of the main culture was 65, suggesting that the growth of E. coli was remarkably poor. 15 hours after the start of the main culture, the OD ₆₀₀ became 65, and the culture solution was lowered to 20 ° C. and the culture was continued without adding IPTG. The OD ₆₀₀ 27 hours after the start of the main culture was 95, and the OD ₆₀₀ after 48 hours was 115, and the culture was completed at this stage.

  During the period from the start of fed-batch (6 hours after the start of main culture) to the end of the culture (after 48 hours), the glucose concentration in the culture solution was maintained at 0.1 g / L or less, and the dissolved oxygen concentration during the same period was The range was from 0.7 mg / L to 4.0 mg / L. In addition, the amount of the fed-batch medium used was 300 mL, the total carbon source (glucose) amount input through the main culture was 148.0 g / L (including the initial input amount), and the total nitrogen source (yeast extract) amount. Was 72.3 g / L (including the initial dose).

  After the completion of the culturing, according to the method described in Example 1, 1600 mL of the obtained culture solution was centrifuged to collect 310 g of bacterial cells. Similarly, the fucose-binding protein 127 was purified from the cells collected by the method described in Example 1 and the productivity of the fucose-binding protein 127 per liter of the culture solution was calculated. As a result, the productivity was 70 mg / L- It was a culture solution.

Test example 1
In Test Example 1, by the surface plasmon resonance method, the fucose-binding protein 127 produced in Example 1, the fucose-binding protein 155 produced in Example 2, the fucose-binding protein 127C72G produced in Example 3, and Example 4 were used. The binding affinity of the fucose-binding protein 127Q39L / C72G produced in 1. and the fucose-binding protein 127Q39L / Q65L / C72G produced in Example 5 to sugar chains was evaluated. Specifically, using a Biacore T100 (T200 Sensitivity Enhanced) device (manufactured by GE Healthcare), the analyte is a recombinant protein, and the solid phase is an H type 1 sugar chain or an H type 3 sugar chain for kinetic analysis. went. As the sensor chip, Sensor Chip CM5 (manufactured by GE Healthcare) coated with dextran was used, and streptavidin (manufactured by Wako Pure Chemical Industries) was immobilized on dextran by an amine coupling method, and then biotin-labeled H type 1 sugar. Chains or H type 3 sugar chains (manufactured by Glycotech) are added, and each sugar chain is immobilized on the sensor chip by the reaction of biotin and streptavidin to fix the H type 1 sugar chains or H type 3 sugar chains. A sensor chip was prepared. HBS-EP + (manufactured by GE Healthcare) was used as a buffer solution for measuring the sugar chain binding affinity, and the measurement conditions were a flow rate of 30 μL / min, a binding time of 6 minutes, and a dissociation time of 3 minutes or 6 minutes. Regeneration of the sensor chip was performed using 25 mM sodium hydroxide at a flow rate of 30 μL / min and a regeneration time of 30 seconds. Analysis was performed using Biacore T100 (T200 Sensitivity Enhanced) analysis software supplied with the instrument (Biacore T100 Evaluation Software, version or Biacore T200 Evaluation Software, version), and 1: calculate 1 Binding fitted by dissociation constants of the _{(K D)} did.

  As a result of calculating the dissociation constants of the fucose-binding protein 127 produced in Example 1 for the H type 1 type sugar chain and the H type 3 type sugar chain, the dissociation constant for the H type 1 type sugar chain was 2.4 nM and H type 3 The dissociation constant for the type sugar chain was 2.5 nM. As a result of calculating the dissociation constants of the fucose-binding protein 155 produced in Example 2 for the H type 1 type sugar chain and the H type 3 type sugar chain, the dissociation constant for the H type 1 type sugar chain was 3.9 nM and H type 3 The dissociation constant for the type sugar chain was 11 nM. As a result of calculating the dissociation constants of the fucose-binding protein 127C72G produced in Example 3 for the H type 1 type sugar chain and the H type 3 type sugar chain, the dissociation constant for the H type 1 type sugar chain was 2.1 nM and H type 3 The dissociation constant for the type sugar chain was 5.3 nM. As a result of calculating the dissociation constant of the fucose-binding protein 127Q39L / C72G produced in Example 4 for the H type 3 sugar chain, the dissociation constant for the H type 3 sugar chain was 3.9 nM. As a result of calculating the dissociation constant of the fucose-binding protein 127Q39L / Q65L / C72G produced in Example 5 for the H type 1 type sugar chain and the H type 3 type sugar chain, the dissociation constant for the H type 1 type sugar chain was 3.9 nM. , H type 3 sugar chain dissociation constant was 4.6 nM.

  From the above results, it was revealed that the fucose-binding protein produced by the production method of the present invention has binding affinity for H type 1 type sugar chains and H type 3 type sugar chains. Table 8 shows the results of evaluation of the binding affinities of the fucose-binding proteins produced in Examples 1 to 5 to the H type 1 type sugar chain and the H type 3 type sugar chain sugar chain.

Test example 2
In Test Example 2, fucose-binding protein 127 produced in Example 1, fucose-binding protein 155 produced in Example 2, fucose-binding protein 127C72G produced in Example 3, fucose-binding protein produced in Example 4. As the thermal stability evaluation of the protein 127Q39L / C72G and the fucose-binding protein 127Q39L / Q65L / C72G produced in Example 5, the denaturation midpoint temperature of each fucose-binding protein was measured. Specifically, a D-PBS (-) solution of each of the fucose-binding proteins was used as a dialysis buffer (50 mM sodium acetate, 50 mM sodium acetate, manufactured by Thermo Fisher Scientific, Inc., molecular weight cutoff 3500). Buffer exchange was performed in 150 mM sodium chloride, pH 5.5). The concentration of the recombinant protein in the dialysis solution was measured by the ultraviolet absorption method, diluted with a dialysis buffer to 500 μg / mL, and then subjected to a differential scanning calorimeter (MicroCalVP-Capillary DSC manufactured by Malvern Panatellitical Co., Ltd.). ) Was used to measure the denaturation midpoint temperature. The denaturation midpoint temperature is the temperature at which half of the protein is denatured, and the higher the denaturation midpoint temperature, the higher the stability to heat. The measurement conditions of the denaturation midpoint temperature were as follows: the amount of each fucose-binding protein solution after dialysis was 400 μL, the temperature rising rate was 60 ° C./h, and the heating temperature was 40 ° C.-110 ° C.

  As a result of the measurement, the denaturation midpoint temperature of the fucose-binding protein 127C72G was 81.9 ± 0.5 ° C., the denaturation midpoint temperature of the fucose-binding protein 155 was 82.3 ± 0.5 ° C., and the fucose-binding protein 127C72G was The denaturation midpoint temperature is 88.3 ± 0.5 ° C., the denaturation midpoint temperature of the fucose-binding protein 127Q39L / C72G is 94.4 ° C., and the denaturation midpoint temperature of the fucose-binding protein 127Q39L / Q65L / C72G is 88.3. It was ± 0.5 ° C. Table 9 shows the measurement results of the denaturation midpoint temperature of each fucose-binding protein produced in Examples 1 to 5.

Claims (7)

A method for producing a fucose-binding protein using Escherichia coli obtained by transforming an expression vector containing a DNA encoding a fucose-binding protein, comprising the following steps (1) to (5) A method for producing a fucose-binding protein, which comprises:
(1) A medium containing a carbon source and a nitrogen source is added to a culture tank, Escherichia coli obtained by transforming with an expression vector containing a DNA encoding a fucose-binding protein is inoculated, and a carbon source in a culture solution is added. A step of culturing with stirring while maintaining the dissolved oxygen concentration in the culture solution at 1.0 mg / L or more until the concentration becomes 5.0 g / L or less.
(2) At any time when the concentration of carbon source in the culture solution becomes 5.0 g / L or less, the weight of the nitrogen source supplied is 0.15 times or more and 2.00 times the weight of the carbon source supplied. Feeding of the carbon source and nitrogen source to the culture solution was started while maintaining the range of less than double, until the density of E. coli in the culture solution (OD ₆₀₀ , turbidity of the culture solution at ₆₀₀ nm) became 60 or more. Process of stirring culture.
(3) At any time when the density of E. coli (OD _{600 nm} , turbidity of the culture medium at 600 _nm ) in the culture medium of step (2) reached 60 or more, the concentration after the addition of the inducer was 0.005 mM. The step of adding to the culture solution so as to be 5.0 mM or less.
(4) After adding the inducer to the culture medium in step (3), the carbon source and the nitrogen source are continuously supplied to the culture medium and the stirring culture is continued until the density of E. coli in the culture medium becomes 90 or more and 180 or less. And a step of stopping the culture at any time.
(5) A step of recovering a fucose-binding protein from the culture obtained by culturing Escherichia coli in steps (1) to (4). The carbon source and the nitrogen source are fed while maintaining the dissolved oxygen concentration in the culture solution in the range of 0.01 mg / L to 5.0 mg / L in the steps (2) to (4). The manufacturing method described in. The production method according to claim 1 or 2, wherein, in the steps (2) to (4), the carbon source and the nitrogen source are fed while maintaining the carbon source concentration in the culture solution at 5.0 g / L or less. . The production method according to claim 1, wherein the carbon source is glucose and the nitrogen source is yeast extract. The production method according to claim 1, wherein the inducer is isopropyl-β-thiogalactopyranoside. The production method according to any one of claims 1 to 5, wherein the fucose-binding protein is the following (a) to (d):
(A) A fucose-binding protein comprising an amino acid sequence from the 1st proline residue to the Xth amino acid residue of the amino acid sequence represented by SEQ ID NO: 1, wherein X is an integer of 120 or more. Sex protein.
(B) an amino acid sequence from the first proline residue to the Xth amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in which one or more amino acids are deleted, substituted or added A fucose, which is a fucose-binding protein containing and has a binding affinity for a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc, and X is an integer of 120 or more. Binding protein.
(C) In the amino acid sequence from the 1st proline residue to the Xth amino acid residue of the amino acid sequence represented by SEQ ID NO: 1, any one of the following amino acid substitutions (1) to (3) A fucose binding protein (1) in which the amino acid sequence including the above is contained and X is an integer of 120 or more, and the 39th glutamine residue of the amino acid sequence represented by SEQ ID NO: 1 is replaced with a leucine residue (2). Substitution of 72nd cysteine residue of amino acid sequence represented by SEQ ID NO: 1 with one kind of amino acid residue selected from glycine residue and alanine residue (3) 65th position of amino acid sequence represented by SEQ ID NO: 1 In the amino acid sequence of the fucose-binding protein of (c) above, wherein the glutamine residue of SEQ ID NO: 39 is replaced with a leucine residue. A structure containing an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted or added in a region other than the second region, and comprising Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc A fucose-binding protein having binding affinity for a sugar chain containing the protein. The production method according to any one of claims 1 to 5, wherein the fucose-binding protein is the following (e) to (h):
(E) To the amino acid sequence from the 1st proline residue to the Xth amino acid residue of the amino acid sequence represented by SEQ ID NO: 1, an oligopeptide containing a polyhistidine sequence at the N-terminus is further added, and the C-terminus A fucose-binding protein consisting of an amino acid sequence to which an oligopeptide containing cysteine is added, and X is an integer of 120 or more.
(F) In the amino acid sequence from the 1st proline residue to the Xth amino acid residue of the amino acid sequence represented by SEQ ID NO: 1, in which one or more amino acids have been deleted, substituted or added On the other hand, a saccharide comprising an amino acid sequence having a polyhistidine sequence added to the N-terminal and an oligopeptide containing cysteine added to the C-terminal, and containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc. A fucose-binding protein, which has a binding affinity for a chain and X is an integer of 120 or more.
(G) To the amino acid sequence from the 1st proline residue to the Xth amino acid residue of the amino acid sequence represented by SEQ ID NO: 1, an oligopeptide containing a polyhistidine sequence at the N-terminus is further added, and the C-terminus In the amino acid sequence to which an oligopeptide containing cysteine is added, an amino acid sequence containing any one or more of the following amino acid substitutions (4) to (6) is included, and X is an integer of 120 or more, Fucose-binding protein (4) Substitution of the 39th glutamine residue of the amino acid sequence represented by SEQ ID NO: 1 with a leucine residue (5) Of the 72nd cysteine residue of the amino acid sequence represented by SEQ ID NO: 1, Substitution with one type of amino acid residue selected from glycine residue and alanine residue (6) The 65th group of the amino acid sequence shown in SEQ ID NO: 1 Substitution of a tamine residue with a leucine residue (h) In the amino acid sequence of the fucose-binding protein of (g) above, one or a plurality of amino acid sequences in regions other than the 39th position, the 65th position and the 72nd position of SEQ ID NO: 1 From an amino acid sequence having an amino acid residue deleted, substituted, inserted or added, an oligopeptide containing a polyhistidine sequence at the N-terminus and an oligopeptide containing cysteine at the C-terminus And a fucose-binding protein having a binding affinity for a sugar chain containing a structure consisting of Fucα1-2Galβ1-3GlcNAc and / or Fucα1-2Galβ1-3GalNAc.