GB2258868A - Microbiological method for the preparation of wheat germ agglutinin protein - Google Patents

Abstract

Disclosed are a maturation-type structural gene of a wheat germ agglutinin (WGA) and a gene coding for a precursor of wheat germ agglutinin expressed by a specific nucleotide sequence, a recombinant plasmid containing the said nucleotide sequence, a microorganism preferably yeast transformed with the said recombinant plasmid and a method for the preparation of a WGA by culturing the said transformed microorganism. The invention provides a possibility of producing, with stability and a large quantity, WGAs of a high purity by applying the techniques of genetic engineering.

Description

MICROBIOLOGICAL METHOD FOR THE PREPARATION OF WHEAT GERM AGGLUTININ PROTEIN The present invention relates to a method for the preparation of a wheat germ agglutinin by means of a method of genetic engineering utilizing a chemically synthesized wheat germ agglutinin gene.

Wheat germ agglutinin, referred to as WGA hereinafter, is a protein occuring in wheat germs which reportedly has an activity specifically to agglutinate cancer cells (see J.C.

Aub, et al., Proc. Natl. Acad. Sci., U.S.A., volume 50, page 613, 1963). Further, WGA has an activity to be specifically combined with oligosaccharides, glycoproteins, glycolipids and the like including N-acetylglucosamine, i.e. GlcNAc, and N-acetyl neuramic acid, i.e. NeuNAc, or sialic acid, so that it is now commercialized as a reagent for isolation and analysis of various kinds of living body constituents by utilizing the activity. Various grades of WGA products are commercially avialable including not only WGA as such but also immobilized WGA products supported on various kinds of carriers. In addition, it is proposed in Japanese Patent Kokai 62-257062 and 62-257063 to utilize WGA for the diagnosis of cancer as a kind of tumor marker because WGA can specifically be bonded to the cancer-related glycoproteins found in the serum or body fluid of cancer patients.

WGA is prepared heretofore from wheat germs as is described in J. Biol. Chem., volume 249, page 3116 (1974) by Nagata and Burger and in Methods in Enzymology, page 611, Academic Press (1974) by Nagata, et al. Commercially available WGA products are each usually a mixture of several kinds of WGA isomers as a reflection of the gene constitution in the wheat germs. The number of the WGA isomers cannot be smaller than 3 or 2 even by using the germs of wheat of a special variety as is reported by Rice in Biochim. Biophys. Acta, volume 444, page 175 (1976). Commercial WGA products now on the market are mostly supplied as a mixture of the WGA isomers without taking any measure for the mutual separation and purification of the isomers because such a treatment is considerably troublesome and lengthy resulting in an increase of the production costs.In addition, production and prices of the WGA products are unavoidably unstable because the available quantity of wheat germs as the starting material of WGA to be obtained from wheat as an agricultural product is naturally under the influences of the climate and other uncontrollable conditions in nature.

The present invention accordingly has an object to provide a novel method for the preparation of a WGA product consisting of a single WGA isomer at low costs and with stability by utilizing the technology of recombinant DNA in order to overcome the above described problems and disadvantages of the conventional WGA production not only in the quality but also in the stability of supply thereof.

In accordance with the present invention there is provided a gene coding for a maturation-type wheat germ agglutinin or a wheat germ agglutinin precursor said gene including at least a nucleotide sequence as shown by the sequence No. 1 below.

In one particular aspect the invention provides a maturation-type structural gene of WGA expressed by the nucleotide sequence shown by the Sequence No. 1 given below; Sequence No. 1 (i) Length of sequence: 513 (ii) Type of sequence: nucleic acid (iii) Number of strands: double strand (iv) Topology: normal chain (v) Kind of sequence: synthetic DNA (vi) Origin: (a) Name of organism: none (synthetic DNA) (b) Name of strain: none (vii) Characteristic of sequence: sequence of a gene to code maturation-form wheat germ agglutinin (WGA) (viii) Sequence: CAA AGA TGT GGT GAA CAA GAA TCC AAC ATG 30 GTT TCT ACA CCA CTT GTT CTT AGG TTG TAC GAA TGT CCA AAC AAC TTG TGT TGT TCT CAA 60 CTT ACA GGT TTG TTG AAC ACA ACA AGA GTT TAC GGT TAC TGT GGT ATG GGT GGT GAT TAC 90 ATG CCA ATG ACA CCA TAC CCA CCA CTA ATG TGT GGT AAG GGT TGT CAA GAC GGT GCT TGT 120 ACA CCA TTC CCA ACA GTT CTG CCA CGA ACA TGG ACT AGT AAG AGA TGT GGT TCT CAA GCT 150 ACC TGA TCA TTC TCT ACA CCA AGA GTT CGA GGT GGT GCT ACT TGT CCA AAC AAC CAC TGT 180 CCA CCA CGA TGA ACA GGT TTG TTG GTG ACA TGT TCT CAA TAC GGT CAC TGT GGT TTC GGT 210 ACA AGA GTT ATG CCA GTG ACA CCA AAG CCA GCT GAG TAC TGT GGT GCT GGT TGT CAA GGG 240 CGA CTC ATG ACA CCA CGA CCA ACA GTT CCC GGC CCA TGT AGA GCT GAT ATC AAG TGT GGT 270 CCG GGT ACA TCT CGA CTA TAG TTC ACA CCA TCT CAA TCT GGT GGT AAG TTG TGT CCA AAC 300 AGA GTT AGA CCA CCA TTC AAC ACA GGT TTG AAC TTG TGT TGT TCT CAA TGG GGC AGC TGT 330 TTG AAC ACA ACA AGA GTT ACC CCG TCG ACA GGT TTG GGT TCT GAA TTT TGT GGT GGT GGT 360 CCA AAC CCA AGA CTT AAA ACA CCA CCA CCA TGT CAA TCT GGT GCA TGC TCT ACT GAC AAG 390 ACA GTT AGA CCA CGT ACG AGA TGA CTG TTC CCA TGT GGT AAG GAC GCC GGC GGT AGA GTT 420 GGT ACA CCA TTC CTG CGG CCG CCA TCT CAA TGT ACT AAC AAC TAC TGT TGT TCT AAG TGG 450 ACA TGA TTG TTG ATG ACA ACA AGA TTC ACC GGT TCT TGT GGT ATT GGT CCC GGG TAC TGT 480 CCA AGA ACA CCA TAA CCA GGG CCC ATG ACA GGT GCT GGT TGT CAA TCT GGT GGT TGT GAC 510 CCA CGA CCA ACA GTT AGA CCA CCA ACA CTG GCT CGA The WGA structural gene described above may include a DNA region to code a signal peptide required for secretion at the 5' terminal and a translation-termination signal TGATAA at the 3' terminal.

Further a recombinant plasmid DNA can be formed containing the WGA gene having, in the WGA structural gene described above, a DNA region to code a signal peptide required for secretion at the 5' terminal and a translation-termination signal TGATAA at the 3' terminal. In such a plasmid the expression promotor may be a yeast promotor with the region to code the signal peptide at the downstream side of the yeast promotor and at the upstream side of the WGA gene, each being in coincidence with the decoding frame.

The recombinant plasmid DNA containing the WGA gene described above can be used to transform a host cell which may be a microorganism such as a yeast.

The transformed microorganism described above may be a yeast selected from the group consisting of the yeast strains belonging to the genus of Saccharomycesand yeast mutant strains belonging to the genus of Saccharomyces having mutation for extracellular secretion of a vacuole-localized protein. In particular the transformed microorganism may be a yeast mutant strain which is a strain of KS-58-2Ddel.

The invention also provides a method for the preparation of WGA comprising the steps of culturing the transformed microorganism described above in a culture medium to cause secretion of WGA into the medium by the microorganism and collecting the WGA from the culture medium.

In another aspect the invention provides a gene to code a WGA precursor expressed by the nucleotide sequence shown in the Sequence No. 2 given below; Sequence No. 2 (i) Length of sequence: 558 (ii) Type of sequence: nucleic acid (iii) Number of strands: double strand (iv) Topology: normal chain (v) Kind of sequence: synthetic DNA (vi) Origin: (a) Name of organism: none (synthetic DNA) (b) Name of strain: none (vii) Characteristic of sequence: sequence of a gene to code wheat germ agglutinin (WGA) precursor (viii) Sequence: CAA AGA TGT GGT GAA CAA GAA TCC AAC ATG 30 GTT TCT ACA CCA CTT GTT CTT AGG TTG TAC GAA TGT CCA AAC AAC TTG TGT TGT TCT CAA 60 CTT ACA GGT TTG TTG AAC ACA ACA AGA GTT TAC GGT TAC TGT GGT ATG GGT GGT GAT TAC 90 ATG CCA ATG ACA CCA TAC CCA CCA CTA ATG TGT GGT AAG GGT TGT CAA GAC GGT GCT TGT 120 ACA CCA TTC CCA ACA GTT CTG CCA CGA ACA TGG ACT AGT AAG AGA TGT GGT TCT CAA GCT 150 ACC TGA TCA TTC TCT ACA CCA AGA GTT CGA GGT GGT GCT ACT TGT CCA AAC AAC CAC TGT 180 CCA CCA CGA TGA ACA GGT TTG TTG GTG ACA TGT TCT CAA TAC GGT CAC TGT GGT TTC GGT 210 ACA AGA GTT ATG CCA GTG ACA CCA AAG CCA GCT GAG TAC TGT GGT GCT GGT TGT CAA GGG 240 CGA CTC ATG ACA CCA CGA CCA ACA GTT CCC GGC CCA TGT AGA GCT GAT ATC AAG TGT GGT 270 CCG GGT ACA TCT CGA CTA TAG TTC ACA CCA TCT CAA TCT GGT GGT AAG TTG TGT CCA AAC 300 AGA GTT AGA CCA CCA TTC AAC ACA GGT TTG AAC TTG TGT TGT TCT CAA TGG GGC AGC TGT 330 TTG AAC ACA ACA AGA GTT ACC CCG TCG ACA GGT TTG GGT TCT GAA TTT TGT GGT GGT GGT 360 CCA AAC CCA AGA CTT AAA ACA CCA CCA CCA TGT CAA TCT GGT GCA TGC TCT ACT GAC AAG 390 ACA GTT AGA CCA CGT ACG AGA TGA CTG TTC CCA TGT GGT AAG GAC GCC GGC GGT AGA GTT 420 GGT ACA CCA TTC CTG CGG CCG CCA TCT CAA TGT ACT AAC AAC TAC TGT TGT TCT RAG TGG 450 ACA TGA TTG TTG ATG ACA ACA AGA TTC ACC GGT TCT TGT GGT ATT GGT CCC GGG TAC TGT 480 CCA AGA ACA CCA TAA CCA GGG CCC ATG ACA GGT GCT GGT TGT CAA TCT GGT GGT TGT GAC 510 CCA CGA CCA ACA GTT AGA CCA CCA ACA CTG GCT GTT TTC GCT GGT GCT ATT ACC GCT AAC 540 CGA CAA AAG CGA CCA CGA TAA TGG CGA TTG TCC ACC TTG TTG GCT GAA AGG TGG AAC AAC CGA CTT The gene described above to code the WGA precursor may include a DNA region to code a signal peptide required for secretion at the 5' terminal and a translation-termination signal TGATAA at the 3' terminal.

Further a recombinant plasmid DNA can be formed containing the gene to code the WGA precursor having, in the gene described above, a DNA region to code a signal peptide required for secretion at the 5' terminal and a translation-termination signal TGATAA at the 3' terminal. In such a plasmid the expression promotor may be a yeast promotor with the region to code the signal peptide at the downstream side of the yeast promotor and at the upstream side of the WGA gene, each being in coincidence with the decoding frame.

A host cell may be transformed with the recombinant plasmid DNA containing the WGA gene described above and the host cell may be a microorganism such as a yeast. The yeast may be selected from the group consisting of the yeast strains belonging to the genus of Saccharomyces and yeast mutant strains belonging to the genus of Saccharomyces having mutation for extracellular secretion of a vacuole-localized protein.

The transformed microorganism may be a yeast mutant strain which is a strain of KS-58-2Ddel.

A method for the preparation of WGA is also provided which comprises the steps of culturing the transformed microorganism described above containing a gene having nucleotide sequence No. 2, in a culture medium to cause secretion of WGA into the medium by the microorganism and collecting the WGA from the culture medium.

Figures 1A, 1B, 1C and 1D are each a scheme of the enzymatic method of ligation and subcloning of the block A, block B, block C+D and block A, B, C+D, respectively.

Figure 2A illustrates an assembly of the secretionexpression plasmid of the WGA genes in the maturation form and precursor form.

Figure 2B illustrates an assembly of the secretionexpression plasmid of the WGA genes in the maturation form and precursor form utilizing the promotor and the prepro region of the a-factor gene in yeast.

Figure 3 shows a Western blot analysis of the secreted WGA protein.

Figure 4A is an elution diagram for the separation of the WGA proteins in the culture of yeast with a Mono S column.

Figure 4B is an elution diagram for the separation and purification of the WGA fraction obtained in Figure 4A with a Mono Q column.

Figure 4C is an elution diagram for the separation and purification of the WGA fraction obtained in Figure 4B with a Mono S column.

Figure 4D is an elution diagram for the separation of a commercial authentic sample of WGA as a mixture of WGA I, II and III with a Mono S column.

Figure 5 is a diagram of electrophoresis with SDS-15% polyacrylamide gel in the purity assay of a recombinant WGA protein.

Figure 6 is an illustration of the measuring method for the sugar-chain binding activity of a WGA protein.

Figure 7 illustrates comparison of the sugar-chain binding activity between a recombinant WGA protein and an authentic sample of WGA protein.

It is known that the method for the preparation of a WGA utilizing the techniques of recombinant DNA by use of a synthetic WGA gene consists basically of the steps including: 1) designing of the gene; 2) chemical synthesis of the gene and introduction of the same into a suitable expression vector; and 3) transformation of a suitable host by means of the thus obtained recombinant plasmid and production and recovery of the WGA protein by culturing the transformant.

In the production of a useful protein by the genetic engineering technology, in general, it is necessary when the protein is accumulated in the cells that the cells are collected and homogenated followed by isolation and purification of the desired protein from the homogenate. It is usual that great difficulties are encountered in the procedure of isolation and purification of the desired protein because of the large amount of contaminant proteins contained in the cells. When yeast cells are utilized, in addition, the efficiency in the homogenation of the cells is also an important problem because of the toughness of the walls of the yeast cells. Accordingly, it would be a very efficient way also in the production of a protein by utilizing the technology of recombinant DNA to have extracellular secretion of the protein.Since the efficiency of protein secretion from cells largely depends on the nature or characteristics of the protein, the process parameters must be selected after experimentation for the respective desired protein.

As is reported by M.A. Mansfield, et al. in Plants, volume 173, pages 482-489 (1988), distribution of the WGA protein in a plant body is localized either in the vacuoles or in the organelles called a protein body in the cells.

Since this protein body is presumably a structural body formed by the fragmentation of vacuoles, it is suggested by N.V. Raikhel, et al. in Curr. Top. Plant. Biochem. Physiol., volume 7, pages 83-89 (1988) that the protein is biosynthesized in the plant body on the membrane of endoplasmic reticulum (ER) followed by transport from the ER to the Golgi apparatus through the secretion passway in the cells and subsequent transport to the vacuoles or protein bodies.

In the case of utilization of yeast cells for the expression of the WGA gene, accordingly, it is presumable that, as in the plant cells, the protein is usually transported to the vacuoles of the yeast or organelles corresponding thereto.

On the other hand, it is reported by T.H. Stevens, et al.

in J. Biol. Chem., volume 102, pages 1551-1557 (1986) that, in the yeast cells, extracellular secretion of the carboxypeptidase Y (CPY) protein, which is inherently localized in the vacuoles, occurs when CPY is highly expressed on the multicopy-plasmid. The reason therefor as understood is that, according to the above mentioned report by T.H.

Stevens, et al., since the amount of the intracellular constituent pertaining to the transport selection from the Golgi apparatus to the vacuoles is limited, failure is caused in the transport selection from the Golgi apparatus to the vacuoles when the production of the CPY protein exceeds this limit but transport of the same proceeds toward the secretory granules finally leading to extracellular secretion. As a consequence, an idea was obtained that it would be possible also for the WGA protein, which should duly be transported to the vacuoles, that secretion of the WGA protein would occur out of the yeast cells by the expression under control of the high-expresson promotor or by the use of a mutant of the yeast having a defective mutation relative to the transport selection from the Golgi apparatus to the vacuole.

While three or four kinds of isomers are known for the WGA protein, the amino acid sequence and the base sequence of the c-DNA have been reported for three of the isomers including WGA I, II and III, of which WGA II has been studied in great detail to give the results including the detailed steric structure obtained by the X-ray diffractometric crystallographic analysis and the steric structure of the complex between the WGA II protein and an oligosaccharide along with accumulation of much information on the types of the bonding thereof with saccharides. Therefore, studies have been undertaken on the secretion expression of the WGA proteins with yeast as the host taking the WGA II protein as a typical case thereof.In particular, designing and chemical synthesis of the WGA II gene were undertaken on the basis of the reported amino acid sequence of the WGA II protein but by using the codon of frequent use in yeast, not the codon of wheat, as the codon on the DNA corresponding to the amino acid. It should be understood, however, that the scope of the present invention is never limited by the above mentioned procedure but the invention is applicable also to the preparation of the genes of WGA I or WGA III by the introduction of a site-specific mutation to the WGA II gene by a known method followed by secretion expression thereof with yeast and to the secretion expression of the above mentioned genes with suitable host cells other than yeast.

(I) Designing of the gene As to the EGA II gene, the base sequence of the c-DNA is reported by Smith and Raikel in Plant Molecular Biology, volume 13, pages 601-603 (1989) pointing out three errors in the amino acid sequence previously reported by Wright and Olafsdottir in J. Biol. Chem., volume 261, pages 71917195 (1986). Namely, corrections were effected from Ser to Phe at the 109th, from Gly to Lys at the 134th and from Gly to Trp at the 150th. Designing of the WGA II gene was performed accordingly by utilizing the amino acid sequence which could be deduced from the sequence of the c-DNA.A difference is still noted at the 37th between the amino acid sequence determined from the WGA II protein and that deduced from the c-DNA since the amino acid by the former method is Asp while that by the latter method is Asn according to the report. As an interpretation of the analytical results relative to the amino acid sequence at the level of protein, there can be a possibility of the deamination reaction from Asn to Asp within the living body but it would be a generally reasonable assumption that the deamination reaction would occur in the course of the preparation of the peptide fragments suitable for the determination of the amino acid sequence from a protein or in the course of isolation and purification of the peptide fragments.This is the reason for the typical assumption of Asn deduced from the c-DNA in the present invention as the 37th amino acid residue in the maturation-form WGA II protein.

In designing the WGA II gene corresponding to this amino acid sequence, however, several codon selections are possible due to the degeneration of the gene codons. In particular, the codon frequently used for plants such as wheat as reported by Smith and Raikel in Plant Molecular Biology, volume 13, pages 601-603 (1989) not always coincides with that in yeast reported by Bennetzen and Hall in J. Biol. Chem., volume 257, pages 3026-3031 (1982).

Accordingly, the designing of the WGA II gene was conducted with consideration of: a) the use of the most probable codon in the yeast which was considered to be the most suitable for the expression of a synthetic gene; b) a specific restriction enzyme-recognizing site provided in the gene to facilitate subcloning; and c) avoidance of a sequence which is self-complementary or complementary with a sequence other than the correct one on one and the same strand or on a complementary strand as far as possible.

A particular example satisfying these various requirements is given by sequence No. 1 or sequence No 2 shown above as the scope of the present invention.

While the present invention primarily relates to the DNA having the synthetic gene for the expression of WGA shown by the above given DNA sequence, the DNA according to the invention includes, in addition to the above mentioned synthetic gene, those having a translation-initiating codon or a DNA sequence to code the signal peptide for secretion ligated to the 5' terminal thereof, those having a translation-terminating codon ligated to the 3' terminal thereof, those with both of them ligated together and those having a restriction enzyme cleavage site at each of the 5' and 3' terminals so as to facilitate expression and the procedure of gene replacement. In addition, the scope of the present invention also includes the plasmid obtained by the introduction of these DNAs.

In wheat germs, the WGA protein possesses, besides the maturation-form WGA protein region, a prepeptide consisting of 28 amino acid residues including Met indispensable for the initiation of translation at the amino end, i.e. N end, as well as an extraneous propeptide consisting of 15 amino acid residues at the carboxyl end, i.e. C end, and is biosynthesized as a so-called prepro-type precursor WGA protein as is deduced from the analysis of the c-DNA sequence thereof by N.V. Raikhel, et al. in Proc. Natl.

Acad. Sci., U.S.A., volume 84, pages 6745-6749 (1987) and by Smith and Raikel in Plant Molecular Biology, volume 13, pages 601-609 (1989).

Accordingly, it would be possible to have expression in the cells of a plant such as tobacco by using the DNA sequence as such corresponding to the protein of the precursor WGA or the prepro WGA but the WGA protein expressed so far would be transported to the vacuoles as is suggested by T.A. Wilkins, et al. in The Plant Cell, volume 2, pages 301-313 (1990). It is accordingly necessary to develop a method leading to a success of secretion expression by utilizing the cells not belonging to a plant A questionable matter there is whether or not the secretion signal inherent in the WGA can properly function in the cells not belonging to a plant.For example, the secretion signal of human interferon consisting of 23 amino acid residues exhibits a heterogeneous signal cleavage point indicating an incomplete function of this signal in a yeast relative to the secretion of human interferon in yeast as is reported by R.A. Hitzeman, et al. in Science, volume 219, page 620 (1983). In most cases, the secretion signal of yeasts in general has a length consisting of 15 to 20 amino acid residues while the signal of WGA has a larger length consisting of 28 amino acid residues. It is reported by Y. Yamamoto, et al. in Biochem. Biophys. Res.Commun., volume 149, page 431 (1987) that, on the other hand, as an effective secretion signal for the secretion of a foreign protein in yeasts, not only those inherent in the yeast but also the signals derived from a foreign organism or artificial signals containing a hydrophobic Leu cluster and consisting of 15 amino acids work efficiently. In the study of expression of secretion of a WGA protein by a yeast, accordingly, the above mentioned various secretion signals working in yeasts can also be utilized. As a particular example in this regard, an example is shown later for the results obtained by the chemical synthesis and use of a DNA fragment to code the above mentioned artificial signal peptide.As the WGA gene ligating to the DNA corresponding to this signal peptide, referred to as the pre-region hereinbelow, the precursor-type WGA gene having a C-end pro-region can be used besides the maturation-type having no C-end pro-region. Therefore, studies have been undertaken for the expression of the respectively synthesized pre-WGA gene, i.e. the DNA region to code the secretion signal plus maturation-form WGA, and the prepro WGA gene, i.e. the DNA region to code the secretion signal plus precursor-form WGA.

While various kinds of host cells can b used to have expression of the DNA containing these WGA genes, a particular example with a yeast as the host is given by the utilization of the ENO 1 promotor which is one of the genes in the glycolytic pathway of yeasts and has a high efficiency for expression, as is suggested by K. Ichikawa, et al. in Agric. Biol. Chem., volume 53, pages 1445-1447 (1989) . On the other hand, the promotor and the prepro region of the a-factor of yeasts are under frequent use as the gene to have a secretion expression of a foreign protein from yeast as is reported, for example, by Brake, et al.

in Proc. Natl. Acad. Sci., U.S.A., volume 81, page 4642 (1984) and by Bitter, et al. in the same journal, page 5330.

As a further particular example of the present invention, preparation has been undertaken of a gene for secretion expression in which the above mentioned maturation-type WGA gene or the precursor-type WGA gene having a C-end proregion is ligated to the downstream side of the DNA region having a promotor and a prepro region of the a -factor gene of yeast.

It is not an easy matter to correctly foresee whether or not these various genes for secretion expression are duly expressed in the yeast and whether or not the recombinant WGA protein secreted in the medium has the same amino acid sequence and sugar-chain binding activity as the WGA II protein isolated from wheat germs. It is also of some interest whether or not a correct cleavage may take place in the pro-WGA protein having a C-end region in the course of secretion in yeast.As is understood from the examples given later, however, the present invention gives a conclusion that correct cleavage of the WGA precursor protein by yeast takes place not only in the signal peptide but also in the C-end pro-region and it has a molecular weight identical with that of the WGA II protein isolated from wheat germs along with the same specific sugar-chain binding activity as that of the WGA II derived from wheat germs.

(II) Synthesis of genes and assembly of plasmid for expression The above mentioned WGA gene can be prepared, for example, by synthesizing a plural number of oligodeoxynucleotides having a strand length of 15-45 nucleic acid bases and ligating them together. For example, synthesis can be performed according to the disclosure by Muraki, et al. in Agric. Biol. Chem., volume 50, pages 713-723 (1986) after dividing into 32 oligonucleotide fragments as is shown in Sequence No. 3 shown below.

Sequence No. 3 (i) Length of sequence: 588 (ii) Type of sequence: nucleic acid (iii) Number of strands: double strand (iv) Topology: normal chain (v) Kind of sequence: synthetic DNA (vi) Origin: (a) Name of organism: none (synthetic DNA) (b) Name of strain: none (vii) Characteristic of sequence: constituted from a part of a yeast ENO 1 promotor, artificial secretion signal and gene to code maturation-form WGA (viii) Sequence::

irCtAC;AC ACAAACAC tAAA TCAAA 1 < TGTGTTTGIGATTTAGTTI saLr S) -15 -io -1 1111 L L L L L L L L P LA L G Sl t g 3 53 ATGAGATTGTTGTTGTTG TGTTGTTGTTGCCATTGGCCtTAGG TACTCTAACAACAACAACAACAACAACAACGGTAACCGGAATCCA 52 t 54 1 10 20 0 R C G E 0 G S NIlE C P N N L c C S a S3 CCA. I 3 CAAAGATGTGGTGAACA GGATC ACATGGAATGTCCAAACAACTTGTGTTGTTCTCAA GTTTCrACACCACrTGTgCCTAG8lTTGTACCTTACAGGATTTGTTGAACAcAAcAAGArT 2 < T 2 1 a 30 tO Y G Y C G n G G 0 Y C G K G C G N G A C + 5 1 7 TACGGTTACTGTGGTATGGGTGGTGATTACTGTGGTAAGGGTTGTCAAAACGGTGCTTGT ATGCCAAT0ACACCATACCCACCACTAAT0ACACCATTCCCAACA0TTYTGCCAC0AACA t 50 6a IllS KR C OS 0 AG GA TC P N N N C q 1 It TGGICTAGT AAGAGATG5GGTTCTCAAGCTGGTGGTGCTACTTGTCCAAACAACCACTGT ACCIGATCA TTcTcTAcACcAAGAGTTCGACCACCtACGATGAAcAGGTTTGTTGGTGAcA t 5QI 70 2 rL 10 C S G Y G H C G f G A E r c G A G C 0 G 13 $ rS 1 TGTTCTCAATACOOTCACTOTGOTTTCOOTOCiOAGTA GAGTACTGTGGTGcTGGrTGTcAAGG G ACAAGAGTTArGCCAGTGACACCAAAGCCACGA CTCATG tCACCACGACCAACAGTTCC v t r Sac t 90 100 t C RA 0 t K C G SOS G G K L C P N 117 9 GGCC TGTAGAGC ATATC AGTGTGGTTCTCAATCTOOTOOTAAGTTGTGTCCAAAC CCGGG ACATCTCG CTATAG#TTCACACCAAGAGTTAGACCACCATTCAACAcAGGTTTG t ç7Rl lg TEECKV 20 4 110 120 N L C C SOW G F C G L G SE F C G G G it T 23 AACTTGTGTTGTTCTCAAfGGGGCTTCTGrGGTTTGGGTTCTGAATTTTGTGGrGGTGGT TTOAACACAACA%G1AGTTACCCcGAAGACACCAfACCCAAOACTTAAAACACCA2CCACCA 22 1 24 130 140 CO SO A CS T OK PC 0K 0 AG OR V TGTCAATCTGG GCArG rCTACTGACAAGCCATGTGGTAAOOA CCGGC GTAGAGTT ACAOTTAGACC COTAC AGATGACTGTTCGGTACACCATTCCTGsC66CCGCATCTCAA sI 26 HatI 150 160 C T N N Y C C S K W G S C G 1 G P G r c 27 TGTACTAACAACTACTGTTOTTCTAAGTOGGGTTCTTGTOGTATTGGr CCGGG ACTOT AATGAT TG TTGATGACAACAAGATTCACCCCAAGAACACCATAAcCA GGCCC TOACA t 2 128 t S?LI(AvaI) GAG C OS GO CO A GGTGcTGGT;TGTcAATCTGGTGGTTGTGACGCTTGATIAt ~ CCACGA3C0CAACAGT TAGACCCdAACACTGCAACT TTCGA 3O r 32 dXt The oligonucleotide fragments can be hybridized and enzymatically ligated together by a known method taught by Agarwal, et al. in Nature, volume 227, pages 27-34 (1970).

Incidentally, the dividing manner into fragments is not limited to the above but various ways of dividing are possible if care is taken to avoid the above mentioned self-association.

The next step to follow is to first conduct introductory subcloning of the thus obtained synthesized genes or fragments thereof into a suitable vector in order to obtain quantitative multiplication. In the following, detailed description is given as a particular example for the above mentioned steps relative to each of the maturation-type WGA gene and the precursor-type WGA gene.

(11-1) Synthesis and cloning of the maturation-type WGA gene.

As is reported by C.S. Wright in J. Mol. Biol., volume 194, pages 501-529 (1987), the maturation-form WGA protein consisting of 171 amino acids are composed of spatially discrete four domains A, B, C and D and each of these domains consists of 41 amino acid residues in a sequence having very high homology with the others as is shown by the result of analyses of amino acid sequence or steric structure. This is the reason for conducting division of the former half portion of the WGA gene in the designing and synthesis of the genes in such a way that the oligonucleotide blocks coincide with the above mentioned domains A and B in order to avoid hybridization of the nucleotide fragments with other ones having incorrect sequence.It was found, however, that, in the assembly of the DNA fragments of the blocks in this way of dividing, the number of steps is increased for the enzymatic ligation consequently resulting in a decrease in the yield of the final product by the ligation so as to decrease the probability of success in the subsequent subcloning. Accordingly, assembly of the DNA fragments was conducted for the latter half portion of the WGA gene with the DNAs corresponding to the above mentioned domains C and D as a block in combination. As to the block A corresponding to the domain A., a design was made that a site of the restriction enzyme BamH I was provided at the position bridging the 7th, i.e. Gly, and the 8th, i.e.Ser, to first prepare the downstream side of this site and coincidence is obtained for the decoding frame at the BamH I site by preparing the side toward the N-end therefrom by the simultaneous preparation with the preparation of the secretion signals mentioned later.

Synthesis of the gene designed in the above described way can be performed by a method in which several fragments obtained by dividing each of the (+) and (-) strands are chemically synthesized separately and these fragments are ligated together. It is preferable that each of the strands is divided into about 32 fragments each consisting of 15-45 bases to have overlappings over at least 6 bases lengths in each fragment with another. Figures 1A to 1D are each a schematic illustration of an outline of the above described procedures.

Figure 1A is for the assembly of the block A corresponding to the domain A. The final product of the enzymatic ligation of this domain A was subjected to cloning by ligating with the BamH I-Xba I fragment of the Phagescript as the vector for subcloning after digestion with the restriction enzymes Spe I. After recovery of the DNA containing the WGA gene fragments from this cloned DNA by the double digestion with BamH I and the Sac I, the BamH I Mae I fragments obtained by the digestion thereof with the restriction enzyme Mae I were taken as the DNA fragments for ligation with the block B and block C+D.For the block B corresponding to the domain B, similarly, double digestion of the final enzymatic ligation product with the restriction enzymes Spe I and Apa I was followed by the subcloning at the site of Spe I-Apa I of Bluescript KS(+). After recovery of the DNA containing the WGA gene fragments from this cloned DNA by the digestion with Apa I and Sac I, the Mae I-Apa I fragments obtained by further digestion thereof with the restriction enzyme Mae I were taken as the DNA fragments for the ligation between the respective blocks (see Figure 1B).

As to the block C+D corresponding to the domains C and D, on the other hand, the strand length of the oligonucleotide fragments was 2 to 3 times larger than that of the blocks corresponding to the domains A and B as is mentioned above so that, despite the substantially the same number of repeated enzymatic ligation as in the above mentioned preparation of the block A or B, the strand length of the final coupling product was about twice larger to correspond to the C+D. Since this block C+D has the Apa I site and Hind III site at the 5' and 3' ends, respectively, subcloning at the Apa I-Hind III site of the Phagescript was followed by the digestion thereof with Apa I and Hind III to recover the DNA containing the WGA gene fragments, which were taken as the DNA fragments for the ligation between the respective blocks (see Figure 1C).

Finally, the above prepared respective DNA fragments of A, B and C+D were ligated together in the order indicated in Figure 1D and the same was cloned at the BamH I-Hind III site of the plasmid vector pUC18. The base sequence of the thus prepared maturation-type WGA gene was identified to be just as designed from the results of the analysis of the base sequence in the whole region of both of the (+) and (-) strands of the DNA fragment conducted according to the known method for the base sequence determination of DNA, i.e. the dideoxy nucleotide method described by F. Sanger, et al. in Proc. Natl. Acad. Sci., U.S.A., volume 74, page 5463 (1977)- (II-2) Synthesis of WGA (pre-WGA) having secretion signal and introduction thereof into vector for expression.

As is described above, the WGA protein is synthesized in wheat as a prepro-form having a secretion signal and an extraneous peptide (CT) consisting of 15 amino acid residues at the N- and C-ends, respectively, of the maturation-form WGA, of which the secretion signal has the indispensable function to be transported to the ER lumen among with the biosynthesis of the protein and thereafter to be transported to the outside of the cells through the secretion passway.

Accordingly, it is necessary also in the secretion expression of the WGA protein with yeast that this secretion signal is added to the maturation-form WGA at the N-end.

Usable secretion signal includes not only the signals inherent in yeasts such as invertase and acid phosphatase as a matter of course but also those foreign signals not belonging to yeasts such as chicken lysozyme and the like.

In addition, the artificial secretion signal having a hydrophobic amino acid cluster, which is proposed by Y.

Yamamoto, et al. in Biochem. Biophys. Res. Commun., volume 149, page 431 (1987) from the results of the analyses of the structure of the secretion signal and the trans location thereof through an ER membrane, can also be used. Description is given in the following as a particular example for the use of these artificial secretion signals although the scope of the present invention is never limited thereto.

The base sequence of the DNA corresponding to the artificial secretion signal is shown at the uppermost part of the sequence of the Sequence No. 3 together with the constitution of the oligonucleotide fragments obtained by dividing the same. In this sequence, the portion corresponding to the signal, i.e. the portion corresponding to the -15 to -1 amino acid residues, has the same sequence in the 5' side thereof as the sequence in the ENO 1 gene of yeast from the initiating point of transcription to the initiating point of translation. Further, it has a site of the restriction enzyme Sal I at the 5' terminal and contains a region at the 3' side corresponding to the 7 to 8 amino acid residues at the N-end side of the maturationform WGA protein as well as a site of the restriction enzyme BamH I at the same terminal. See the region corresponding to S1 to S4 in the sequence shown in Sequence No. 3. A known method was applied to the chemical synthesis and enzymatic ligation of these oligonucleotide fragments. On the other hand, the cloned WGA gene described in the above given Section II-1 is devoid of 7 to 8 amino acids of the maturation-form WGA at the N-end side but has a BamH I site at the 5' terminal and a 171st amino acid as well as a Hind III site after the two translation-terminating codons at the 3' terminal. Then, the signal portion and the WGA gene portion were ligated together at the BamH I site followed by cloning to the Sal I-Hind III site of the plasmid vector pNJ1053, which was obtained by replacing the HLY gene portion of the plasmid pESH taught by K. Ichikawa, et al.

in Agri. Biol. Chem., volume 53, pages 1445-1447 (1989) with the Sal I-Hind III fragment of the plasmid pBR322 (see Figure 2A). These authors also have indicated that, since this plasmid vector pNJ1053 has the Sal I site at the 3' side of the transcription-starting point in the ENO 1 promotor of yeast, insertion of a gene starting from the translation-initiating codon ATG results in efficient proceeding of the transcription and translation. This plasmid is named as pSW, i.e. abridgement of SIGNAL-WGA (see Figure 2A) The signal sequence of the pre-WGA gene having the thus prepared signals and the base sequence in the ligating portion thereof were found to be just as designed from the results of the base sequence determination undertaken according to a known method.

(II-3) Synthesis of precursor-type WGA (prepro-WGA) gene having secretion signal and introduction thereof into vector for expression.

In the next step, synthesis was performed of the prepro WGA gene having an extension peptide for the C-end consisting of 15 amino acid residues found in wheat. germs at the 3'-terminal of the maturation-type WGA gene having the above described secretion signals. Although the role played by the C-end pro-region, i.e. CT peptide, is unclear, it may be indispensable in order that the WGA protein is assembled to have an appropriate steric structure or may be indispensable for the efficient transport of the same in the secretion passway. At any rate, it would be. an interesting matter whether or not this WGA protein precursor plays a role for the improvement of the secretion efficiency in the yeast cells and whether or not this WGA precursor is correctly recognized in the yeast to be converted into the maturation-form WGA.

Sequence No. 4 given below shows the base sequence of the DNA to code the CT peptide as designed and the constitution of the synthetic oligonucleotide fragments.

Sequence No. 4 (i) Length of sequence: 94 (ii) Type of sequence: nucleic acid (iii) Number of strands: double strand (iv) Topology: normal chain (v) Kind of sequence: synthetic DNA (vi) Origin: (a) Name of organism: none (synthetic DNA) (b) Name of strain: none (vii) Characteristic of sequence: sequence of a gene to code a part of C-end region of a maturation-form wheat germ agglutinin (WGA) and a prepro region of WGA (viii) Sequence:

Y C GAG C Q S G G CO A V F AG A ; CT I A lCCGGG1TACrGTGGTGCTGGTTG I CAATCTGGTGGTrGTGACGCrGTTTTCGCrGGrGCT naI(AvI) TG Ska I(A v a T) CT 2 P t TANS T L LA C3 ATTACCGCTAACTCCACCTTGTTGGCTGAATGA TAATGGCGATTGAGGTGGAACAACCGACTTACTf TTCG4~ c - 4 The 5'-terminal in this region has a site of the restriction enzyme Sma I or Ava I and is designed so as to be ligated to the maturation-type WGA gene after Ava I cleavage. The four fragments indicated as CT1 to CT4 were hybridized by the above described method followed by the enzymatic ligation.Since this ligation product has an Ava I site at the 5'-terminal and a Hind III site at the 3'-terminal, it was ligated at the Ava I site to the Sal I Ava I fragment containing the WGA gene on the previously cloned plasmid pSW and then inserted into the Sal I-Hind III site of the vector M13mp19. Correctness of the ligation between the maturation-type WGA gene containing the signals and the CT portion (on the cloned DNA fragment) could be confirmed by checking the base sequence according to the above mentioned known method. The above described Sal I Hind III fragments prepared in this manner had a structure of the prepro WGA gene having a DNA corresponding to the CT ligated to the 3'-terminal of the maturation-type WGA gene having secretion signals.The plasmid to cause expression of this gene was prepared by inserting the above mentioned Sal I-Hind III fragment into the Sal I-Hind III site of the plasmid pNJ1053 having a yeast ENO 1 promotor (see Figure 2A). This plasmid is named as pSWC, i.e. abridgement of SIGNAL-WGA-CT. Figure 2B shows the detailed procedure for the assembly of the secretion-expression plasmids pPW, i.e. pMF a-prepro-WGA, and pPWC, i.e. pMF a-prepro-WGA-CT, by utilizing the pro-motor and the prepro region of the a-factor gene of yeasts. Reference should be made to Example 7 for further details of this assembly method.

(III) Transformation and secretion production of WGA protein by yeast.

The "cloning" of the synthetic DNA fragments described in the foregoing section can be achieved practically by the transformation of a host such as Escherichia coli as a species of colon bacillus and the like for the vector containing the thus obtained synthetic gene. The method for the transformation per se is known and the applicable methods for the transformation include, when colon bacillus is used as the host, for example, the method disclosed by S.N. Cohen, et al. in Proc. Natl. Acad. Sci., U.S.A., volume 69, page 2110 (1972) and, when a yeast is used as the host, the method disclosed by A. Hinnen, et al. in Proc. Natl.

Acad. Sci., U.S.A., volume 75, page 1927 (1978) and the method disclosed by Ito, et al. in J. Bacteriol., volume 153, pages 163-168 (1983). Particular examples of the strains suitable as the host include E. coli C600 taught by Nelson, et al. in Virology, volume 108, pages 338-350 (1981) among strains of E. coli and S. cerevisiae KK4 taught by Nogi, et al. in Mol. Gen. Genet., volume 195, pages 29-34 (1984) and S. cerevisiae KS-58-2D having ssl 1 mutation for extracellular secretion of a large amount of human lysozyme taught by Suzuki, et al. in Mol. Gen. Genet., volume 219, pages 58-64 (1989) among yeasts.Detailed disclosure is given in the above mentioned literatures as well as in Japanese Patent Kokai 64-47377 by some of the inventors on the method for the preparation of the yeast mutant for high secretion of human lysozyme and the method for the preparation of human lysozyme by using the same. The above mentioned hosts are not the only ones for the transformation by the recombinant plasmid having the WGA gene introduced thereinto but various kinds of known S. cerevisiae derivatives listed, for example, in the catalogue of Yeast Genetic Stock Center can be used. Strains of these microorganisms are deposited in Yeast Genetic Stock Center and other authentic organization such as American Type Culture Collection and available therefrom.

The host microorganisms for the expression are not limited to yeast but other microorganisms such as fungi and the like can be widely used. Usable hosts also include animal cells and insect cells as well as viruses capable of multiplication replication therein. A desirable vector for expression is a plasmid having a Sal I cleavage site for insertion of the pre WGA gene or prepro WGA gene in the downstream of the expression promotor suitable for the respective hosts.

Particular examples of the transformant in yeasts include the transformant obtained by the transformation of the wild-type S. cerevisiae KK4 or KS-58-2Ddel having the ssl 1 mutation with the above mentioned plasmid pSW or pSWC or the transformant obtained by the transformation of the same yeast with the plasmid pPW or pPWC. In the present invention, they are respectively named as S. cerevisiae KK4 (pSW) or KS-58-2Ddel (pSW) , S. cerevisiae KK4 (pSWC) or KS-58-2Ddel (pSWC) , S cerevisiae KK4 (pPW) or KS-58-2Ddel (pPW) and S. cerevisiae KK4 (pPWC) or KS-58-2Ddel (pPWC) respectively.

The WGA protein can be produced by secretion when the transformant obtained in this manner is cultured according to a conventional procedure known in the field of molecular biology and fermentation science. A suitable culture medium by using a yeast is, for example, the minimal medium of Burkholder disclosed in Proc. Natl. Acad. Sci., U.S.A., volume 77, page 4505 (1980). Culturing is performed usually at 20 to 40 C or, preferably, at 25 to 37 C for 24 to 144 hours or, preferably, for 36 to 120 hours with optional aeration or agitation of the medium and supplemental supply of a carbon source according to need. After completion of culturing, the yeast cells and the supernatant are separated by a known method.The crude WGA protein obtained by the extracellular or intracellular production in this manner can be purified by a known purification method for proteins including salting-out, isoelectric precipitation, ionexchange chromatography, gel-permeation chromatography, high-performance liquid chromatography (HPLC, FPLC) and the like to give the desired purified WGA protein. The biological activities of the thus obtained WGA protein, such as the specific sugar-chain binding activity, can be confirmed by checking the binding activity to a glycoprotein containing N-acetylglucosamine to which it is known that WGA binds specifically. In particular, for example, ovoalbumin is adsorbed on a microtiter plate and the WGA protein bound thereto can be detected by using a commercially available anti-WGA antibody according to the sandwich ELISA method described by N.Shibuya in Bioscience and Industry, volume 47, pages 1301-1302 (1989). The activity of WGA for cell agglutination can be confirmed by the visual observation of the agglutination of human or horse red blood cells in the presence of the WGA protein.

In the following, examples are given to illustrate the present invention in more detail although the scope of the invention is never limited thereby in any way. The restriction enzymes employed in the examples described below were supplied by Takara Shuzo Co. or Toyobo Co..

Example 1. Synthesis of oligonucleotide A DNA synthesizer (Model 391 manufactured by Applied Biosystems Co.) was used in the synthesis of the respective oligonucleotide fragments corresponding to S1 to S4, 1 to 32 and CT1 to CT4 by the solid-phase synthetic method. The reagents for synthesis including the phosphoamidite reagent and others were all products of the same company. The operator's manual given in the book provided by the company was followed. The oligonucleotides as synthesized were recovered by cleaving from the silica gel carrier in the state having the nucleic acid bases and 5' hydroxy groups protected and the phosphate protecting groups deprotected.

An oligonucleotide entirely deprotected excepting the dimethoxytrityl groups at the 5'-terminal was obtained by admixing 1 ml of the protected deoxyoligonucleotide with 3 ml of a 28% ammonia water and keeping the mixture for 8 to 16 hours at 60 C in a tightly stoppered vessel. The mixture was freed from ammonia by blowing nitrogen gas thereinto followed by filtration through a Millex-GS filter having a pore diameter of 0.22 pm (manufactured by Millipore Co.) and the filtrate was subjected to high-performance liquid chromatography using a C18 reversed-phase column (Cosmosil 5 C18, a product by Nakarai Chemical Co.) to recover the last-eluted fraction. This fraction was concentrated into a volume of 2 ml and kept standing for 30 minutes with addition of an equal volume of acetic acid to remove the dimethoxytrityl groups.After removal of acetic acid by extraction with ether, the liquid was purified by the C18 reversed-phase chromatography in the same manner as described above to give about 148 to 481 pg of the completely deprotected oligonucleotide corresponding to 4 to 13 OD 260 units.

Example 2. Hybrid formation of oligonucleotide and enzymatic ligation thereof Figures 1A to 1C illustrate the outline of the enzymatic ligation method for the preparation of the blocks A, B and C+D in the double strand of the maturation-type WGA II gene. Following is a detailed description thereof.

Excepting Nos. 1, 18, 19 and 32 having a restriction enzyme site at the 5' terminal, a solution of 40 pg of each of the oligonucleotides was prepared in an overall volume of 100 ul having compositions of 50 mM Tris-HCl (pH 7.5), 10 mM MgCl21 15 mM DTT, 0.4 mM of Na2-EDTA and 1 mM ATP.

The solution was incubated for 1 hour at 37 'C with addition of 1 pl of T4 polynucleotide kinase (EC 2.7.1.78) (16 units/pl). Thereafter, the solution was washed with watersaturated phenol, kept standing for 10 minutes at -80 C with addition of 3M sodium acetate (pH 5.2) and ethanol to precipitate the oligomer and further admixed with 100 pl of a buffer solution containing 20 mM of Tris-HCl (pH 7.5) and 10 mM of MgCl2. Equimolar amounts of the n-th and (n+1)-th oligomers, n being an odd number, phosphorylated at the 5' terminal were mixed together and incubated first at 65 C for 10 minutes and then at 37 C for 20 minutes followed by standing and annealing at room temperature for 10 minutes to form the double strand. According to the ligation schemes illustrated in Figures 1A to 1C, the adjacent double strands were mixed together to make up an overall volume of 60 pl of the solution having compositions of 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM DTT and 1 mM ATP and admixed with 2 pl of T4 DNA ligase (EC 6.5.1 .1) (5 units/pl) followed by incubation at 20 C for 30 minutes to effect ligation. Completion of the reaction and occurrence of side reactions were checked at each stage of ligation by subjecting a small portion of the reaction mixture to the electrophoresis with a polyacrylamide gel.

A large number of bands due to side reactions were detected at the final stage of ligation in the procedure with the block C+D so that the electrophoresis with a 8% polyacrylamide gel was undertaken and the gel containing the target band taken by sectioning was comminuted followed by elution using a centrifugal ultrafilter (Model Ultrafree C3HV manufactured by Millipore Co.).

The fragments of the block A after completion of the reaction in the final stage and the fragments of the block C+D recovered by elution from the gel were washed with phenol, admixed with 3M sodium acetate (pH 5.2) and ethanol and kept standing at -80 C for 10 minutes to effect precipitation followed by phosphorylation at the 5' terminal with T4 DNA kinase in the same manner as above.

The terminal portions of the blocks A and B were cleaved with the restriction enzymes Spe I and Apa I to expose the cohesive ends for subcloning. As to the block B, in particular, 2.18 pg of the DNA fragments were reacted for 1 hour at 37 C in 300 p1 of a solution containing 10 mM of Tris-HCl (pH 7.5), 10 mM of MgCl2 , 1 mM of DTT and 18 units of Apa I followed by the addition of 26.1 pl of 1M NaCl and 2 pl of Spe I (10 units/pl) to effect further reaction at 37 'C for 1 hour. As to the block A, 3 pg of the DNA fragments were reacted for 1 hour at 37 C in 50 pl of a solution containing 10 mM of Tris-HCl (pH 7.5), 50 mM of NaCl, 10 mM of MgCl2, 1 mM of DTT and 10 units of Spe I.The blocks A and B after the restriction enzyme treatment in this manner were each washed with phenol and subjected to precipitation with ethanol.

Example 3. Subcloning of each block fragment A 10 pg portion of the Phagescript SK (a product by Stratagene Co., code No. SC221201 by Toyobo) was admixed with 50 units of Apa I and, after reaction for 1 hour at 37 C in 50 pl of a solution of the compositions of 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT, further admixed with 6.95 pl of 1M KCl and 5 pl of Hind III (10 units/pl) to be reacted for another 1 hour. After deproteinization with phenol and precipitation with cold ethanol, electrophoresis with a 0.7% agarose gel was undertaken to take the gel portion containing the target band by sectioning, which was enclosed in a dialysis tube and immersed in a buffer solution for electrophoresis to effect electrical elution.

The eluate was subjected to a treatment with phenol and precipitation with cold ethanol. The DNA was mixed with 19.2 pg of the DNA fragments of the block C+D prepared in Example 2 and the DNAs were ligated by the reaction for 1 hour at 16 C in 100 pl of a ligating solution having a composition of 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 1 mM DTT and 1 mM ATP and containing 12.5 units of T4 DNA ligase.

A half volume thereof was used for the transformation of a strain of E. coli XL1-Blue (produced by Stratagene Co.

code No. SC212301 by Toyobo) according to the method disclosed by S.N. Cohen, et al. in Proc. Natl. Acad. Sci., U.S.A., volume 69, page 2110 (1972). The target transformant was selected by utilizing the fact that, while the plaque of the target E. coli on the plate was blue in color by the decomposition of X-Gal in the absence of insertion of the DNA fragments, the plaque was colorless with insertion of the DNA fragments. The plasmid was isolated from this transformant by the alkali extraction method taught by H.C. Birnboim, et al. in Nucl. Acids Res., volume 7, page 1513 (1979), which was subjected to the determination of the molecular weight and the disintegration pattern by the restriction enzyme to give a plasmid having the block C+D inserted into the Apa I-Hind III site of the Phagescript SK (see Figure 1C).

By undertaking a similar method to the above, the DNA fragments of the blocks A and B were inserted respectively to the BamH I-Xba I site of the Phagescript SK and to the Spe I-Apa I site of the Bluescript KS(+) (a product by Stratagene Co., code No. SC212207 by Toyobo) , respectively.

In view of the BamH I site and Spe I site located at both ends of the DNA fragments of the block A, an attempt was first made for insertion into the BamH I-Spe I site in the multicloning sites of the Phagescript SK without success.

The reason therefor is presumably that the BamH I site and the Spe I site are located in adjacency to each other so as not to cause cleavage by both of the restriction enzymes BamH I and Spe I. Accordingly, insertion was performed into the BamH I-Xba I site by utilizing the Xba I site having the same cohesive ends as the Spe I site (see Figures 1A and 1B). The base sequence of the DNA fragment of each block could be identified to be just as designed by conducting the analysis of the DNA base sequence for the whole regions of the two strands according to the known deoxynucleotide method taught by F. Sanger, et al. in Proc. Natl.

Acad. Sci., U.S.A., volume 74, page 5463 (7977).

Example 4. Subcloning of WGA genes A 500 pg portion of the Phagescript SK after introduction of the DNA fragments of the block C+D was reacted for 2 hours at 37 C in 1.2 ml of a reaction solution having a composition of 10 mM Tris-HCl (pH 7.5), 10 mM of MgCl2 and 1 mM DTT and containing 50 units of the restriction enzyme Apa I followed by the admixture of 86.1 pl of 1M KCl and 38.5 pl of Hind III (10 units/pl) to effect further reaction overnight at 37 C. After deproteinization with phenol and precipitation with ethanol, electrophoresis with a 88 polyacrylamide gel was undertaken to recover the DNA fragments of the block C+D of 0.27 kb.

The procedure for the DNA fragments of the block A was basically the same as for the block C+D. Since the fragment was introduced into the BamH I-Xba I site of the Phagescript SK, the Sac I site at the outside of the Xba I site of the multicloning sites was utilized in the first place for conducting double digestion with BamH I and Sac I involving the DNA frangmens of the block A to obtain the DNA fragments by cleavage. These fragments were further digested with Mae I recognizing the same base sequence as the cohesive end portion of the four bases produced by Spe I to give the DNA fragments of the block A having the BamH I and Mae I sites at both ends.

Since the DNA fragments of the block B should have the same section of the Mae I at the end in order to be ligated with the DNA fragments of the block A, the DNA fragments of the block B having Mae I and Apa I at both ends were obtained from the Bluescript KS(+) after introduction of the DNA fragments of the block B by first conducting the double digestion with Sac I and Apa I followed by further digestion thereof with Mae I.

The respective DNA fragments of the blocks A, B and C+D were ligated together as is illustrated in Figure 1D.

The DNA fragments of the blocks B and C+D were mixed together and reacted overnight at 16 C in 350 pl of the reaction solution containing T4 DNA ligase as described in Example 2. After the treatment with phenol and precipitation with ethanol, reaction was effected for 1 hour at 45 C in 150 pl of a reaction solution having a composition of 20 mM Tris-HC1 (pH 8.0), 250 mM Nail, 6 mM MgCl2 , 7 mM 8-mercaptoethanol and 100 pg/ml BSA and containing 30 units of Mae I followed by the electrophoresis with a 8% polyacrylamide gel to recover the DNA fragments of the block B+(C+D) of 0.39 kb.

In the next place, the DNA fragments of the block A and the DNA fragments of the block B+(C+D) were mixed together to effect the ligation reaction in the same manner as above and then double digestion was performed with BamH I and Hind III followed by the treatment with phenol and precipitation with ethanol to prepare the DNA fragments of the block A+B+(C+D) or, namely, the maturation-type WGA gene. On the other hand, 10 pg of the pUC18 plasmid (code No. PUC-018 by Toyobo) were reacted overnight at 37 C in 100 pl of a reaction solution having a composition of 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT and 65 mM KCl and containing 30 units of Hind III and the plasmid DNA converted into a linear form was recovered by the electrophoresis with a 0.7% agarose gel.A 5 pg portion thereof and 45 pg of the above described maturation-type WGA gene of A+B+(C+D) were mixed together and subjected to the ligation reaction in the same manner as described above.

The strain JM109 of E. coli (code No. DNA-900 by Toyobo) was subjected to the transformation by using the DNA according to the method of Cohen, et al. The plasmid DNA was isolated from the transformant selected by the ampicillin resistance as an index by the alkali extraction method and it was subjected to the tests of the molecular weight and the disintegration pattern with restriction enzymes to give a plasmid having the maturation-type WGA gene introduced thereinto.

Example 5. Assembly of the plasmid for secretion expression of the maturation-type WGA gene Figure 2A illustrates the outline of assembly of which particulars are described below. The method described in Example 1 was applied to the synthesis of the oligonucleotides S1 to 54 to code the artificial signal sequence modified for the secretion expression of a foreign protein using a yeast disclosed by Y. Yamamoto, et al. in Biochem.

Biophys. Res. Commun., volume 149, page 431 (1987). In these fragments, S2 and S3 were phosphorylated according to the method described in Example 2 to effect annealing and ligation followed by further phosphorylation of the terminals. On the other hand, 200 pg of the.pUC18 plasmid after insertion of the maturation-type WGA gene prepared in Example 4 were subjected to double digestion with the restriction enzymes BamH I and Hind III in the same manner as in Example 4 followed by the electrophoresis with a 8% polyacrylamide gel to recover the fragments of the maturation-type WGA gene of 0.5 kb.

The DNA fragments of the signal portion (S1 to S4) and the fragments of the maturation-type WGA gene were mixed in an equimolar proportion to effect ligation according to the method described in Example 1. After a phenol treatment and ethanol precipitation, the reaction was effected in 100 pl of a reaction solution (65 mM KCl, 10 mM Tris-HCl with a pH of 7.5, 10 mM MgCl2 and 1 mM DTT) containing 40 units of Hind III at 37 'C for 1 hour and then 9 pl of 1M KCl, 8 pl of dH2O and 3 pl of Sal I (15 units/pl) were added thereto to effect the reaction at 37 C for additional 1 hour.Thereafter, the phenol treatment and ethanol precipitation were repeated to give 6 pg of the WGA gene (SIG-WGA) ligated with the secretion signal.

On the other hand, the plasmid pNJ 1053 was subjected to double digestion with the restriction enzymes Hind III and Sal I in the same manner as above and the reaction mixture was subjected to the electrophoresis with a 0.7% agarose gel to recover the DNA fragments of 8.6 kb. A half amount of the above mentioned SIG-WGA gene was mixed with 0.04 pg of this plasmid DNA and a ligation reaction was undertaken according to the method described in Example 3.

The same was subjected to the transformation to the E. coli strain of XL1-Blue according to the method of Cohen, et al.

The plasmid was isolated from the transformant selected by taking the ampicillin resistance as an index by the alkali extraction method, of which the molecular weight and the disintegration pattern with a restriction enzyme were examined to obtain 1.145 mg of the plasmid pSW for secretion expression of the maturation-type WGA gene with yeast having the SIG-WGA gene inserted into the Sal I-Hind III site at the downstream of the yeast ENO 1 promotor of pNJ1053.

Example 6. Assembly of the plasmid for secretion expression of precursor WGA protein gene An outline of the assembly is shown in the latter half part of Figure 2A. The oligonucleotides CT 1 to CT 4 coded with the CT peptide were synthesized according to the method described in Example 1. In the next place, CT 2 and CT 3 were phosphorylated according to the method described in Example 2 and CT 1 and CT 2 as well as CT 3 and CT 4 were subjected to annealing to effect ligation followed by second phosphorylation of terminals to give the block DNA fragments to code the CT peptide.On the other hand, 330 pg of the pSW plasmid prepared in Example 5 were subjected to double digestion with the restriction enzymes Sal I and Hind III according to the method described in Example 5 followed by the electrophoresis with a 88 polyacrylamide gel to recover the fragments of the SIG-WGA gene of 0.58 kb. A 18 pg portion of this SIG-WGA gene fragments was subjected to the reaction at 37 C for 2 hours in 100 pi of a reaction solution (10 mM Tris-HCl with a pH of 7.5, 10 mM MgCl21 1 mM DTT and 25 mM NaCl) containing 20 units of the restriction enzyme Ava I followed by the electrophoresis with a 8% polyacrylamide gel to recover the DNA fragments of 0.53 kb.After ligation of a mixture of 6.4 pg of the above obtained DNA fragments and 1.6 pg of the DNA fragments to code the above mentioned CT peptide according to the method described in Example 2, double digestion was performed with the restriction enzymes Sal I and Hind III in the same manner as described above. Thereafter, electrophoresis with a 5% polyacrylamide gel was undertaken to recover 2.1 pg of the WGA gene of 0.63 kb (SIG-WGA-CT gene) having the secretion signal and the CT peptide.

In the next place, 20 pg of the plasmid M13mp19 (code No. M13-019 by Toyobo) were subjected to double digestion with the restriction enzymes Sal I and Hind III in the same manner as above followed by the electrophoresis with a 0.8% agarose gel to recover the plasmid DNA fragments of 7.5 kb.

A 0.4 pg portion of this plasmid DNA and 2 pg of the above mentioned SIG-WGA-CT gene fragments were mixed together and subjected to ligation according to the method described in Example 2. The same was transformed to the E. coli strain XL1-Blue according to the method of Cohen, et al. The plasmid was isolated by the alkali extraction method from the phage exhibiting no blue color by the X-gal disintegration but turned colorless by the insertion of the DNA fragments as is described in Example 3, of which the molecular weight and the disintegration pattern by the restriction enzymes were examined to obtain the plasmid having the SIG-WGA-CT gene fragments introduced into the Sal I-Hind III site of M13mp19.The ssDNA was recovered from this phage and the base sequence of the SIG-WGA-CT gene was confirmed to be just as designed from the DNA base sequence by the known dideoxynucleotide method.

Further, double digestion was conducted of the above mentioned M73mp19 having the above mentioned SIG-WGA-CT gene introduced thereinto with the restriction enzymes Sal I and Hind III in the same manner as above followed by the electrophoresis with a 5% polyacrylamide gel to recover the SIG-WGA-CT gene fragments of 0.63 kb. This DNA fragment and the pNJ1053 devoid of the Sal I-Hind III site as prepared in Example 5 were subjected to ligation.This was transformed to the E. coli strain XL1-Blue according to the method of Cohen, et al. and the plasmid was isolated by the alkali extraction method from the transformant selected by taking the ampicillin resistance as an index, of which the molecular weight and the disintegration pattern with the restriction enzymes were examined to obtain the plasmid pSWC for secretion expression of the precursor WGA protein having the SIG-WGA-CT gene inserted into the Sal I-Hind III site at the downstream of the yeast ENO 1 promotor of pNJ1053.

Example 7. Assembly of the plasmid for secretion expression of maturation-type WGA gene and pro WGA gene utilizing the promotor and prepro region of the a-factor gene of yeast An outline of the assembly is shown in Figure 2B.

The particular procedure was as follows. Thus, 260 pg of the pLS0l plasmid disclosed by K. Inokuchi, et al. in Mol.

Cell. Biol., volume 7, pages 3185-3193 (1987) having the DNA fragments of 1.7 kb and containing the whole region of the structural gene from the promotor of the a-factor at the EcoR I site were subjected to the reaction at 37 C overnight in a reaction solution (10 mM Tris-HCl with a pH of 7.5, 10 mM MgCl2 , 1 mM DTT and 75 mM KCl) containing 300 units of the restriction enzyme EcoR I and 600 units of Hind III followed by the electrophoresis with a 0.7% agarose gel to recover the DNA fragments of 1.2 kb containing the prepro region from the promotor of the a-factor of yeast.In order to convert the EcoR I site at the end of this DNA fragment into the Sal I site, the Sal I-EcoR I adaptor (5'TCGACCACAACG3' 3'GGTGTTGCTTAA5') was synthesized according to the method described in Example 1. A 20 pg portion of the above mentioned adaptor was reacted with 40 units of the T4 polynucleotide kinase (a product by Toyobo) at 37 C for 1 hour in 100 pl of a reaction solution (50 mM Tris-HC1 with a pH of 7.5, 10 mM MgCl21 15 mM DTT, 0.4 mM Na2-EDTA and 1 mM ATP) to phosphorylate the 5' terminal.A 1 pg portion of this adaptor was mixed with 40 pg of the above mentioned DNA fragments of the prepro region from the promotor of the a-factor to effect ligation by the activity of the T4 DNA ligase under the conditions described in Example 2 followed by digestion with the restriction enzymes Sal I and Hind III. In the next place, electrophoresis with a 8% polyacrylamide gel was undertaken to recover the DNA fragments of the prepro region from the promotor of the a -factor having the Sal I site and the Hind III site at the terminals.

In order to effect in-frame ligation of the prepro region of the a-factor of the above mentioned fragments and the maturation-type and precursor WGA genes, the synthetic adaptor (5' AGC TCA AAG ATG TGG TGA ACA kO 3' 3'GT TTC TAC ACC ACT TGT TCC TAG 5') was synthesized according to the method described in Example 1. Each a 40 pg portion of the oligonucleotides was reacted with the T4 polynucleotide kinase in the same manner as above to be phosphorylated at the 5' terminals and then 8 pg of this DNA and 25 pg of the above mentioned DNA fragments containing the prepro region from the promotor of the a-factor were mixed together to effect ligation by the activity of the T4 DNA ligase under the conditions described in Example 2.The same was subjected to double digestion with the restriction enzymes Sal I and BamH I followed by the electrophoresis with a 0.8% agarose gel to recover the DNA fragments of about 1.2 kb having a ligating site with the WGA gene and containing the prepro from the promotor of the a-factor.

On the other hand, 200 pg of the pUC18 plasmid having the maturation-type WGA gene inserted thereinto as prepared in Example 4 were reacted at 37 C for 30 minutes in 200 pl of a reaction solution (10 mM Tris-HCl having a pH of 7.5, 10 mM MgCl2, 1 mM DTT and 65 mM KC1) containing 200 units of the restriction enzyme Hind III followed by the addition of 18 pl of 1M KCl, 6 pl of dH2O and 16 pl of BamH I (200 units) to effect further reaction for additional 1 hour. The same was subjected to the electrophoresis with a 8% polyacrylamide gel to recover the maturation-type WGA gene of 0.5 kb.A 30 pg portion of this DNA fragment was admixed with 10 pg of the DNA fragments having the ligating site with the above mentioned WGA gene and containing the prepro region from the promotor of the a-factor to effect ligation by the activity of the T4 DNA ligase under the conditions described in Example 2. After double digestion of the same with the restriction enzymes Sal I and Hind III, electrophoresis was undertaken with a 0.8% agarose gel and the DNA was recovered from the gel to obtain the maturationtype WGA gene (MF a-WGA gene) ligated in-frame with the prepro region under the promotor of the a-factor. This DNA fragment was inserted between the Sal I-Hind. III sites of M13mp19 by the same method as in Example 6. The ssDNA was recovered from this phage and the base sequence of the MF a-WGA gene was confirmed by the analysis of the DNA base sequence according to the known deoxynucleotide method to find that it could be given by the Sequence No. 1 shown before. The expression vectors of the MF a-WGA and MF a -WGA-CT genes in the yeast were assembled as described below.Thus, 100 pg of pNJ1053 were reacted at 30 'C for 1 hour in 200 pl of a reaction solution (10 mM Tris-HCl with a pH of 7.5, 7 mM MgCl2 , 20 mM KCl, 7 mM 2-mercaptoethanol and 100 pg/ml BSA) containing 105 units of the restriction enzyme Sma I followed by the admixture with 0.3 pg of the Sal I linker (5' CGGTCGACCG 3' 3' GCCAGCTGGC 5') synthesized according to the method described in Example 1 and phosphorylated at the 5' terminal to effect ligation by the activity of the T4 DNA ligase. After further digestion with Sma I, it was transformed to the E. coli strain XL1-Blue according to the method of Cohen, et al.The plasmid was isolated by the alkali extraction method from the transformant selected by taking the ampicillin resistance as an index and formation of the DNA fragments of 0.95 kb was confirmed by the Sal I digestion to obtain pNJ1054 having the Sal I linker introduced into the Sma I site of pNJ1053. Then, 37.5 pg of this pNJ1054 were subjected to double digestion with the restriction enzymes Sal I and Hind III under the conditions described in Example 5 followed by the electrophoresis with a 0.8% agarose gel to obtain a DNA of 7.3 kb devoid of the ENO 1 promotor.A 2 pg portion of this vector side DNA and 1.5 pg of the DNA fragments taken by cleavage of the MF a-WOA between the Sal I-Hind III sites of the above mentioned M13mp19 by the double digestion with Sal I and Hind III were mixed together to effect ligation by the activity of the T4 DNA ligase according to the method described in Example 2. This DNA was transformed to the E. coli XL1-Blue according to the method of Cohen, et al. followed by the isolation of the plasmid by the alkali extraction method from the transformant selected by taking the ampicillin resistance as an index and the disintegration pattern with the restriction enzyme was examined to obtain 182.5 pg of the plasmid (pPW) for secretion expression of the maturation-form WGA protein having the prepro region of the yeast a-factor.

As to the precursor WGA having the CT peptide, on the other hand, 245 pg of the expression plasmid (pPWC) having the prepro region of the yeast a-factor were prepared by just the same method as above.

Example 8. Preparation of yeast transformant By using four kinds of the plasmids for secretion expression of the maturation-type and precursor WGA genes pSW, pSWC, pPW and pPWC, the wild yeast strain Saccharomyces cerevisiae (MAT a, ura3, hisl or his3, trip1, leu2, gal80) or the high-secretion yeast variant KS-58-2Ddel (MAT a, leu2, ura3, hisl or his3, ssl1) was transformed by the known lithium acetate method disclosed by Ito, et al. in J.

Bacteriol., volume 153, page 163 (1983) to obtain transformants holding the same plasmid and requiring no leucine.

The names given to these transformants were S.c. KK4 (pSW) S.c. KK4(pSWC), S.c. KK4(pPW) and S.c. KK4(pPWC) and S.c.

KS-58-2Ddel(pSW), S.c. KS-58-2Ddel(pSWC), S.c. KS-58- 2Ddel(pPW) and S.c. KS-58-2Ddel(pPWC), respectively. As a control to check appearance of expression, KK4 strain and KS-58-2Ddel strain having the plasmid pNJ1053 without the WGA gene were prepared by the same method and named as KK4(pNJ1053) and KS-58-2Ddel(pNJ1053), respectively.

The transformants obtained here are deposited in Fermentation Research Institute, Japan, under the Deposition Nos. of: S.c. KS-58-2Ddel (pSW), FERM-P 12444; S.c. KS-58-2Ddel (pSWC), FERM-P 12442; S.c. KS-58-2Ddel (pPW), FERM-P 12443; and S.c. KS-58-2Ddel (pPWC) , FERM-P 12.441.

Example 9. Culturing of wild yeast strain and high-secretion variant having secretion-expression plasmid The yeast transformant having various kinds of plasmids as prepared in Example 8 were each subjected to shake culture at 30 'C for 5 days in a minimal medium containing various amino acids excepting leucine (final concentration 20 to 375 mg/liter), an excess amount of histidine (final concentration 400 mg/liter), adenine sulfate (final concentration 20 mg/liter) and an excess amount of uracil (final concentration 400 mg/liter) (a product by Difco Co., a bactoyeast nitrogen base without amino acids) disclosed by Sherman, et al. in Method in Yeast Genetics, page 62, Cold Spring Harbour (1982) with supplemental addition of 8% glucose as the carbon source.

Example 10. Analysis of the WGA protein produced by secretion by the Western blot technique A 40 ml portion of the culture medium after 5 days of culturing at 30 C according to the method of Example 9 was taken and freed from the yeast cells by centrifugation to give a supernatant of the medium, which was freeze-dried and then dissolved in 5 ml of deionized water and the aqueous solution was thoroughly dialyzed against deionized water. Freeze-drying and dialysis were repeated and the solution was concentrated to have a 333 times concentration.

A 15 pl portion of this concentrated solution corresponding to 5 ml of the supernatant of the culture medium was admixed with the same volume of a Laemmli's buffer solution (80 mM Tris-HCl with a pH of 6.8, 2% SDS, 10% glycerol, 100 mM DTT, 2 mM PMSF and 0.001t BPB) and boiled for 10 minutes.

After rapid cooling, the whole volume of the solution was subjected to SDS-15% PAGE and then to the electrophoresis for 3 hours at 150 volts.

After the electrophoresis, the secreted protein was transferred to a PVDF membrane filter (Immobilon-P, a product by Millipore Co.). This transfer was performed by using a transblot apparatus manufactured by Atto Co. for 1 hour at 10 volts in a transblotting buffer solution (25 mM Tris-HCl with a pH of 7.5, 192 mM glycine, 0.1% SDS and 15% methanol). The thus obtained membrane filter was dipped for 10 minutes in a TBS buffer solution (100 mM Tris-HC1 with a pH of 7.5 and 150 mM Narc1) and then shaken for 30 minutes in a blocking solution prepared by dissolving 3% of gelatin in TBS.In the next step, the membrane filter was put into a 500 times diluted solution of a rabbit anti WGA antibody (a product by SIGMA Co., Lot No. 128-8810) and gently shaken overnight followed by twice of washing each by gentle shaking for 10 minutes in TTBS prepared by the addition of 0.05% of Tween 20 to TBS. The thus obtained membrane filter was put into a solution of 125 I-protein A (NEX 146L10, a product by New England Nuclear Co.) and lightly shaken for 1 hour to effect the reaction followed by washing by gentle shaking first in TTBS for 10 minutes and then in TBS for 10 minutes. The membrane filter was dried and subjected to detection by the autoradiography.

The result was as shown in Figure 3 which clearly indicates a single clearly defined band having reactivity with the anti-WGA antibody at the position showing the same mobility as a commercially available authentic sample of WGA (a product by Honen Corp.). Formation of the WGA of the same size as the commercially available authentic sample of WGA with the yeast having the plasmid pSW indicated that the artificial secretion signal could be correctly cleaved in the yeast at the fused portion with WGA. Also in the yeast having the plasmid pSWC for secretion expression of the precursor WGA having the CT peptide, furthermore, a band was detected at the same position as the authentic WGA sample and it is a remarkable fact that the amount of secretion thereof is about twice of the amount in the pPW having no CT peptide. This fact suggests that addition of the CT peptide is effective in increasing the amount of secretion and also that the CT peptide portion can be cleaved from the prepro WGA even in the yeast cells as in the wheat germs.

Further, the above mentioned result indicates that the WGA protein fused to the prepro region of MFa can be correctly cleaved at the fused site to give the same molecular weight as the authentic WGA sample. It is noted, however, that the amount of secretion of WGA was significantly larger in the combination of the ENO 1 promotor and artificial secretion signal than in the prepro form of MFa . A still further remarkable fact is that the amount of the WGA protein secretion of KS-58-2Ddel, which is a yeast mutant isolated as a mutant to highly secrete the human Xysozyme and is a strain having a mutation ssl 1 to missort the vacuole protein out of the cells, is at least about 20 times larger than that of the wild strain KK4.This result clearly indicates that utilization of a mutant having a defect in the protein trasnsport to the vacuoles is advantageous for the secretion production of WGA. Incidentally, the largest amount of secretion of the WGA protein was obtained by using KS-58-2Ddel (pSWC) reaching about 200 g/liter.

Example 11. Isolation and purification of WGA protein produced by secretion The transformant of yeast KS-58-2Ddel (pSWC) was cultured for 5 days at 30 C in 6 liters of the culture medium used in Example 9. The medium after culturing was freed from the yeast cells by centrifugation to give a supernatant. The supernatant after freeze-drying was dissolved in 100 to 150 ml of deionized water and thoroughly dialyzed against 20 mM acetate buffer solution having a pH of 4.0.

After the dialysis, the concentrated and desalted sample was subjected to separation on a cation-exchange column Mono S HR5/5 (manufactured by Pharmacia Co.) by using a FPLC system manufactured by the same company. The fraction having the sugar-chain binding activity described later in Example 12 was obtained as the eluate in the period of about 20 to 40 minutes when elution was performed at a flow rate of 1 ml/minute in the presence of a 20 mM acetate buffer solution having a pH of 4.0 with a linear concentration gradient of 0.01M NaCl/minute. Figure 4A shows the elution diagram and the position of the active fraction in the above mentioned chromatography.

The thus collected active fraction was subjected to separation on an anion-exchange column Mono Q HR5/5 (manufactured by Pharmacia Co.) after buffer exchange to a 20 mM Tris-HCl having a pH of 9.5 by using a centrifugal ultrafilter (Ultrafree 20 manufactured by Millipore Co.

having a regenerated cellulose membrane). The elution was performed at a flow rate of 1 ml/minute in the presence of a 20 mM Tris-HC1 buffer solution having a pH of 9.5 with a linear concentration gradient of 0 to 0.3M NaC1 over 60 minutes. Figure 4B shows the elution diagram in this case and the activity of the respective fractions. The fraction having activity was replaced with a 20 mM citrate buffer solution having a pH of 3.8 by using the same centrifugal ultrafilter as above and again subjected to separation on a cation-exchange column Mono S HR5/5 (supra) at a flow rate of 1 ml/minute in the presence of a 20 mM citrate buffer solution having a pH of 3.8 with a NaC1 linear concentration gradient of 0.01M NaCl/minute.Figure 4C shows the elution diagram, of which the sugar-chain binding activity was detected in the fraction corresponding to the principal peak at about 27 minutes of the elution time.

Separately, a commercially available authentic sample of WGA, which was a mixture of WGAs I, II and III, was subjected to the procedure of separation and purification in the same manner as above. Figure 4D shows the separation diagram of the WGA isolectin proteins in this case, from which it was understood that the elution position of the recombinant WGA protein was in coincidence with the elution position of the WGA II eluted at about 27 minutes. The fraction corresponding to the main peak shown in Figure 4C in the separation and purification of the recombinant WGA by the above described chromatography was collected and subjected to SDS-15% PAGE to give the result shown in Figure 5.

A single band showing the same mobility as the authentic sample of WGA II protein alone was detected from the WGA protein sample produced by secretion of the recombinant yeast by staining with Coomassie Brilliant Blue. The yield of the thus purified recombinant WGA protein II was about 100 g/liter.

Example 12. Detection of sugar-chain binding activity of WGA protein produced by secretion Figure 6 illustrates the method for the detection of the sugar-chain binding activity of the WGA proteins.

A 100 ul portion of a PBS solution of 1% ovoalbumin (5X Crystals, a product by SIGMA Co.) was put on each of the wells of a 90-wells microtiter plate (ELISA Plate F-FORM, manufactured by Gleiner Co.) and kept standing for 1 hour at room temperature to form a coating layer of the ovoalbumin on the plastic surface. After three times of washing each with 100 pl of PBS containing 0.05% of Tween 20, a 50 pl portion of a PBS solution, which contained a purified recombinant WGA II protein or the WGA II protein separated and purified from the commercially available authentic WGA sample according to the method shown in Example 11 in varied concentrations, was added thereto and kept standing for 1 hour at room temperature.After three times of washing each with 100 p1 of a PBS solution with addition of 0.05% of Tween 20, a 50 pl portion of a 150times diluted PBS solution of an anti-WGA antibody (a product by SIGMA Co., Lot No. 128F8810) was added thereto and kept standing for 1 hour at room temperature. After washing repeated in the same manner as above, a 50 pl portion of a 300-times diluted PBS solution of a conjugate of an antirabbit IgG antibody and an alkaline phosphatase (a product by E.Y. Laboratories Co.) was added thereto as a secondary antibody and kept standing for 1 hour at room temperature.

After washing, a 50 pl portion of a diethanolamine buffer solution (9.7% by volume diethanolamine, 0.5 mM MgCl2 3 mM NaN3 and pH 9.5) containing 1 mg/ml of p-nitrophenyl phosphate was added thereto to observe coloration of p-nitrophenol at room temperature. The coloration was interrupted by the addition of 50 pl of 1N NaOH solution and the absorbance at a wavelength of 415 nm was measured with a microtiter protoreader (Model MTP-32, manufactured by Corona Co.).

The results were as shown in Figure 7 which indicates that the recombinant WGA has the same specific activity for sugar-chain binding, i.e. the sugar-chain binding activity per unit amount of the protein, as the authentic WGA II sample. Quantitative determination of the protein was conducted by using a BCA protein determination reagent (a product by Pierce Co.) according to the procedure of the supplied protocol. The sensitivity of the BCA method was about 70 times higher than the Coomassie Brilliant Blue method at least when WGA was concerned. The above described results support the conclusion that the sugar-chain binding activity of the WGA II protein produced by the secretion of yeast was the same as that of the WGA derived from wheat.

Claims (15)

CLAIMS:

1. A gene coding for maturation-type wheat germ agglutinin or a wheat germ agglutinin precursor, said gene including at least a nucleotide sequence as shown by the sequence No. 1.

2. A gene as claimed in claim 1 which is a structural gene of maturation-type wheat germ agglutinin which is expressed by the nucleotide sequence shown by the sequence No. 1.

3. A gene as claimed in claim 1 which codes for a wheat germ agglutinin precursor which is expressed by the nucleotide sequence shown in the sequence No. 2.

4. A gene as claimed in claim 2 or claim 3 which has a DNA region to code a signal peptide required for secretion at the 5' terminal and a translation-termination signal TGATAA at the 3' terminal.

5. A recombinant plasmid DNA containing a gene according to claim 4.

6. The recombinant plasmid DNA as claimed in claim 5 in which the expression promotor is a yeast promotor with the region to code the signal peptide at the downstream side of the yeast promotor and at the upstream side of the maturation-type wheat germ agglutinin gene or wheat germ agglutinin precursor gene, each being in coincidence with the decoding frame.

7. A transformed microorganism obtained by the transformation of a host cell with the recombinant plasmid DNA containing a gene according to claim 5 or claim 6.

8. A transformed microorganism as claimed in claim 7 in which the host cell is a yeast.

9. A transformed microorganism as claimed in claim 8 in which the yeast is selected from the group consisting of the yeast strains belonging to the genus of Saccharomyces and yeast mutant strains belonging to the genus of Saccharomyces having mutation for extracellular secretion of a vacuole-localized protein.

10. A transformed microorganism as claimed in claim 9 in which the yeast mutant strain is a strain of KS-58-2Ddel.

11. A method for the preparation of a wheat germ agglutinin which comprises the steps of culturing the transformed microorganism according to any one of claims 7 to 10 in a culture medium to cause secretion of the wheat germ agglutinin into the medium by the microorganism and collecting the wheat germ agglutinin from the culture medium.

12. A gene coding for maturation-type wheat germ agglutinin or a wheat germ agglutinin precursor substantially as described herein with reference to the Examples 1 to 4 and Figures lA, 1B, 1C and 1D of the accompanying drawings.

13. A recombinant plasmid substantially as described herein with reference to Examples 5 to 7 and Figures 2A and 2B of the accompanying drawings.

14. A transformed microorganism substantially as described herein with reference to Example 8.

15. A method for the preparation of wheat germ agglutinin substantially as described herein with reference to Examples 1 to 12 and the accompanying drawings.