JPS62502025A - DNA sequences useful for secreting peptides and proteins from yeast - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] 34. The yeast cell according to paragraph 30, which is of the species S. cerevisiae.

35, a promoter region that is functional in yeast cells, a promoter region that is functional in yeast cells, Sig encodes a signal peptide that is functionally connected to the promoter region. null array, and A polypeptide encoding a polypeptide and operably linked to the signal sequence. Propagating Yeast Cells Transformed with DNA Constructs Containing the Petide Coding Region A method for secreting polypeptides from yeast cells, which consists of growing them in a promoting medium. A method in which a small number of the signal sequence and the promoter region (one of which is A method characterized in that it is a segment of a lyase gene.

36, the third polypeptide whose coding region encodes something other than melibiase The method described in Section 5.

37. Item 36, wherein the promoter region is a promoter region of melibiase. How to put it on.

38. The method according to item 37, wherein the signal sequence is a melibiase signal sequence. .

39. The coding region of the polypeptide was derived from Cellulomonas fuimi. , encoding an endoglucanase.

38. The method of paragraph 37, wherein the method is regulated by changes in conditions in the growth medium that affect the growth medium.

41, signal sequence of yeast-specific melibiase gene.

42, the signal sequence according to paragraph 41, which is unique to S. Carlsbergensis.

43, substantially the formula: %formula% The system according to item 42, which has a DNA sequence encoding the amino acid sequence shown in Gunar array.

44, substantially the formula: %formula% 44. The signal sequence according to item 43, having a DNA sequence represented by:

45, a DNA construct containing a melibiase secretion signal sequence; Restrictions on the sides of the GNAR array for inserting additional NA components into the assembly. A DNA construct that contains DNA segments that define positions.

46, the DNA segment having a nucleotide sequence substantially as shown in FIG. 4A. 46. The assembly according to clause 45, having:

47, promoter region of yeast-specific melibiase gene.

48, S. The promoter region according to paragraph 47, which is unique to S. carl spergensis.

49, item 47 having a DNA sequence substantially as shown in Figure 1 of the accompanying drawings. promoter region.

5Q, a DNA construct containing the promoter of melibiase and , a control for inserting an additional NA component into the vector downstream of the promoter. A construct containing DNA segments that define a restriction site.

Specification Peptides and proteins from yeast NA sequence useful for secretion The present invention relates to recombinant DNA technology.

More specifically, the present invention functions, on the one hand, to promote gene expression; In this case, the function is to signal the secretion of the protein encoded by the expressed gene. It concerns the DNA sequence. Such sequences are useful for manipulating yeast cells It is.

Background of the invention Secretion, the process by which specific gene expression products are released (exported) from cells into the culture medium , a natural property of living cells. Appears to express foreign peptides and proteins The property of genetically modified cells to secrete these products is that they Because the product is placed outside the cell, it is in a partially purified form and is easy to obtain. This is a desirable property since it will be produced at

Proteins and polypeptides produced within cells either remain within the cell or exit outside the cell. Whether it is secreted or not is determined in advance. The two types of proteins in these classifications are The fate-determining mechanism is a functional “system” encoded by the nucleotide sequence. 'gnal' peptide, i.e., in the majority of known cases, the protein that is actually to be secreted. The signal sequence that precedes the sequence of the gene encoding the protein. It was experimentally found that.

Based on that fact (and as translated and secreted from complementary nucleotide sequences) Defined immature proteins direct immature proteins to the secretory pathway from the beginning. and receptors present in the secretory channels on the intracellular membrane that allow the passage of It has a precursor-like sequence of amino acids that is recognized as a "signal." So When bound, the signal sequence of the precursor protein is proteolytically After being cleaved and structurally modified (e.g. glycosylated), it is secreted (released) from the cell. It provides the active protein that will be absorbed.

By decoding and utilizing such specific signal sequences, constant secretion from cells can be achieved. It becomes possible to genetically engineer cells that can produce the proteins that are produced. genetics To recover proteins from artificially engineered cells, it is usually necessary to extract the translated proteins from the sequence. the appropriate nucleotide sequences necessary to confer secreted properties on the protein. Since the column is not included in the vector, lyse the engineered cells and add the desired protein. It is necessary to refine the quality.

Although cells are destroyed in this step, according to the method of the present invention, as described below, Keep the cells supplied with sufficient growth medium while secreting the desired protein. can be kept alive. Additionally, the engineered cells were incubated with nutrients. Continuously extract proteins by known chemical methods directly from the broth can be extracted.

As is known, products expressed in yeast from genetically modified expression vehicles Secretion is, in principle, coated in the vehicle with a signal peptide suitable for the yeast. This is achieved by inserting the native nucleotide sequence and the foreign gene together. It will be done. This signal peptide is in contact with a peptide containing the desired gene product. , and must be in contact with the N-terminus of the peptide.

Expression of a foreign protein in genetically engineered (genetically engineered) yeast Therefore, expression itself in which the expressed molecules are not secreted has often been attempted. It's here. In some cases these genes have been expressed in yeast, but in many cases Together, they are not expressed at all, or at least not expressed at the required levels. stomach. In order for a specific foreign gene to be expressed, it is necessary to change certain properties (characteristics). There is a point. This means that the part of the gene unique to the organism and the part of the foreign gene are fused. by reducing the difference between the foreign gene that is not expressed and the native gene that is expressed. will be held.

The most common feature to change is the upstream region of the gene's coding sequence. It is an array. This region normally contains sequences necessary for transcription initiation, as well as often , increases transcription levels by binding to specific proteins present in cells, and / or the sequences necessary to suppress (i.e., regulate) the transcription and translation of the gene. It is included along with other sequences that affect ``. This area is, as mentioned above, Although it includes sequences other than those strictly defined as regions that promote copying, Usually referred to as a "promoter".

Usually added or substituted when you want to secrete a specific foreign gene product from yeast. Another feature is the signal or leader sequence. This signa A sequence is usually a short sequence encoded by the sequence at the beginning of the translated portion of a gene. It is a polypeptide.

In this way, in order for a specific foreign gene product to be expressed and secreted from yeast, Immediate addition of the above promoter and/or signal sequence or similar It is common practice to replace foreign sequences in yeast with sequences that are functional in yeast.

Attempts to secrete foreign proteins from yeast without using yeast signal peptides have been successful. It was performed by Hitzemanes et al. (1). interest A certain protein, namely human interferon, is found in its gene within human cells. It has its own signal sequence suitable for secretion in yeast, but when expressed in yeast, Although this interferon is efficiently expressed, it is hardly secreted. In a recent report uses yeast signal peptides to secrete foreign gene products from yeast. It has been tested for use. Emr et al. (2) , a secreted yeast peptide, a nucleotide encoding the leader region of α-factor. The normal yeast veliplasmink gap (the area between the yeast cell membrane and cell wall) The nucleotide encoding invertase, a yeast protein secreted by the Gene fusion was performed with the code sequence.

As a result, invertase was secreted into the periplasmic space, which caused α-factor. Whether it is by the child leader or not, the invertase gene in the fusion It is not obvious, as it contains a segment of the tase signal peptide sequence. . In fact, α-factor is secreted into the medium and invertase The localization of invertase in Emul et al.'s experiment was determined by the genetic This may be due to the invertase component in the child fusion. Brake et al. keetal, ) (3) is the leader region of alpha factor and human epidermal growth factor ( A gene fusion with a synthetic NA sequence encoding EGF) is constructed, and this fusion The protein injects a limited region within the sequence of the region encoding the α-factor leader. When altered by in vitro mutagenesis, yeast effectively secrete mature EGF. I discovered that. Bitter et al. (4) is the α factor Child leader region β-endorphin and “consensus” human interface These foreign proteins are secreted by constructing fusions with each protein. However, at the same time, it is expected that it will be somewhat destroyed by proteolysis during the secretory event. I found it. However, at least 95% of the ink proteins are recovered intact. It was. At least in the case of alpha factors, exogenous peptides and proteins Yeast secretory signals, whether or not they are secreted gene products, in their natural host. The key is information that facilitates the secretion of these foreign peptides and proteins from yeast. It seems clear that it contains information.

Melipiase, or α-galactosidase (E, C03, 2, 1, 22), Yeast S. carl spergenesis (a multiplication product of some strains and a naturally secreted It is a protein. Among these yeast strains obtained by cloning into yeast plasmids, A DNA fragment derived from one of the cells is introduced into the cell by transformation. and Bost, which confers a melibiase-positive phenotype to melibiase-negative yeast. - Baitenmiller et al. (Po5t-al,.

Beittenmiller et Δ) (5).

The present invention provides the amino acid sequence and sequence of precursor premelibiase i and its play region. provide the nucleotide sequences encoding each of these, and Vector assembly (construct) containing a nucleotide sequence encoding a region The purpose is to provide the following.

Furthermore, the present invention provides a signal sequence capable of signal secretion of melibiase and other of a gene or portion of a gene, and extract this gene or gene from a yeast host cell. In order to secrete the protein by the gene parts, these are transformed into cloning vectors. The purpose of this invention is to provide a method for introducing the vector into the cell via a vector.

Furthermore, it is an object of the present invention to protein unique to Charomyces carlsbergensis) A regulatable promoter capable of promoting transcription of the DNA region encoding melibiase. The goal is to provide a data center area.

Furthermore, the present invention also provides the above promoter region, a signal sequence and a polypeptide. The polypeptide encoded by the coding region is secreted by cloning the coding region. It is also an object of this invention to provide a method that can be used to

That is, the present invention first provides a signal sequence of a yeast-specific melibiase gene, Preferably, the sequence of the gene derived from S. carlsbergensis is provided. This is what we provide.

Second, the present invention provides yeast-specific, preferably Saccharomyces karlspergen The regulatable promoter region of the cis premeribiase gene or the synthetic or semi-synthetically obtained equivalent substances.

Third, the present invention encodes yeast-specific premelibiase and melibiase. cipher region, preferably derived from Satucharomyces carlspergensis. The goal is to provide the best possible results.

Fourth, the present invention provides at least one protein used to transform yeast cells. A vector assembly having a motor region and a signal sequence is provided. . In a preferred form, the promoter region and signal sequence are specific to or suitable for the host yeast cell to be transformed. It is functionally (operably) associated with a foreign cryptographic domain. Promoter region and signal originating from premelibiase specific to S. rugensis Vector constructs assembled from each of the sequences and coding regions can be used to transform a foreign host. Used to convert.

Brief description of the drawing Figure 1 shows the first BamH part from the Hindl site of plasmid PMP550. The nucleotide sequence of the premelibiase gene including the promoter (including the 5' flank region and premelibiase gene sequence) . The amino acid sequence of premelibiase is at the bottom and the corresponding nucleotide sequence is at the top. and the sequence leading to the 3' untranslated sequence up to the first BamH restriction site. is shown.

FIG. 2 is a diagram schematically showing the division of the DNA segments shown in FIG. 1.

Figure 2a shows the nucleotides of the secretion signal of the natural melibiase shown in Figure 2. It is a figure showing an arrangement.

FIG. 2b shows that the natural melibiase secretion signal sequence shown in FIG. A modified melibiase secretion signal nucleotide linked to a car sequence. It is a figure showing an array.

Figure 3 is a schematic representation of the assembly of plasmid P4, shown by a plasmid map. .

FIG. 4 is a schematic diagram of the assembly of plasmid P5 shown by a plasmid map.

Figure 4a shows the nucleotides of one segment of plasmid P5 shown in Figure 4. It is a figure showing an array.

FIG. 5 shows plasmid P6 and plasmid P7 shown by plasmid map. This is a schematic diagram of the assembly.

In the present invention, a preferred signal sequence is a signal sequence for secretion of melibiase. It is obtained from S. Carlsbergensis by the method described below. be. The specific amino acid sequence encoded by this signal sequence is shown below. (Schematically shown.

me t-phe-a l a-phe-t yr-phe-1eu-t hr -a 1 a-c ys -i l e-ser-1eu-1ys-gl y- Similarly to val-phe-gly, the nucleotide sequence corresponding to the above amino acid sequence The columns are represented as follows.

5' ATG m C; CT TTCTACm CTCAeCGCA TGCA TCTr AGT TTCAAG GGCGTT α℃3’△ The first six nucleotides 3' from the reference number □□□ in Figure 1 are pMP. 550 Hindl [sites are shown. Reference number 25 is Hjnct[[site This is the first BamH site downstream of BamH. This 5' Hi nd l[- The Bam H engineering 3' region contains the promoter region of the melibiase gene and the gnal sequence as well as the melibiase coding region. premelibiase dark It is shown in the number area column. The corresponding nucleotide sequence is this specific amino acid It is shown above the column indicating the array. In Figure 1, 3 is the adenine nucleotide. t represents a thymine nucleotide and g represents a guanine nucleotide. , C represents a cytosine nucleotide. The amino acid that corresponds to each codon is They are indicated using the usual three letter abbreviations. Premeribise starting methionine Starts from the atg codon corresponding to the tga translation termination sequence and ends immediately before the tga translation termination sequence. I'm tied up.

This sequence consists of 1.413 nucleotides as shown in FIG. Reference number 1 Translation of amino acids does not occur before 0 or beyond reference number 12. In addition , - Tree rows indicate nucleotide sequences. For naturally occurring substances, atg open The start codon 16 is preceded by a promoter DNA sequence (between reference numbers 20 and 10). ), which we will refer to later.

The secreted protein contained within the nucleotide sequence for premelibiase. The nucleotide sequence encoding lyvase is shown in Figure 1 with reference number 1. 4, starting from gtg, which corresponds to the N-terminal valine of secreted melibiase. , ending in a tct codon corresponding to serine 12. The length of this array is 1.3 It is 59 nucleotides. The molecules of the total protein shown in Figure 1 are shown in Figure 1. The nucleotide sequence with reference numbers 10-14 is the pre-region of premelibiase, That is, the atg codon corresponds to the starting methionine 16 of Signa's premeribiase. starting at glycine residue 18, which is adjacent to the start of the secreted melibiase. Consists of corresponding reference nucleotides.

This melibiase signal sequence is co-coordinated by a region associated with this MEL sequence. The predicted role in directing secretion from yeast cells of coded gene products It turned out that he had great outside abilities. The present invention is not limited to any particular theory or is not intended to be limited or bounded by its method of operation. , MEL signals can better differentiate the gene products produced under its influence. It is predicted that it will lead to the secretory pathway or have other efficient manipulation capabilities. Ru.

Preferred promoters, associated with or separate from the above signal sequences, are of the premelibiase gene unique to S. carlsbergensis in a separable manner. A promoter that can promote transcription. The promoter region is defined herein as Preferably, those derived from yeast identified in this yeast according to the method; Nucleotide sequences similar to those that exist may be synthesized.

Selecting this particular promoter as a component in vector construction is clearly beneficial.

The promoter region of premelibiase (B) contains a certain protein (GAL4 gene). GAL80 gene and the product of the GAL80 gene). Especially this When the expression level of the GAL4 gene increases in a host that has a promoter region, this The promoter region of It has been shown to increase the expression level of structural genes. Also, this promotion When normal GAL80 product production is disrupted in the host containing the target region, this protein The expression of genes associated with the motor region is promoted, and the GAL8Q protein is Unlike in the presence of proteins, expression of such genes is no longer possible in the presence of bacteria in the medium. It was found to be independent of the presence of lactose.

This promoter sequence is shown in FIG. 1 between reference numbers 20 and 10.

Based on knowledge of the complete and complete sequence of the promoter and promoter regions, this identifying and isolating all or part of these sequences from yeast cells; and/or it can be produced by synthetic means.

Then, for example, if you want only one expression, insert this promoter region into a vector. be incorporated and functionally linked to the polypeptide coding region itself, or secreted. In order to place the null sequence in the same leading sequence as the coding region encoding the desired protein. It is functionally combined with the cryptographic region within the frame.

In the latter assembly, the signal sequences and those described herein are particularly useful. preferable.

Examples of host cells that can be used in the method of the invention include S. carlsbergensis, S. Levische, Saccharomycobsis and Lipolyteica sislipolytica) [Candida lipolytica lipolytica) or Yarrowia lipolytica (Yarrowi Also known as a1ipolylica). S, car It will be appreciated that Lus bergensis is also known as S. ubarum.

The gene used in the construct of the present invention is a gene that is heterologous to the above yeast species. Yes, these include interferon, insulin, hormones, growth factors, and enzymes. human proteins and peptides such as, bacterial and fungal proteins such as cellulases. , plant proteins, peptide flavors, peptide enhancers, antigens, etc. This includes human, animal, and bacterial genes.

Expression vehicles or plasmids suitable for introducing the constructs of the invention are suitable genes or genes to maintain and select them in yeast and E. coli. part of; like a strong yeast promoter, the inserted nucleotide sequence to express the inserted foreign sequences to ensure that they are well transcribed. Suitable yeast sequences; as well as locations downstream from the insert to ensure termination of transcription. It contains the sequence of the yeast that is located in the yeast. No specific examples of such vehicles are known. It can be purchased commercially [see Reference Example (6)].

Additionally, other than the promoters and signal sequences described herein, Currently suitable sequences include transcription termination sequences as well as the desired assembly to form the object into a functional form. This includes the modifications necessary to do so.

Methods of introducing these into host cells are known and can be accomplished using conventional techniques in the art. be.

Embodiments of the invention are illustrated in more detail by the following examples.

Example 1 Melibiase and premelibiase Bost-Bitenmiller et al. 5), a protein containing additional fragments of yeast DNA. Plasmid pMP550, the lasmid Yep24 (7 and 26), makes it When introduced into melibiase-negative yeast by transformation, melibiase is introduced into those yeasts. confers a positive phenotype. This means that approximately 2 ．． This is a 9 kilobase EC R1w BamHl fragment. to pMP550 The included DNA insert contains the structural gene for premelibiase. This shows that. J, E, Ho of Pennsylvania State University The plasmid pMP550 obtained from Mr. For sequence determination, ◎pMP5502μg and phage M13m prepared as follows. The replicable form (RF) of p18 (Norander et al. 1), 8] to EcORl and BamHI in a New England jar. Digest with Iolabo x (New England Biolabs)), 1 % (v/v) of low melting point agarose. pMP of approximately 2.9 kilobases 550 fragment and the approximately 7.2 kilobase M13mp18 fragment. Warm the sections to 65°C to release the DNA, mix a few μl of each and add 70mM ) Squirrel, 'HC! Buffer (PI-17,5), 7mM MgC/, 7 0. IJM in 20 μj containing ATP and 0.9 units of T4 DNA ligase Ligation was allowed for 3 hours at 12°C. Using this ligated DNA, a The cells were made competent in advance using Hanahn's method (10). Escherichia coli JM109 strain (9) was transformed. 6 plaques generated by transformation Select a block, isolate the DNA, and check for the presence of the pMP550 DNA fragment. I checked. All had appropriate structure. Run one of them as follows: selected for sequencing. Colon transfected with selected phages RF DNA was prepared from fungus JM109, and E, oRI and pstI were injected with 20 μg was completely digested using BioLabos) and ethanol precipitated to give 0.0 μg. 66mM) squirrel HC/buffer (pH 8, containing 66mM MgC11) 0). Hen1koff (Hen1koff) Correct the method of (11) Following the exact procedure, the DNA was digested with exonuclease l [(PL-Pharmacia (PL-p hermacia)), take specimens at intervals, and incubate with nuclease 5. (BRL) Escherichia coli, NA bolimerase (Flenow fragment) [Berry Boehringer Mannheim A) and Treated with T4 DNA ligase (Boehringer Mannheim), the M13mP18 sequence was A series of circular DNA sections of decreasing size of the pMP550 insert adjacent to the column. generated a child.

DNA ligated by transfection of large intestine TL M 109 After collecting the pMP55Q DNA, the size of the remaining pMP55Q DNA was determined by electrophoresis. and sequenced by the dideoxy method (12) described in the instructions for use (13). . The resulting sequences were subjected to learning and meshing (Larson and Messi ng), and further concatenated according to the program α shown by Dev. The analysis was performed using the program (15) of ereuxet az, ). First pM 2 contained in all 7 fragments of E5:p, R-K-BamH, obtained from P550. The sequence of 864 nucleotides was determined. Figure 1 shows the complete Eco, Ri sites ( This sequence is shown extending from (28) to the complete amH1 site (25).

Figure 1 also shows a translation based on a long open reading frame. The deduced amino acid sequence of melibiase is also shown. calculated from the nucleotide sequence The molecular weight of premelibiase is 52.101. secreted melibiase Based on the known N-terminal amino acid sequence (see Example 2), it is designated by reference number 16. Amino acids from methionine to glycine shown with reference number 18 are melivia This is the signal peptide of ZE.

Therefore, the calculated molecular weight of secreted melibiase is 50.104. this The values are obtained by purifying the culture supernatant of yeast transformed with plasmid pMP550. Example 3), molecular weight of deglycosylated melibiase, i.e. approximately so, ooo matches.

The remaining sequences on both sides of S. carlsbergensis were determined as follows. 2μg pMj550 and 2μg of M13mP1B were added to Xma1 and 5ph, respectively. Digested with 1C New England Biolabs), 0.8XW/v low melting point a Electrophorese in garose. The approximately 1.8 kilobase pMP550 fragment and the approximately The gel section containing the 7.2 kilobase M1'3mp18 fragment was melted and a few microbases 1 of each were mixed, ligated, and transformed into JM109 in substantially the same manner as above. It was used as a replacement. After proving the presence of the desired insert, one plas- ty was prepared for further experiments. selected.

Substantially following the method of Dale et al. (1985) A series of deletion clones were created extending through the insert. Briefly, - heavy chain D After hybridizing 5 μg of NA with the primer (RD29), the mixture was incubated at 50°C. , - Digested with Bind [at night. - The 31 end of the heavy chain DNA was merase to various extents by New England Biolabs), Terminal transferase (InI) and dATP (Sigma) The mixture was cured, annealed with RD29 primer, and ligated. JM109 pMP remaining in some phage clones after transfection of After analyzing the size of 55Q DNA by electrophoresis, -heavy chain D NA was prepared and sequenced. Using this method, 929 nucleotides of the sequence of PMP550 Chido was further determined. Therefore, by these two methods, the complete PMP550 S, force-Rusbergensis induced sequences were obtained.

261 Purification of melibiase 2, i, 1. Preparation of yeast that secretes melibiase Ito et al. al) (16) f) Transform the plasmid according to the method described. PMP550, J, E, Hopper (J, E, Hopper) (Pencil Melibiase-negative yeast strain S supplied by Vanier State University).

agar to select cells that proliferate independently of exogenous uracil. It was spread on a plate and incubated at 30'C for 2-3 days. It is called selective medium. The composition of this medium is 20g of glucose, 17g of agar, and no amino acids. Yeast nitrogen base 6.7g, succinic acid 5.8g, dipotassium hydrogen phosphate 8.7g% and adenine, arginine, histidine, inleucine, leucine, methionine , tryptophan and tyrosine each 0.02g, lysine 0.03g, phenyla Contains 0.05 g of ranine, 0.10 g of threonine and 0.15 g of valine, pI-1 was 4.6-4.8. Renew the colonies that appeared on selective medium 1. Transfer to a new selective medium and grow at 30'C, then add selective medium (lactic acid 20 g/ L, glycerol 30g/L, and galactose instead of glucose 20g/L The cells were transferred to an agar plate containing a selective medium containing 20 g/L of water. Hidden, Gala Coutose induced melibiase synthesis (5). Next, this colony It was used to inoculate a liquid medium consisting of selective medium (■) without agar. Cell growth at 30℃ After that, the secreted melibiase was analyzed using the following method. Culture 500 μj The cells were pelleted by centrifugation, and then 10 μl of the supernatant was diluted with 12.5 mM Nitrophenyl α-logalactodide (p-Npc: Sigma Chemical Co , ) containing pH 4 buffer (3Q mM citric acid, 38 mM dibasic phosphorus) Add 190 μl of sodium chloride and 800 μl of sodium to change the yellow color to 407 nm. It was measured by spectrophotometry. The uracil-independent transformants tested All produced melibiase, but S. cerevisiae strain 284 itself did not. No melibiase was produced. pMP 55Q, for purification of Scase I chose it for this purpose.

2,1.2-Melibiase purification Agar-free selective medium If IOL containing yeast transformants pMP550, SC284, 1 and incubated at 30°C until maximum optical density at 660 nm was reached. The cells were incubated under shaking. Centrifuge the culture at 3,000×g to remove cells. , Amicon (Δm1con) limit offshore passage unit equipped with PM-IQ Menplan. The supernatant was concentrated to 100% in a vacuum chamber. This concentrated solution was heated at 40,000×g for 30 minutes. The mixture was centrifuged for a while to clarify the solution. This material was then diluted with 5Q mM Tris HC/buffer. Dissolve in buffer (pI-17,5) (buffer) 25- Furthermore, dialysis was performed against 2.5 L per change). This substance was heated for 0.4 s micro A solution containing 16.619 protein and 3534 melibiase units was obtained (17). Next, a Mono Q anion exchange column (HR515 ) 7/L/7'i7 (Pharmacia) FPLC system The melibiase activity was separated from some other proteins on the system.

Melibiase, which remains bound to the column, is removed by a linear gradient of sodium chloride. Diendo (125-245 mM in buffer) was applied to elute. 1f The nl fractions were collected, analyzed and the active fractions were pooled. protein ! 7.61■ (containing 3437 units of melibiase) was recovered. Freeze this substance Dry, dissolve in buffer 6.8-, divide into two parts, and mix with Biogel. l) A-' Chromatograph with 1.5 M column (1,5X 90.0Ql) It was rough. The fractions containing the highest melibiase activity were pooled; contained 3.8 μ protein and 2302 units of melibiase.

SDS acrylamide according to the method of Laemml (18) When electrophoresed in a dog gel, this substance.

It contained two bands representing 98% of the protein. one band is diffuse and includes a size range of 75 to 100 kilodaltons (centered at 82 kilodaltons) The second band is sharp, and its calculated molecular weight is 70 kilodaltons. Ta. Determine the molecular weight of the melibiase polypeptide chain and also whether it is pure To determine whether melibiase secreted from S. carlsbergensis Covalently bound carbohydrates that are known to be part of [Lazo et al. Lazo et al) l 9: Lazo et al., 20] and end it as follows: - Using β-N-acetylglucosaminidase H (endo H) , was oxygenally removed from the sample. 40μg of protein, 0.0.5M citric acid and 0.1 M sodium dihydrogen phosphate at 37°C in 40 μl of reaction solution. for 25 hours, Endo H (Behringer Mannheim, Canada) in 5 mm increments and one incubated together. This sample was then lyophilized and 5DS-polyacrylic The results were analyzed using Lumid gel electrophoresis. A single compound with a calculated molecular weight of 50 kilodaltons A protein band was observed.

2.2 Determination of amino acid sequence Pure melibi without Entodo I treatment to remove carbohydrate side chains. Derivatization of Aase with phenylthiohydantoin Therefore, the first 10 amino acids were sequenced. Department of Biochemistry, University of Toronto part of Biochemistry.

University of Toronto) amino acid sequencing device The array determined by the formula: %formula% We do not know why the first two residues are unclear. Nevertheless, the plasmid Predicted from the nucleotide sequence of the DNA insert in pMP550 (Figure 1) By identifying the remaining 8 residues, the amino acid sequence of secreted Nori-1ase was determined as follows. The conclusion was reached that it starts from the lower N-terminus.

val-ser-pro-ser-tyr-asn-gly-1eu-gly- 1eu +++2.3 Structure of melibiase signal sequence Secreted melibiase The prediction was made from the N-terminal amino acid sequence of the enzyme and the nucleotide sequence of the premelibiase. Based on the measured amino acid sequence, the signal sequence of melibiase starting from the N-terminus is determined. Column formula: %formula% It was decided that it would be indicated by

Use of expression cassettes This expression cassette is schematically shown in FIG. The basic elements are (1) both sides restriction site (in this example, Bam II engineering) and a suitable E. coli/S, S. A site that facilitates insertion into the Viche Shuttle vector, an activation sequence upstream of (It). ) and the DNA sequence of melibiase signal peptide 36, The coding sequences of substances other than revivase (as shown in 38) can be combined with these in functional (operational) A DNA sequence in a form that can be arranged in association (possible). In this example, this means that Bg is adjacent to the sequence encoding the signal peptide of melibiase. This was achieved by creating a l■ site. This allows the signal peptide to It is possible to create a fusion between a codon that specifies a protein and a codon that specifies a new protein. Become. Therefore, the functional association is promoter: signal peptide codon: new Consists of regular protein codons. The segment indicated by 34 in FIG. Contains additional sequences to specify transcription, termination, cleavage and polyadenylation There is. This expression cassette was prepared using standard recombinant DNA techniques, as described below. It was then assembled as shown in Figure 3.

Plasmid PMP550 (Figure 3) contains the melibiase gene and the DNA on both sides of it. Sequence [Po5t, Beittenmiller et al. t al), 5 and Sumner-Sm1th et al. 4.3 kilobase DNA fragment containing 40) Plasmid PBR22 (Boliver et al., 22 ]. The nucleotide sequence of fragment 4o is shown in FIG. ndl [[ site 3o and RamH1 site 25 are the boundaries. first 346 nuku It should be noted that Resmid pB was derived from R322. This is This is due to the fact that a DNA fragment of 4.3 kilobases was used. The function of the expression cassette derived from Plasmid PMP550 ind-1 [and BamHI], the 4.3 kilobase fragment 4o was added to plasmid P [Jc8 [Vieira] and Messing (Me ssing):24] between the Hindl[ and BamH sites.

The resulting plasmid P1 was restricted with Hindl, and the DNA end was inserted into E. coli DNA. Filling-in using the Flenow fragment of A polymerase Following the method of Lathe et al., the formula: 5' CCGGATCCGC, 3' 3' (,CCCTAGGCC5' This ligation product is ligated to the synthetic oligonucleotide 42 shown in One plasmid, P2, contains PMP550, which is restricted on both sides at the Bam1 site. pUc8 contains the original 4.3 kilobase fragment 40. P lasmid P2 within the DNA sequence encoding the melibiase signal sequence. It was restricted with 5phl, which specifically cuts with . This linearized plasmid was then transformed into the formula : Synthetic oligonucleotide 42 with the sequence shown was added by the method of Latte et al. (25). I made Reigate. Across (beyond) the position of the inserted oligonucleotide The recombinant plasmid was sequenced and this oligonucleotide was sequenced in the orientation shown in Figure 2. A plasmid containing P3 was selected. Next, plasmid P3 Restrict with BamHl and transform the 4.3 kb fragment into shuttle plasmid Yep 24 (26)'s BamH1 site Niclonite plasmid P was generated.

Plasmid P contains a 4.3 kilobase fragment of pM P2S5 origin However, at this point, I, which encodes the melibiase signal peptide, ) The NA sequence has been modified as shown in FIG. 4.3 in P4 (Figure 3) The kilobase BamHI fragment 40 is an example of a preferred embodiment of the invention. It constitutes an expression cassette.

Use of Expression Cassettes to Manipulate Production of Lucanase or 1, Endo-1,4- Known to encode β-D-glucanase, EC3,2,1,4) Utilization of expression cassettes in S. cerevisiae using DNA sequences proved. As shown in Figure 4, the coding region of this endoglucanase is , Dr. Rob C. Miller, University of British Columbia Plasmid pE02.2 (Figure 4, ski It is included in BamHI fragment 46 derived from Per et al.; 27). This cipher The region contains bacterial proteins, including a 31 amino acid bacterial signal peptide (28). It was found that the codon for the first 767 amino-terminal amino acids of the protein was lost. There is. According to previously conducted experiments, the 2.4 kilobase B derived from PEC2,2 The am I-II fragment was inserted into the yeast alcohol dehydrogenase 1 promoter. Satsuka Roma transformed with a plasmid containing in functional association with the tar Ises cerevisiae produces extremely low levels of extracellular endoglucanase activity. [5kipper et al. ) ;27]. Codon specific for the amino terminus of the yeast preprotoxin protein addition to a functional signal peptide increases the level of extracellular endoglucanase activity. (Skipper et al.: 27).

The 2.4 kilobase Bam HI 75 component derived from pEC2.2 was added to the plasmid. By sticky end ligation with the Bgl ■ site of p4 (Figure 4), the protein is inserted into the lasmid. Recombinant plastic containing this endoglucanase insert The same is true for those containing inserts in the appropriate orientation by restricting the sumid with Bgl fl. some of them across the melibiase/endoglucanase junction. were sequenced and those containing the predicted gene fusion were identified. for further analysis , one plasmid P5 was selected. It is the correct translation reading frame (leading Endoglucanase fused with the signal sequence of melibiase in frame) (See Figure 4a). As expected from the assembly drawing (Figure 5) As shown, the terminal codon (GGG glycine) of the signal peptide of melibiase and the There is an extra link between the 77th codon (ATC, inleucine) of ndoglucanase. codons (AAG, lysine) are present. 4.3 kilobase expression cassette and 6.7 kilobases of P5 containing a 2.4 kilobase endoglucanase coding sequence. The 13amI-11 fragment 48 of the lower base has a functional Melibiase: Contains an endoglucanase gene fusion.

Plasmid P5 contains the Yep melibiase:endoglucanase gene fusion. It is 24. Yep24 can be heard. Endoglue from melibiase elements A range of receptor Satucharomyces cerevisiae strains for the purpose of evaluating the expression of canases. To expand P5, two shuttle vectors, Yep21 and Yep 24.

P6 and P7 were obtained (Figure 5). Plasmid Yep Mid Yep 24 (26 ) and Yep (26).

It is useful for

to test the expression of endoglucanase in yeast. Rebiche strain 20B12 (α, trp 1 pep 4.3; Jones (Jo nes) 29) was transformed with plasmid P7 in a tryp- and tophane-independent manner. The extracellular endoglucanase activity was examined. Two assay methods were used: transformation Direct staining of agar plates (containing the substrate carboxymethylcellulose), which has been rumored Qualitative analysis by color and equivalent to reducing sugars, carboxymethyl cellulose ice melt Based on the determination of the amount (qualitative analysis method). 0).

1, 2% agar, 0.9% carboxymethyl cellulose (high viscosity, Sigma Chemica Le Co,), 2% glucose, and 59mM sodium phosphate buffer. Transformants were grown at 30°C on agar plates with a final pH of 6.8-7.0. . Rinse the yeast colony with water, then wash it on a flat plate, Cong o Red) (After staining with 2"9/mO for several minutes, 1M sodium chloride I rinsed it off.

Under these conditions, β-1,4 glucans consisting of 7 or fewer glucose residues This is because Congo Red does not combine with Congo Red (Wood, 31). , endoglucanase activity is indicated by uncolored areas.

In this assay, all transformants produced extracellular endoglucanases. Ta. For quantitative analysis, transformants were grown in 2% glucose and 5QmM sodium phosphate. (pH 6.8-7.0). This culture The cells were removed by centrifugation, the supernatant was dialyzed against water, and the dialysate was lyophilized. . After reconstitution in water, the sample was prepared as described by Miller et al. 2) and carboxymethyl as described by Skipper et al. (27). Regarding the production of reducing sugars and other damaged substances from cellulose (low viscosity, Sigma Chemical C00) It was measured by According to this measurement, all transformants contained extracellular endoglucana. It was producing ze. A typical transformant contains 6.4 units of endoglucan per culture. Glucanase was produced (1 unit is 1 μmol reduced per minute at 37°C). produce sugar).

In common with CAL7 and GALIQ, the MEL-1 (melibiase) gene expression is induced by galactose and repressed by glucose. this carbon In the absence of galactose, binding of the DNA action site product is inhibited. damaged and all genes are expressed.

The binding site of the CAL4 product is within the 5'-flanking region of each target gene. , it is called UAS or upstream activation sequence [Sumner-Smith et al. urnner+　Sm1th　et al　;　see brief review in 23]. follow By manipulating the CAL4 and GAL13Q genes, promoter-dependent protein expression (herein used as an example It is anticipated that the regulation of the use of glucanases (using glucanases) could be improved.

Three Saccharomyces cerevisiae strains, 5c284, Sc295 and Sc300 (Table 1) was extracted from a plasmid containing functional melibiase. To examine the effects of CAL4 and GAL80 modification on doglucanase expression. It was used for. These strains were developed by Dr. J. E. Hopper of Pennsylvania State University. Obtained from.

Therefore, galactose is required to fully express the melibiase promoter. do not need. 5C300 strain (lcu 2-3, leu 2-11211a del, gal 1801: cAL4) lacks CAL3Q, and in addition , intended to increase the activity of GAL4 protein on the melibiase promoter Since then, two types of modifications have been made and gene groups have been deleted. Second, yeast alco This is due to the strong promoter of the alcohol dehydrogenase l gene; It is predicted to increase the synthesis of normal single chain chromosomal GAL protein. Meri Increased phenotypic production of strain 5c300 regarding biase promoter function It is reasonably expected to have both increased maximal function due to It will be done.

Each of the three yeast strains was incubated with Escherichia coli/S-cerevisiae suitable for selection of transformants. Included in the shuttle vector.

Transformation with a functional (operable) expression cassette/endoglucanase fusion in (Table 1). Next, the transformants were transformed into normal GAL14 and GAL8Q strains. within the melibiase promoter, whether non-inducible, inducible or repressive. After growth in liquid medium containing either carbon source, extracellular endoglucan activity was measured. The results are shown in Table 1 and discussed below.

The absolute amount of extracellular endoglucanase produced by this transformant is low; It is difficult to evaluate with any degree of accuracy the effect of adjusting carbon sources in various combinations. Met. However, the induction by nigalactose in response to the carbon source , consistent with inhibition by glucose.

Endoglucanase expression was still observed in the absence of galactose, and 2% glucose was significantly reduced by the course, which was consistent with normal glucose suppression. . In the presence of galactose, endoglucanase synthesis is actually partially inhibited. This effect was also observed in the GAL8o deletion strain that expresses extracellular melibiase (not yet Observations of the presentation).

Endoglucanase expression in this strain is among those observed in all yeast receptors. , it was the best. Surprisingly, galacinyl chloride helps maximize endoglucanase expression. requires tosose and also uses glucose as a repressor in this synthesis. The effect is likely to be partial and due to increased availability of the product.

These experiments are known to affect the function of the melibiase promoter. The expression of extracellular endoglucanases can be influenced by manipulating a certain gene. In this way, in s, cereviche, this or Demonstrate the utility of the expression cassette in producing other novel proteins. It provides further evidence of this.

ATC Deposited in C. These are represented by the following abbreviations and given the deposit numbers shown below. I got it. All of these are contained in a suitable E. coli host.

P' NAS-153360 p5 NAS-253361 pa NAS-353362 p7 NAS-453363 Cultures containing these assemblies are also currently available in allelics, (Alyleix) ) Inc, 585 Q Boreway Drive ve), Mississauga, Ontario, Canada It is maintained in a permanently viable state in the laboratory and is designated by the abbreviation shown here. Identified.

... I jl! )J ＋n’6”! K total -L6+-+. Gen -p 41 ＼ Arrival S4! Encampment of feet n Ling - ('1 e and be p To 6th F5 and 1 bowl \+　--v-'-xmouth Frozen - Ichiro, -, 1゜ 8 amAm+8(' Takashi & 1) Ball Table of reference examples included in the specification 1) Hilaraman, Lang, Berry, Cole, Levin, Geladel ('Hitzem) an, RlA, Leung, J, W, Perry.

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J.) (1983). Proceedings of the National Academy My of Science (Proc, Natl, Aead, Sci,) USA 80.7080-7084゜3) Blake, Merryweather, Koito, Heperlein, Masialut, Mullenbach, Uldea, Parenzuela, Baal ( Brakj A, J, Merryweather, J.

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Gaithersburg, Me7! J-57 de (Maryland) 20J37? .

14) Larson, Messing (Larson, RoMessing, J.) (1982). Nuclitsk Asits Research (Nucl, Ac1ds) Res, ) 10, 39-49゜15) Dehelyou, Heberli, Sumire x (Devereux, J.

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22), T'River, Rodriquets, Glenn, Betratsuno), Heinetzker , Boyer, Klosa, Falkow (Bolivar, F., Rodriqu ez, RoLlGrene, P, J+, Betlach.

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29) Jones, E. (1978). Syenetics ( Genetics) 85, 23-33゜30)''C tryptophan medium is , 6.7 g of yeast nitrogen base; adenine, arginine, inleucine, Rosine, uracil and leucine each 0.02g; lysine 0.03y; phenyla Lanin o, osy; Threonine 0.10 f: Barrier 0.15 y; Na2HPO43,09; KH2PO43,Of; KH2PO41,5F including.

31) Wood (1980) Carbohydrate Research (C arbobydrate Re5search) 85, 271-287゜3 2) Mi5, Plumb, Brennon, Bart 7 (Miller, G, L,.

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Biochem, ) 2.127-132゜33)" Uracil medium in 1 l, Yeast nitrogen base 6.7g, adenine, arginine, inleucine, tyrosine, tri Ptophan, and 0.02r each of leucine; lysine O, o:llp; phenyla Ranine 0.05g; Threonine 0.109: Bali 70.15y; Na2H PO43, □y; Contains KH2PO41,5V.

34) Leucine medium contains 6.71 yeast nitrogen bases per liter! ;adenine, algi 0.02g each of nin, isoleucine, tyrosine, tryptophan, and uracil ! ; lysine 0.03P; phenylalanine 0.05P; threonine 0.10r; Valine 0.15y; Na2HP04a. Of　;　KH2PO41,59 including.

891 I+ -1indl11 international search report 0 shots clear Sumner-Smith, Martin Canada's wide bottom 0 shots by Bozat, Richard Paul, Canada; N, Chat 0 shots: Davis, Roger Uniin, Canada, Nis, Arno 1 L.0.Pete.0 33rd Hot Crescent, Porton, Ontario M.9.B.5.K.9 Ontario, Eislind Doprtsk Dry 3am L. O. Pete H. O. Ontario, Limebaur R. No. 1

Claims (50)

[Claims] 1. a DNA construct expressible in transformed yeast can be, The promoter region of the melibiase gene and the points that are functionally compatible with it. Cryptographic region of lipeptides An assembly containing. 2. The first term in which the coding region of the polypeptide encodes something other than melibiase Assembly as described. 3. The coding region of the polypeptide is Cellulomonas. Fuimi's endoglucanase genetics The assembly according to paragraph 2, which is derived from a child. 4. The coding region is followed by a transcription termination region, and these regions are functionally associated. The assembly described in Section 2. 5. The promoter region is S. The second term is derived from Carl Bergensis. Assembly as described. 6. The DNA sequence of the promoter region is substantially as shown in Figure 1 of the accompanying drawings. The assembly according to paragraph 2, which is a product. 7. A method for modifying the expression characteristics of yeast cells, the method comprising: A DNA segment containing the promoter region of the melibiase gene that is operably linked to the melibiase gene. A method comprising introducing an agent into said yeast cells. 8. The polypeptide coding region of the DNA segment encodes something other than melibiase. The method described in paragraph 7. 9. Yeast cells are S. cerevisiae and S. cerevisiae. selected from Carlspergensis 9. The method according to item 8. 10. promoter region. Nucleo substantially as shown in Figure 1 of the accompanying drawings. 9. The method according to item 8, having a tide sequence. 11. The coding region of the polypeptide is the endoglucanase residue of Cellulomonas fuimi. The method according to item 9, which is derived from a gene. 12. Functionally linked to coding regions encoding polypeptides other than melibiase melibiase, which has the effect of controlling the transcription of this coding region. A yeast cell containing the promoter region of a gene. 13. Saccharomyces cerevisiae and Saccharomyces 'Karlsperge' 13. The yeast cell according to clause 12, which is selected from P. chinensis sp. 14. The coding region of the polypeptide is the endoglucanase residue of Cellulomonas fuimi. 13. The yeast cell according to item 12, which is derived from a gene. 15. Functional promoter region in yeast cells, functional signals in yeast cells A signal sequence that encodes a peptide and is functionally connected to the promoter region. a federated array, and A DNA set comprising a coding region of a polypeptide operably linked to the signal sequence. at least one of the signal sequence and the promoter region Construct that is a melibiase gene segment. 16. The assembly according to paragraph 15, wherein the signal sequence is a melibiase signal sequence. Things. 17. The signal sequence is effectively the formula: (N-terminus) [Sequence available] 17. The construct according to claim 16, having the amino acid sequence shown in 18. The signal sequence is effectively the formula: (5') [There is an array] (3') 17. The construct according to claim 16, having a nucleotide sequence of: 19. The signal sequence is S. No. 16 derived from Carlsbergensis Assemblies described in section. 20. Item 15, wherein the promoter region is a promoter region of melibiase. assembly. 21. The promoter region comprises a nucleonucleotide substantially as shown in Figure 1 of the accompanying drawings. 21. The assembly according to claim 20, having a tide sequence. 22. Item 16, wherein the promoter region is a promoter region of melibiase. assembly. 23. The coding region of the polypeptide encodes a polypeptide other than melibiase. 23. The assembly according to paragraph 22. 24. The coding region of the polypeptide was derived from Cellulomonas fuimi Assembly according to paragraph 23. 25. The promoter region is the promoter region of melibiase, and is essentially the promoter region of melibiase. The set according to item 16, which has the nucleotide sequence shown in Figure 1 of the accompanying drawings. A structure. 26. The coding region of the polypeptide is followed by a transcription termination region, and these are functional An assembly according to paragraph 15 associated with. 27. introducing the construct according to any one of paragraphs 15 to 26 into the yeast cell; A method for modifying the expression and secretion properties of a yeast cell, comprising: If the motor region and the signal sequence are both segments of the melibiase gene, If so, do the coding regions of the polypeptide encode something other than melibiase? , or characterized in that the construct is foreign to the yeast cell. How to do it. 28. 28. The method of paragraph 27, wherein the yeast cell is Saccharomyces cerevisiae. 29. Item 27, wherein the yeast cell is Satucharomyces Carlsbergensis sp. How to put it on. 30. Polypeptide coding region and functional coding for something other than melibiase A yeast cell containing a melibiase signal sequence bound to a yeast cell. 31. Item 30, wherein the promoter region is the promoter region of melibiase. yeast cells. 32. The coding region of the polypeptide was derived from Cellulomonas fuimi Yeast cell according to paragraph 31. 33. S. 31. The yeast cell according to paragraph 30, which is of the species Carlsbergensis. 34. S. 31. A yeast cell according to paragraph 30, which is of the species S. cerevisiae. 35. A promoter region that is functional in yeast cells, a promoter region that is functional in yeast cells, Sig encodes a signal peptide that is functionally associated with the promoter region. null array, and A polypeptide encoding a polypeptide and operably linked to the signal sequence. Propagating Yeast Cells Transformed with DNA Constructs Containing the Petide Coding Region A method for secreting polypeptides from yeast cells, which consists of growing them in a promoting medium. at least one of the signal sequence and the promoter region is A method characterized in that it is a segment of a lyase gene. 36. The third polypeptide whose coding region encodes something other than melibiase The method described in Section 5. 37. Item 36, wherein the promoter region is a promoter region of melibiase. How to put it on. 38. The method according to paragraph 37, wherein the signal sequence is a melibiase signal sequence. . 39. The coding region of the polypeptide was derived from Cellulomonas fimi , encoding an endoglucanase. 40. Transcription of the signal sequence and coding region occurs in GAL4 and/or GAL8. 0 by changing conditions in the growth medium that affect the activity of the 0 gene and its products. 38. The method of paragraph 37, wherein the method is regulated. 41. Signal sequence of yeast-specific melibiase gene. 42. S. 42. The signal sequence according to paragraph 41, which is specific to Carlsbergensis. 43. In effect, the formula: (N-terminus) [Sequence available] The system according to item 42, which has a DNA sequence encoding the amino acid sequence shown in Gunar array. 44. In effect, the formula: (5') [There is an array] (3') 44. The signal sequence according to item 43, having a DNA sequence represented by: 45. A DNA construct containing a melibiase secretion signal sequence and comprising a melibiase secretion signal sequence. Restrictions flanking the signal sequence for inserting additional DNA components into the construct. A DNA construct that contains DNA segments that define positions. 46. The DNA segment has a nucleotide sequence substantially as shown in FIG. 4A. 46. The assembly according to clause 45, having: 47. Promoter region of yeast-specific melibiase gene. 48. S. 48. The promoter region according to paragraph 47, which is specific to Carlsbergensis. 49. Item 47 having a DNA sequence substantially as shown in Figure 1 of the accompanying drawings. promoter region. 50. A DNA construct containing a melibiase promoter and , a control for inserting additional DNA components into the vector downstream of the promoter. A construct containing DNA segments that define a restriction site.