KR0171160B1 - Expression Vector for Secretion Production of Salmon Calcitonin Precursor and Method for Preparing Salmon Calcitonin Precursor Using Same - Google Patents

Abstract

The present invention relates to an expression vector for the production and secretion of salmon calcitonin precursor (sCT-Gly) and to a method for producing sCT-Gly using the same, sCT-Gly linked to a polynucleotide encoding a ppL or inulinase secretion signal sequence According to the method for culturing yeast transformed with an expression vector containing a gene, sCT-Gly can be secreted with high efficiency.

Description

Expression vector for secretory production of salmon calcitonin precursor and method for producing salmon calcitonin precursor using the same.

1 shows the construction of plasmid pYSCAL1,

2 shows the construction of plasmid pYSCAL2,

Figure 3 shows the results of urea polyacrylamide gel electrophoresis of the cytoplasmic protein and culture protein of the transformed yeast cells of the present invention,

Figure 4 shows the result of Western blotting analysis of the cytoplasmic protein and culture protein of the transformed yeast cell of the present invention.

The present invention relates to a method for producing and secreting salmon calcitonin precursor (hereinafter abbreviated as 'sCT-Gly') from yeast, and more particularly, to a polynucleotide encoding a ppL or inulinase branch signal sequence. An expression vector comprising a linked sCT-Gly gene, a yeast transformed with the vector and a method for culturing the yeast to produce and secrete sCT-Gly.

Calcitonin is a peptide hormone that regulates calcium metabolism in the body and is secreted from secretory cells called C cells. Calcitonin suppresses the loss of calcium from bones and reabsorbs calcium from the kidneys. It is derived from eight kinds of animals including humans, pigs, cattle, sheep, rats, salmon, eel, and chicken. Eleven types of calcitonin have been isolated and purified to determine the primary structure (three types of salmon and two types of calcitonin in eel) (Japan, BIO INDUSTRY., 3, 3 (1993)). These calcitonins are short-chain polypeptides composed of 32 amino acids, each having a cyclic structure in which the N-terminus and the 7th cysteine are connected by disulfide bonds, and the C-terminus is an acid amide (like the C-terminus of glucagon or secretin). Proline amide).

On the other hand, Escherichia coli and yeast, which are mainly used as host cells for mass production of recombinant proteins, do not have alpha-amidating enzymes. It is impossible to produce it yourself.

Therefore, Tajima et al. Produced human calcitonin precursors with glycine bound to the C-terminus in recombinant E. coli, and then converted to mature calcitonin using alpha-amidase (Tajima et al., J. Ferment.Bioeng., 72, 362 (1991). This method has a disadvantage in that the separated purification process is difficult because the expressed human calcitonin precursor accumulates in E. coli.

On the other hand, Saccharomyces cerevisiae is a yeast in which the host-vector system is best established at present, and much research has been carried out physiologically and molecularly, and there is no pathogenicity in the human body. It is a microorganism generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA) that does not produce non-pathogenic endotoxins. Therefore, its importance as a host cell for heterologous protein production using genetic recombination technology is increased, and the use of yeast secretion signal can secrete the expressed heterologous protein into cells, thereby reducing the cost of separation and purification.

Secretion signal sequences currently used to secrete heterologous proteins from yeast include the invertase signal sequence, the acid phosphatase signal sequence, and the preproleader sequence of the cross-factor α (weakly referred to as ppL: U.S. Patent No. 4,558,684). In this case, double ppL is most commonly used. In addition, the secretion signal sequence of Cluj Veromaises marcianus (Laloux et al., FEBS Lett. 289, 64 (1991)) is also used for the purpose of secreting heterologous proteins from yeast.

Therefore, the present inventors continued to develop a method for producing and secreting sCT-Gly from yeast, and thus, an expression vector comprising an sCT-Gly gene linked to a polynucleotide encoding a secretion signal sequence of yeast was prepared. Then, the present invention was completed by culturing yeast transformed with the vector to produce and secrete sCT-Gly with high efficiency.

Accordingly, an object of the present invention is to provide an expression secretion vector capable of producing and secreting sCT-Gly from yeast with high efficiency.

Another object of the present invention is to provide a recombinant yeast transformed with the expression secretion vector.

Still another object of the present invention is to provide a method for producing and secreting sCT-Gly by culturing the recombinant yeast.

In order to achieve the above object, the present invention provides an expression vector comprising a polynucleotide and a salmon calcitonin precursor gene encoding a preproleader sequence (ppL) or inulinase secretion signal sequence of a yeast cross-factor α.

In order to achieve the above another object, the present invention provides a recombinant yeast cell transformed with the expression vector.

In order to achieve the above another object, the present invention provides a method for producing sCT-Gly in yeast cells, comprising culturing the recombinant yeast cells, and recovering sCT-Gly from the culture medium.

The present invention will be described in detail as follows.

Expression vectors of the invention used to produce and secrete sCT-Gly from yeast include polynucleotides encoding the secretory signal sequence of yeast; A DNA sequence capable of promoting transcription in yeast, located upstream of the secretion signal-coding sequence; An sCT-Gly gene positioned downstream of the secretion signal-coding sequence and inserted therein into an open reading frame; And transcription termination sequences downstream of the sCT-Gly gene.

As the yeast secretion signal sequence, ppL, Inuriase INU1, Invertase signal sequence, acid phosphatase signal sequence, and the like can be used. It is preferable to use ppL and INU1, and most preferably, ppL.

Examples of such vectors include plasmids pYSCAL1 (KCTC 0171BP) prepared by inserting ppL and sCT-Gly genes into plasmid YEGα-HIR525 (KCTC 8518P), and plasmids prepared by inserting INU1 and sCT-Gly genes into plasmid YEGα-HIR525. pYSCAL2 (KCTC 0172BP) and the like. In addition to the above vectors, there may be various recombinant expression vectors depending on the type of promoter and transcription termination sequence used.

Using such recombinant expression vectors, conventional methods are described, for example, in the Laboratory Course Manual for Methods in Yeast Genetics, ed, F. Sherman, GR Fink, and JB Hicks, Cold Spring Harbor Laboratory (1986). The yeast cells can be transformed according to the conventional method. Host cells include Saccharomyces, Schizosaccharomyces, Kluyveromyces, Hansenula, Yarrowia and Pichia. Various yeast cells belonging to can be used, preferably Saccharomyces cerevisiae can be used. In particular, Saccharomyces cerevisiae SEY2102 (MAT α, ura3-5, leu2-3, -112, his4-519; Carlson Botstein, Cell, 28, 145 (1982)) is most preferably used.

Transformed yeast cells can be produced under yeast conditions on medium suitable for the production of sCT-Gly protein, selected according to the host cell and the expression system used, and purified from the yeast to produce sCT-Gly. have.

Production and secretion of sCT-Gly can be confirmed by SDS-polyacrylamide gel electrophoresis and western blotting. That is, sCT-Gly was found in the culture protein fraction of the transformed yeast induced protein expression and sCT-Gly was not found in the bacterial protein fraction. have.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1

Production of sCT-Gly Expression Secretion Vector for Yeast Using ppL

(Step 1)

To prepare polynucleotides encoding sCT-Gly, six oligonucleotides C1, C2, C3, A1, A2 and A3 having the following sequence were synthesized by a DNA synthesizer (Applied Biosystems, USA, Model 391);

C1: 5'-CTA GAT AAG AGA TGT TCT AAC TTG TCT ACT TGT GTT-3 '

C2: 5'-TTG GGT AAG TTG TCT CAA GAA TTG CAC AAG TTG CAA-3 '

C3: 5'-ACT TAC CCA AGA ACT AAC ACT GGT TCT GGT ACT CCA GGT TAA G-3 '

A1: 5'-AGG CAA AAC ACA AGT AGA CAA GTT AGA ACA TCT CTT AT-3 '

A2: 5'-GTA AGT TTG CAA CTT GTG CAA TTC TTG AGA CAA CTT-3 '

A3: 5'-TCG ACT TAA CCT GGA GTA CCA GAA CCA GTG TTA GTT CTT GG-3 '

1 μl (100 pmoles) of the oligonucleotides C1, C2, C3, A1, A2 and A3 are respectively mixed and phosphorylated with a polynucleotide kinase (T4 polynucleotide kinase, Boehringer Mannheim), followed by double chaining by an annealing reaction. Formed. Each double-stranded oligonucleotide was linked with a ligase (T4 DNA ligase, Boehringer Mannheim) followed by agarose gel electrophoresis to separate about 100 base pairs of fragment 1 encoding sCT-Gly from the gel.

(Step 2)

Plasmid YEGα-HIR525 (KCTC 8518P) was digested with BamHI and XbaI to isolate fragment 2 of about 700 base pairs containing the GAL10 promoter and ppL. In addition, YEGα-HIR525 was digested with BamHI and SalI to isolate fragment 3 of about 5,700 base pairs. The sCT-Gly gene of fragment 1 and ppL of fragment 2 were linked to the yeast vector of fragment 3 to construct plasmid pYSCAL1 (see Figure 1).

Plasmid pYSCAL1 contains the GAL10 promoter :: ppL :: sCT-Gly gene :: GAL7 terminator in this order. The ppL starting with the ATG start codon consists of a total of 85 amino acids, and amino acids 84 and 85 at the C-terminus are lysine and arginine, respectively. Following this lysine-arginine sequence is the complete sCT-Gly gene, starting from TGT, the N-terminal Cys codon of sCT-Gly. The protease ysc F (expression of the KEX2 gene), which recognizes the lysine-arginine sequence, is present in the trans Golgi apparatus of yeast, and the synthesized polypeptide of ppL :: sCT-Gly is processed in the Trans Golgi apparatus to release the secreted signal portion. Will go out. Therefore, sCT-Gly secreted into the medium is a complete form of sCT-Gly whose N-terminus begins with Cys.

Example 2

Production of sCT-Gly Secretion Vector for Yeast Using Inulinase INU1 Secretion Signal

(Step 1)

Oligonucleotides C4 and A4 having the following sequences were synthesized with a DNA synthesizer (Applied Biosystems, USA, Model 391):

C4: 5'-T AAT TAC AAG AGA TGT TCT AAC TTG TCT ACT TGT GTT-3 '

A4: 5'-ACC CAA AAC ACA AGT AGA CAA GTT AGA ACA TCT CTT GTA AT-3 '

1 μl (100 pmoles) of each of the oligonucleotides C2, C3, C4, A2, A3 and A4 were mixed and phosphorylated with a polynucleotide kinase, and then double chained by an association reaction. Each double-stranded oligonucleotide was linked with ligase followed by agarose gel electrophoresis to separate fragment 4 of about 100 base pairs encoding sCT-Gly from the gel.

(Step 2)

Six oligonucleotides C5, C6, C7, A5, A6 and A7 having the following sequence were synthesized by a DNA synthesizer (Applied Biosystems, USA, Model 391) to prepare polynucleotides encoding the inulinase INU1 secretion signal. Was:

C5: 5'-AA TTC GGA TCC ATG AAG TTA GCA-3 '

C6: 5'-TAC TCC CTC TTG CTT CCA TTG-3 '

C7: 5'-GCA GGA GTC AGT GCT TGA GTT AT-3 '

A5: 5'-GGA GTA TGC TAA CTT CAT GGA TCC G-3 '

A6: 5'-TCC TGC CAA TGG AAG CAA GAG-3 '

A7: 5'-T AAT AAC TCA AGC ACT GAC-3 '

1 μl (100 pmoles) of the oligonucleotides C5, C6, C7 A5, A6 and A7 were respectively mixed and phosphorylated with a polynucleotide kinase, followed by association to form a double chain. Each double-stranded oligonucleotide was linked with ligase followed by agarose gel electrophoresis to separate fragment 5 of about 60 base pairs encoding the inulinase INU1 secretion signal.

(Step 3)

Plasmid YEGα-HIR525 was digested with EcoRI and SalI to isolate fragment 6 of about 6,300 base pairs containing the GAL10 promoter. The sCT-Gly gene of fragment 4 and the INU1 secretion signal of fragment 5 were linked to the yeast vector of fragment 6 to prepare plasmid pYSCAL2 (see also Figure 2).

Plasmid pYSCAL2 contains the GAL10 promoter :: INU1 secretion signal :: sCT-Gly gene :: GAL7 terminator in this order. The INU1 secretion signal, which begins with the ATG initiation codon, consists of a total of 23 amino acids and amino acids 22 and 23 of the C-terminus are lysine and arginine, respectively. Following this lysine-arginine sequence is the complete sCT-Gly gene, starting from TGT, the N-terminal Cys codon of sCT-Gly. The protease ysc F (expression of the KEX2 gene), which recognizes the lysine-arginine sequence, is present in the trans Golgi apparatus of yeast, and the synthesized INU1 secretion signal :: sCT-Gly polypeptide is processed in the Trans Golgi body to produce a secretion signal portion. This will come off. Therefore, sCT-Gly secreted into the medium is a complete form of sCT-Gly whose N-terminus starts from Cys.

Example 3

Transformation of Saccharomyces cerevisiae SEY2102

According to the method of Ito et al., J. Bacteriol., 153, 163 (1983), Saccharomyces cerevisiae SEY2102 (MATα, ura3) using the plasmids prepared in Examples 1 and 2, respectively -5, leu2-3, -112, his4-519; Carlson Botstein, Cell, 28, 145 (1982)).

50 μl of Saccharomyces cerevisiae SEY2102 culture medium obtained by shaking overnight at 30 ° C. in 10 ml of YPD medium (1% yeast extract, 2% bacterium peptone, 2% glucose) was inoculated again in 10 ml of YPD medium to OD ₆₀₀ = Shaking was incubated at 30 ° C. until 1.0. The medium was centrifuged at 2,000 rpm for 5 minutes to remove the medium, and the cells were washed with 5 ml of TE buffer (10 mM Tris-Cl, pH 8.0, 1 mM EDTA). 0.5 ml of TE buffer and 0.5 ml of 0.2 M lithium chloride (Sigma) were added and suspended well, followed by shaking at 30 ° C. for 1 hour, and 100 µl of the shaking solution was transferred to an Eppendorf tube. 20 μl of the plasmid solution obtained in Examples 1 and 2 were added to each tube, and shaken at 30 ° C. for 30 minutes, 150 μl of a 70% polyethylene glycol 4,000 solution was added, and shaken at 30 ° C. for 1 hour, followed by Thermal shock was applied for 10 minutes. The supernatant was discarded by centrifugation at 15,000 rpm for 1 minute, and the precipitate was washed with 0.8% saline. Suspension suspended in 100 μl of saline was incubated at 30 ° C. for 4 days on minimal selection medium (yield nitrogen substrate 0.67% without amino acid, 2% glucose, Leu 0.003%, His 0.002%) to transform the two strains Romeis cerevisiae SEY2102 strains were obtained, ie Saccharomyces cerevisiae pYSCAL1 / SEY2102 and pYSCAL2 / SEY2102, respectively.

Saccharomyces pYSCAL1 / SEY2102 and pYSCAL2 / SEY2102 were deposited on June 9, 1995 as the accession numbers KCTC 0171BP and KCTC 0172BP, respectively, to the Genetic Bank (KCTC) affiliated to the Biotechnology Research Institute of Korea Institute of Science and Technology.

Comparative Example 1

Saccharomyces cerevisiae SEY2102 (host cells) without plasmids was inoculated in 10 ml of YPD medium, respectively, and incubated at 30 ° C. for 72 hours.

[Examples 4 and 5]

Culture of Transformed Yeast Cells

PYSCAL1 / SEY2102 and pYSCAL2 / SEY2102 obtained in Example 3 were cultured under the same conditions as in Comparative Example 1.

Comparative Example 2

Saccharomyces cerevisiae SEY2102 (host cells) was inoculated in 10 ml of YPDG medium (1% yeast extract, 2% bacterium peptone, 0.4% glucose, 2% galactose) and shaken at 30 ° C. for 72 hours. Incubated.

EXAMPLES 6 AND 7

Culture of Transformed Yeast Cells

Saccharomyces cerevisiae pYSCAL1 / SEY2102 and pYSCAL2 / SEY2102 obtained in Example 3 were cultured under the same conditions as in Comparative Example 2.

Example 8

1 ml of each of the culture solutions obtained in Comparative Examples 1, 2 and 4, 5, 6, and 7 was centrifuged at 4,000 rpm for 5 minutes to recover the culture supernatant and cells. The culture supernatant was lyophilized and dissolved in 10 μl of urea lysis buffer (50 mM Tris-Cl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 5% mercaptoethanol, 0.1% bromophenol blue). The resulting solution by heating at 100 ° C. for 2 minutes was termed the culture protein fraction.

Meanwhile, according to Hoffman Winston's method (Hoffman Winston, Gene, 57, 267 (1987)), the recovered cells were crushed in a buffer solution (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-Cl, pH). 8.0, 1 mM EDTA), suspended in 2 ml of glass beads of 0.4-0.5 mm in diameter, and then 0.5 ml of 5-fold concentrated urea lysis buffer was added thereto and heated at 100 ° C. for 5 minutes. The resulting solution was named cell protein fraction.

Each 10 μl of the culture protein fraction and the cell protein fraction were placed on a 1 mm thick 10% separating gel (pH 8.8, 20 cm, 10 cm) with a 1 mm thick 5% stocking gel (pH 6.8). , 20 cm wide and 3.0 cm long) were loaded onto a urea polyacrylamide gel, followed by electrophoresis at 100-200 volts and 30 mA for 6 hours and stained with Coomassie stain. The result is shown in FIG.

In FIG. 3, column 1 is the standard molecular weight labeling protein (80, 50, 33 and 27 kDa from above) and column 2 is the standard molecular weight labeling protein (16.9, 14.4, 10.6, 8.1, 6.2, 3.4 from above, respectively). And 2.5 kDa), rows 3 and 4 are the cell protein fractions of Comparative Examples 1 and 2, respectively, and columns 5, 6, 7 and 8 are the cell protein fractions of Examples 4, 5, 6 and 7, respectively. Columns 9 and 10 are the culture protein fractions of Comparative Examples 1 and 2, and columns 11, 12, 13 and 14 are the culture protein fractions of Examples 4, 6, 5 and 7, respectively, and row 15 Is purified calcitonin.

No sCT-Gly band was visible in the cell protein fraction and the culture protein fraction of the host cells of Comparative Examples 1 and 2 (columns 3, 4, 9 and 10).

In the case of Saccharomyces cerevisiae pYSCAL1 / SEY2102 cultured in the YPD medium of Example 4, no sCT-Gly band was found in both the cell and culture protein fractions (columns 5 and 11). Saccharomyces cerevisiae pYSCAL1 / SEY2102 of Example 6, which induced protein expression with galactose in YPDG medium, showed no sCT-Gly band in the bacterial protein fraction (column 6), but sCT- in the culture protein fraction. Gly bands were detected near 4 kDa (column 12). This result indicates that when the ppL signal was used, the expressed sCT-Gly was completely secreted into the medium. Saccharomyces cerevisiae pYSCAL2 / SEY2102 showed no sCT-Gly bands in both the cell protein fraction and the culture protein fraction when cultured in YPD medium (Example 5) (columns 7 and 13). ). However, when protein expression was induced by galactose in YPDG medium (Example 7), the sCT-Gly band was not found in the bacterial protein fraction (column 8), but the sCT-Gly band was found in the culture protein fraction (Example 8). Column 14). This result means that the intracellularly expressed sCT-Gly secreted most of the cells.

Example 9

According to Burntle's method (Burnetle, Anal, Biochem., 112, 195 (1981)), electrophoretic transcription apparatus (Hoefer Co., Ltd.) without Coomassie staining of proteins separated by SDS-PAGE as in Example 8 , Tank Transfer unit, model TR-22) was transferred to nitrocellulose membrane at 150 mA, 80 V for 1 hour. Membrane-transcribed proteins were reacted with rabbit anti-calcitonin-polyclonal antibody (Sigma) for 30 minutes diluted 5,000-fold, and the calcitonin band was reacted with Western blotting AP system (secondary antibody: goat's anti-rabbit IgG (H). + L or Fc) -alkali phosphate conjugate, Promega, USA) and the results are shown in FIG.

In FIG. 4, columns 1 to 15 each represent the same sample as that of FIG. 3, with FIG. 4 more clearly showing the results of FIG.

As can be seen in the above example, when the sCT-Gly gene is linked to the inulinase secretion signal of ppL or Kluyveromyces marcianus and expressed in yeast, the produced sCT-Gly can be secreted extracellularly with high efficiency. Can be. In addition, it was confirmed that the inulinase secretion signal of Cloberomyces martianus can secrete heterologous proteins having low molecular weight including sCT-Gly with high efficiency.

Claims (5)

An expression vector comprising a salmon calcitonin precursor gene linked to a polynucleotide encoding an inulinase secretion signal sequence. The plasmid pYSCAL2 of claim 1. A recombinant yeast cell transformed with the vector of claim 1. The recombinant yeast cell of claim 3, which is Saccharomyces cerevisiae pYSCAL2 / SEY2102 (KCTC 0172BP). A method for producing sCT-Gly comprising culturing the recombinant yeast cell of claim 3 and recovering salmon calcitonin precursor (sCT-Gly) from the culture medium.