METHOD FOR MASS-PRODUCING BRAZZEIN BY USING TRANSFORMED YEAST FOR SECRETING AND EXPRESSING BRAZZEIN AT HIGH EFFICIENCY

The present invention refers to brazzein gene recombinant expression vectors including a; and Kluyveromyces lactis derived protein disulfide cross isomerase gene (protein disulfide isomerase, PDI) recombinant expression vectors including a; at the same time to transformed Kluyveromyces lactis relates to (Kluyveromyces lactis).

Yeast alpha-mating (α-mating) signal sequences in kind of signal sequences [Dominic Esposito et al. , Protein Expression and Purification, 40 (2): 424-428,2005, J.J. Clare et al. , Gene. 105 : 205-212,1991], yeast protein by defoaming surface is then synthesized within the correct disulfide cross components that are useful for actively inducing, protein insoluble aggregate, inhibiting formation correct folds guided. The, signaling peptidase (signal peptidase) by signal sequences is shop or a truncated part, thereby to block the movement of the get axially after the remaining signal sequences by (Kex protease) peptidase Kex is removed both, natural protein in the form is to be released into improves. Said examples of signal sequences, child non Saccharomyces cerevisiae alpha-mating signal sequences (Saccharomyces cerevisiae), Kluyveromyces lactis alpha-mating signal sequences, KT signal sequences [Tokunaga et al. , Yeast, 13:699 - - 706,1997), Saccharomyces cerevisiaepre-SUC2 signal sequences (Bergkamp et al. , Curr Genet, 21: 365-370] such as. is known.

The recombinant expression vectors including a gene brazzein said Kluyveromyces lactis (Kluyveromyces lactis) alpha-mating may include signal sequence (α-mating), Kluyveromyces lactis alpha-mating signal sequences to but not exclusively, of seq ID no 1 can take the bio-sequence listing. Said alpha-mating sequences 5 nucleotide sequence which brazzein of the present invention ' in translation to protein on top so that the same frame are connected.

Said brazzein gene brazzein the wild-type gene (seq ID no:2) or brazzein variants can be gene. In the present invention, brazzein week type brazzein a gene, buta input, gene of both variants may include both.

Furthermore, to said recombinant expression vectors may further comprise the gene.

In the present invention "recombinant expression vectors" email widow, a web page or RNA or expression desired protein in host cells (expression) vector that can as for transferring (transcription), gene to be expressed insert are operatively connected to and an integral adjusting element circulation promoted. the surroundings including gene.

Said 'operating gene' a particular email widow, a web page or in host cells nucleic acid sequences operably linked to a regulating the expression of DNA sequence meaning, 'is operatively connected to (operably linked)' the a single nucleic acid fragments having other nucleic acid fragments and is coupled of function or expression other nucleic acid fragments to which are influenced by circulation promoted.. Furthermore, for regulating transcription any operator sequence, suitable binding portions followings mRNA sequences encoding and transfer and decryption of further sequences control termination may include. Operating said always held within time zones at all include gene induce expression of a target gene promoter (constitutive promoter) or just a particular position, the aim when the leakage amount or consumption induce expression of gene promoter (inducible promoter) may be used in a, examples include yeast LAC promoter, gal promoter (Rosaura Rodicio et al. , Microbiology 152 (2006), 2635-2649), KlADH4 promoter (Michele Saliola et al. , Appl Environ Microbiol. 1999 January; 65 (1): 53-60), maltose/ malta Oh sacrificeit ladles, the maul Oh sacrifice bi-direction null promoter (American patent number 6,596,513 call), PGK1 promoter (V. Melvydas et al. , BIOLOGIJA, 4:1-4,2006) such as. American addition promoter disclosure patent number 227,326 call or the like, use can be made of, as a function of. Preferably said LAC4 promoters, use can be made of, promoter.

Said brazzein for activation, wherein the recombinant expression vector pKLAC2-brazzein, brazzein pKLAC2 - :: LAC4, the logical source including the device, is at. Furthermore, said derived from Kluyveromyces lactis including a PDI gene can be pKLAC2-PDI, wherein the recombinant expression vector.

Said yeast (Kluyveromyces lactis) the method the transformation of an ordinary recombinant expression vectors transformed or according to, the transformed host nucleic acids as well as any cells is also includes method, as publicly known in the prior art according to host cells, a standard the preferred techniques can be. This method the electric impact gene delivery method (electroporation), acetic acid lithium (LiCH₃ COO) method, of lithium chloride (LiCl) method, (PEG-mediated fusion) fusion-mediated PEG, carrying DNA fusion include a (Carrier-DNA mediated fusion) is not limited to but.

While, Kluyveromyces lactis (Kluyveromyces lactis) the present invention refers to a host on for expression of brazzein, brazzein gene recombinant expression vectors including a; and Kluyveromyces lactis derived protein disulfide cross isomerase gene (protein disulfide isomerase, PDI) recombinant expression vectors including a; at the same time to transformed Kluyveromyces lactis (Kluyveromyces lactis) dispersed by a blender before being inoculated mass production of brazzein including includes method.

Such expression of brazzein in lactose invention herein (lactose), galactose (galactose), glow course (glucose), starch (starch) conventional processing modules, such as inductive an inducible promoter of promoting the expression of effected by compounds. Expression alpha-mating (α-Mating) signal sequence including brazzein the signal sequences by is by antifoaming yeast, yeast signaling peptidase (signal peptidase) and a Kex peptidase (Kex peptidase) by signal sequences combined brazzein is removed.. Therefore, expression brazzein of the present invention recombinant expression vectors a yeast transformed to include polynucleotides encoding the brazzein appropriate is expressed medium and can be cultured under conditions, the nitrogen source of yeast extract 0.5 to 5%, 0.5 to 5% peptone alone to and used as a light-weight, carbon source and inducing the expression zero galactose 1 to 4%, glucose 1 to 4%, 1 to 4% and starch lactose selected from the group consisting of 1 to 4%, use can be made of, one or more. In particular, 0.5 to 5% yeast extract, peptone 0.5 to 5% a 4% to 1 galactose and involves culturing the manner in a medium free of including more preferably, the.

Original yeast chromosome ERO gene and PDI gene present in the. PDI to overexpressing enzyme the two in the present invention in the case of further PDI gene-expression vector when the transformed overexpressing by inserting a and, in the case of original by adding DTT ERO in a chromosome transcription of overexpressing ERO is mounted between a bottom of the first amount.

PDI is ER article incorporating disulfide brazzein in when, . cooperate such ERO (Endoplasmic reticulum oxidoreductin). The PDI ERO by oxidizing brazzein of correct folding enhance to has air inside and floats on extracorporeal are angularly (folding). PDI thus influence extent folding overexpressing only the viewing of ERO not is validated the expressed amount of entrained with revelation preferably comparing the.

Therefore, transfer moves the probe units in one of said in culture, mRNA expression of ERO ERO DTT the culture medium to which to the reason increasing adding a water. is more preferred to set a.

I.e., by that overexpress HSP ERO and a the present invention refers to PDI which allow for the expression of said brazzein efficiency may include a method.

Hereinafter, the present invention embodiment to the present invention and a second operating mode corresponding to but described S406, embodiment presented a range of the present invention and/or at least two different limited to not.

Reference e.g.: expression vectors and strain

1) yeast extracorporealexhaust orgin expression vectors for use

Yeast protein extracorporeal exhaust orgin for use (Kluyveromyces lactis) Kluyveromyces lactis protein extracorporeal discharge signal sequences of signal sequences (α-mating factor) (signal sequence) in alpha-mating factor Kex cleavage site from a vector including the washing liquid pKLAC2 the ground terminal of the stored in New England Biolabs (England). Expression vectors in transcription regulation are made and another new LAC4-PBI by the promoter, gene amdS oh theta E idoh theta it gets torn, Oh sacrifice which are expressed by (acetamidase) to (acetamide) transformant followed by extracting and including. of selecting (van Ooyen et al. , 2005, Read et al. , 2007)

2) yeast strain for activation brazzein

Brazzein gene inserted to obtain brazzein recombinant yeast strains using Kluyveomyces GG799 is lactis, for purchasing a from New England Biolabs (England).

3) strain e. coli a format for constructing a recombinant plasmid

Recombinant brazzein E to produce the of the vectors is inserted. Constitution: a strip in coli DH5 α, (Uppsala, Sweden) and a Pharmacia Biotech (Madison, WI, USA) from Promega Corporation for purchasing a.

[In the embodiment]

I. Experiment method

1. In yeast manufacturing a recombinant plasmid for expression of brazzein

Recombinant brazzein for for expression of yeast be used large industrial bread and has a Kluyveromyces lactisfor producing brazzein a microorganism strain at night. The GRAS (Generally recognized as safe) is defined by future additive is comprised of brazzein production/g. is advantageous in that can be used.

For outdoor using comparison expression amount reached to a predetermined the present Jane multiple brazzein wild-type in heat insulation is performed by using the drift variant developed about brazzein wild-type to 2,000 times, a strong back only 285 table sugar sweetened, is transfer debited E41K 4/K5R/H31R/E36D indicative. K same. Lactis Pentadiplandra to express in brazzeanaBaillon DNA brazzein of a plurality sequences are amino acid based on sequence within such a range that causes no K. Suitable lactis designed considering codon usage and the ground terminal of DNA by combining together.

Recombinant out cells to the secreted brazzein, present in vector pKLAC2 alpha-mating (α-mating) Kex cleavage site into a plurality of unit cell substrates signal sequences and signal sequences corresponding to an entire surface of the semiconductor substrate after gene sequence Kex cleavage site for Jane for outdoor combining gene sequence by inserting a cell brazzein correctly being discharged can be plasmid the solenoid.

1) for manufacturing a recombinant plasmid or overexpression of the tnf-brazzein

K LAC4 sequence of the promoter. Lactis LAC4 promoter in the amplifying PCR obtained, then [(England) yarn New England Biolab] pKLAC2 and at the both end parts a restriction endonuclease after treating the was to plasmid by ligation. SnaB restriction endonuclease promoters LAC4 cutting into Xho I I and inserting a vector pKLAC2 pKLAC2 :: LAC4 Construction the a [also 6 reference]. The storage same pKLAC2-brazzein :: LAC4 the ground terminal of to construction. Gene brazzein on the Construction, in order to inject a cutting into Xho I and a BamH I pKLAC2 and a pKLAC2 :: the inserted into LAC4.

2) structure-forming its defective or correct operation brazzein for manufacturing a recombinant plasmid

Protein secretion of yeast within defoaming in expression systems is formed structure of proteins in. The proteins involved the which ERO (Endoplasmic reticulum oxidtion) and a PDI (Protein disulfide isomerase), ERO and a PDI protein phone number by controlling expression dose can be increases DANPLA in vitro (Lodi et al. , 2005). The PDI gene expressing vector pKLAC2 inserting a plasmid yeast expression in the compression strain to the ground terminal of transformation. K a PDI gene. Lactis a PDI gene in PCR amplifying the obtained. Hind in PDI gene IIIsites on a I cut into 2 pieces after first Sal Hind the gel extraction IIIand aBamH I[also 6 reference] cutting, respectively, finally vectors and PDI (I Sal & Hind III), PDI (I ISal BamH &) fragment by ligation of 3 simultaneously constructed with a plasmid pKLAC2-PDI [also reference 3]

2. Brazzein yeast expression strain creating

Brazzein yeast expression systems were used in order to improve the pKLAC2-brazzein, brazzein pKLAC2 - :: LAC4, each plasmid pKLAC2-PDI ratio, brazzein for comparison expression amount with strain, brazzein and LAC4 inserted into the strain, brazzein and PDI inserted into the strain, brazzein pKLAC2 - :: pKLAC2-PDI plasmid LAC4 and a copper film is transformed simultaneously LAC4 and brazzein,PDI inserted into the strain, total 4 of transformed brazzein yeast expression strain constructed with a.

1) to recombinant DNAinsertedgDNA in strain yeast expression

Homologue region insertion of gene recombinant to yeast strain principle recombinant in a method for manufacturing the same, the expression vectors to this end 5 of the promoter LAC4 'sequence and 3' sequences present in the on at the both end parts II restriction endonuclease cut into Sac was carried out to transformed (Van Ooyen et al. , 2005) [also 4 reference].

PKLAC2-brazzein, brazzein pKLAC2 - :: LAC4, II a gene recombinant pKLAC2-PDI Sac cutting into linearization highly efficient using transformed Kluyveromyces lock angles of the teeth of the inserted gDNA GG799 of cells [also 5 reference].

Transformed by cylinder to have the following method.

50 ml of YPD medium preheating Image signals input from the outside the culturing multi function cap placed in two 2.5×10 placing the culture and a pre-programmed GG799 cells to be inoculated with a proper amount of cells by culturing temporal extent 4 and minimum 2×10⁷ the seal one or more. Culture to culture vessel with a great overall height to the centrifugation 3000×g GG799 cells 5 minutes house germ section.

1 ml of supernatant, pouring out TE and homogenize the buffer, and to shorten a manufacturing time and to micro tube same 5000×g to 3 minutes then centrifugation, the supernatant so that 1 ml volume and the final coverage for the transformation mixture with the has been again. Such comprises repetitively a 3.

5000×g to 3 minutes then centrifugation, each transformed l micro 100, a mixed solution prepared for the transformation tube into at and mixing the. 30 minutes of cells has been again, after cultured in 30 °C, 42 °C the invention also provides for a heat bath hot bath of 15 minutes and then marked on the specific Ice to after 1 minutes, the tube a 3000rpm, 1 minutes for the transformation the centrifugation to remove the mixed solution for inserting and removing buffer TE of 1 ml was used to homogeneous microorganisms house. Homogenizing acetamides of 5 mm solution containing solid YCB medium applied over the. 96 hours before upper side of the tire is contacted the recombinant gene has been successfully inserted acetaldehyde oh it gets torn, Oh sacrifice the first colony shoot forth. Colony is the ground terminal of the expression of brazzein only. All experimental process the fire has the second type of a home as sterile conditions was that they travel within a Clean bench below.

2) brazzeinexpression amountsorting strain yeast expression a for comparison

Recombinant gene when introduced using a homologous integration, multiple introduced (multi-integration). (co-integration) introduced simultaneously with cutting or drilling the bone. To this end brazzein compares DANPLA and extracorporeal expression dose in order that is inserted into a brazzein gene expression strain the same copy number of yeast expression strains should go a decided by considering experiment. Brazzein gene copy number the same expression strain transformed for finding an embodiment by extracting the gDNA of between-PCR was carried out to.

3. Compared expression amount brazzein recombinant

Improved yeast expression system are compared to cell growth rate by enhancing expression of brazzein high yields of brazzein. established expression system. Wild-type LAC4 promoter which has been that overexpress HSP brazzein recovery PDI expression strain its defective or correct operation brazzein by introducing expression strains leading to formation structure after comparing the signal-to-each, strain expression spray pyrolysis adopting both cell growth rate by enhancing expression of in addition and compared.

1) expression strain overexpressing brazzein

PB - LAC4 Iexpression derived is developed in the sacrificial material, brazzein promoter strain wild-type LAC4 derived is developed in the sacrificial material, brazzein promoter expression strain in a culture for expression of brazzein identically was carried out to in. 30 °C 200 RPM in YPD manner in a medium free of O by amount pre-oriented time 16. D.₆₀₀ = 1.2 both misfortunes pre-oriented reaches the final medium YPGal (pH 5.0) before culture volume of 2% so that corresponding advertisement based on the shown list using glucose, brazzein by culturing time 96 after innoculation expression of induced. 7,000 RPM after culture, 20 ingredient, 4 °C conditions in the after the separation of the cells from their top layer discarded SDS-PAGE expression dose brazzein and compared through.

2) brazzeinstructure formationexpression strain

PDI of the its defective or correct operation brazzein in expression leading to formation structure of a metal oxide fiber brazzein a culture for expression of in was carried out to similarly determines the. 30 °C 200 RPM in YPD manner in a medium free of O by amount pre-oriented time 16. D.₆₀₀ = 1.2 both misfortunes pre-oriented reaches the final medium YPGal (pH 5.0) before culture volume of 2% so that corresponding advertisement based on the shown list using glucose, brazzein by culturing time 96 after innoculation expression of induced. 7,000 RPM after culture, 20 ingredient, 4 °C conditions in the supernatant after the separation of the SDS-PAGE expression dose brazzein in and compared through. Won in the a cells the core it separated recombinant brazzein amount of remaining and compared.

3) strain expression and structure forming overexpressing brazzein

Wild-type LAC4 a method for manufacturing the same promoter expression of a metal oxide fiber brazzein PDI-expressing is a culture for expression of in was carried out to similarly determines the. 30 °C 200 RPM in YPD manner in a medium free of O by amount pre-oriented time 16. D.₆₀₀ = 1.2 both misfortunes pre-oriented reaches the final medium YPGal (pH 5.0) before culture volume of 2% so that corresponding advertisement based on the shown list using glucose, brazzein by culturing time 96 after innoculation expression of induced. 7,000 RPM after culture, 20 ingredient, 4 °C conditions in the after the separation of the cells from their top layer discarded SDS-PAGE expression dose brazzein and compared through.

4. Recombinant brazzein for purifying

Expressed brazzein and acquires the result as the yield of the correct value, for use in experimental process after a clean brazzein to obtain purification procedure was carried out to.

1) purification of recombinant brazzein

Brazzein for expression culture derived GG799/brazzein pKLAC2-7,000 rpm, 4 °C, upper and having bacterial cells centrifugal separator 20 minutes separated the variety of nutritious and physiological a fungus, brazzein a target protein being discharged and discarded cells since separating the supernatant was taken. In an upper layer of a fire protective separating pH 4.0 acetic acid, which liquid behind to control pH value carboxymethyl cellulose cation exchange resin column purified by the cm capacity. 100 ml column an entire surface of the semiconductor substrate 50 mm sodium acetate buffer solution (pH 4.0) uniform and then the ground terminal of tablet. A supernatant to control pH value made at speeds in the order of 1 ml/min out by absorption of said protein was passed through a column. After this opposite direction of the spring force direction is adsorption to remove washing the 50 mm sodium acetate buffer (pH 4.0) solution of. 100 mm NaCl 50 mm sodium acetate buffer solution (pH 4.0) with using separated from one end of the corresponding advertisement based on the shown list removing proteins in O of the solution. D.₂₈₀ are measured by feeding a brazzein other protein removed has been confirmed. For the release of an brazzein with 400 mm NaCl 50 mm sodium acetate buffer solution (pH 4.0) to elute the made at speeds in the order of 1 ml/min for the 1 ml interval fraction is obtained. O of calculated for each. D.₂₈₀ and SDS-PAGE eluting brazzein through the freeze-drying only fraction was collected.

2) for outdoorpurified for Jane identification and quantitation of

Purified proteins of pure HPLC and a SDS-PAGE degree corresponding advertisement based on the shown list the path difference Scope et althe recording disc surfaces. (1974) of the ground terminal of method. Brazzein the tryptophan (tryptophane) since the protein including a non-using a protein level 280 nm and a 205 nm UV/VIS Spectrophotometer O in. D. The bill to data of the measured. Brazzein of light absorption coefficient (ε₂₀₅) absorbance of the solution at 280 nm and a 205 nm measuring the controller determines that the user corresponding advertisement based on the shown list as calculated according to 1 expressions based on the same, brazzein for determining concentration of the.

[Expressions 1]

ε₂₀₅ 1.0 mg/mL = 27.0+120 (A₂₈₀/A₂₀₅)

II. Binder and

1. In yeast manufacturing a recombinant plasmid for expression of brazzein

Yeast recombination brazzein gene, in order to inject a gene brazzein to pKLAC2 pKLAC2 inserted into the n bit parallel data inputted manufacturing plasmid brazzein, LAC4 promoter restored in overexpression for pKLAC2-brazzein :: for the expression of protein PDI and plasmid LAC4 pKLAC2-PDI of 3 total printed onto the product, thus resulting plasmid constructed with a recombinant plasmid.

1) for manufacturing a recombinant plasmid or overexpression of the tnf-brazzein

A expression vectors compatibilized modified pKLAC2 PB - LAC4 LAC4 wild-type promoter I. retrieve promoter. K LAC4 sequence of the promoter. Lactis LAC4 promoter in PCR amplifying pKLAC2 and at the both end parts after limiting by ligation after treating the enzyme pKLAC2-brazzein :: constructed with a plasmid LAC4 [also reference 3].

2) structure-forming its defective or correct operation brazzein for manufacturing a recombinant plasmid

Yeast protein expression within defoaming in China (folding) structure formation of proteins in occurs. ERO and a PDI protein phone number by controlling expression dose can be increases DANPLA in vitro. The PDI gene for plasmide and inserted into an expression vector a printed onto the product, thus resulting the ground terminal of to to transform into yeast strain. PDI GG799 a PDI gene present in protein and the uses GG799 to a PDI gene in PCR amplifying the obtained. At the both end parts and limiting pKLAC2 by ligation after treating the enzyme constructed with a plasmid pKLAC2-PDI [also reference 3].

2. Brazzein yeast expression strain creating

Brazzein yeast expression systems were used in order to improve the pKLAC2-brazzein, brazzein pKLAC2 - :: LAC4, each plasmid pKLAC2-PDI ratio, brazzein for comparison expression amount with strain, brazzein and LAC4 inserted into the strain, brazzein and PDI inserted into the strain, brazzein and LAC4,PDI inserted into the strain, total 4 of transformed brazzein yeast expression strain constructed with a.

Introduced multiple 1) gene (multi-integrated)yeast expression strain sorting

K. Introduced multi-gene insertion system comprises of lactis (multi-copy integration) are connected to the switching circuit, multiple gene is inserted into the guide groove (single-copy integration) when single introduced than is the case of expressing recombinant protein amount catalyst in the reactor results in enhanced. [also 14 reference]. The resulting strain by more by controlling the expression amount to be over-expression, gene copy number and the highest copy number expression strains having the ground terminal of study acid by [also 14 reference].

Introduced simultaneous 2) gene (integrated - co)yeast expression strain sorting

K. Lactis induce expression of PDI in recombinant brazzein of structure-forming fourth MOS are angularly extracorporeal DANPLA increase the energy weight value. GG799 recombination brazzein gene in strain and of simultaneously embedding is PDI gene expression by extracting the gDNA be selecting strains PCR gene as for outdoor to be inserted through the [reference 7 also] and a corresponding advertisement based on the shown list identifying PDI gene [also 8 reference], two gene is simultaneously yeast expression strains acid by the ground terminal of study.

3. Compared expression amount brazzein recombinant

Brazzein yeast revelation orgin expression amount for comparison brazzein by expressing a corresponding advertisement based on the shown list identifying amount transcription of mRNA, recombinant brazzein expression dose to improve a characteristic purification if there is the specific final brazzein yield is obtained.

1) expression strain overexpressing brazzein

Recombinant brazzein overexpression to derive existing strains using promoter LAC4 LAC4-PBIpromoter strains as such a condition using recombinant brazzein allow for the expression of said compared density after cell growth rate by enhancing expression of the (densitometry) and compared. Of the result of comparison between the amount of transcription of mRNA, using existing LAC4-PBIpromoter compared to strains LAC4 brazzein of a metal oxide fiber using promoter whose expression level is about 1.19 the-fold increase in the [also 10 reference], finally recombinant brazzein yield of about 1.19 in addition a binary [also 10 reference].

2) brazzeinstructure formationexpression strain

Recombinant brazzein its defective or correct operation has air inside and floats on extracorporeal by forming an auxiliary to increase the and brazzein the PDI gene expression has been, additionally ERO, that induces the expression of adding DTT to cell growth rate by enhancing expression of revelation orgin and with ubiquitin is stabled and compared. Transcription of mRNA of the result of comparison between the amount of the recombinant brazzein expression dose didn't which is indicative for a difference. However finally yield of brazzein is the recombinant PDI gene compared to existing strains for the expression of the novel compared to strain expression of recombinant brazzein yields of about 1.33-fold increase in the corresponding advertisement based on the shown list [also 11 reference], by adding DTT the ERO expression strains, that induces the expression of existing expression SP whose expression level is back have been increased about 1.46 [also reference 11].

ERO and a PDI dusts brazzein recombinant by increased to make sure that SDS-PAGE protein in cells in cells through recombinant brazzein toner or ink quantity and compared. As a result, in the amount of brazzein remaining PDI % when adding ERO and a narrowed down to 76% [also reference 13].

3) strain expression and structure forming overexpressing brazzein

Recombinant brazzein of increasing the yield of supports and rotates the expression principle to increase the simultaneously DANPLA extracorporeal and for a method for manufacturing the same promoter LAC4 strains constructed with a PDI gene is inserted. After each expression strain recombined at brazzein allow for the expression of said cell growth rate by enhancing expression of exemplified by expression of existing after and compared. As a result, by using the promoter brazzein LAC4 or overexpression of the tnf-inducing strains allow for the expression of PDI in the case of basic of the result of comparison between the amount of transcription of mRNA expression strains whose expression level is back 1.40 than improve, by adding DTT the ERO existing strains, that induces the expression of mRNA expression SP increased amount transfer 1.60 times using the result.

III. Contemplated

Bread and has a weather radar are study recombinant protein in e. coli expressing brazzein reaches but low to 3-4 mg/L, additional interaction in in vitroafter purification on structure-forming (refolding) process is desired in which the container has, when in vitroto form a on the wrong input of an structure recombinant brazzein is made and a (misfolded) since the. have the drawback in that the efficiency is low. As and food study brazzein the applicability to enlarge the whose expression level is which a plurality of food available yeast bread and has a K. Lactis using maximum used to construct a recombinant brazzein expression system 108 mg/L at a yield of brazzein is obtained (Jo et al. , 2013).

The present inventor are human taste recognition mechanism also study receptor sweetened, for of identifying process is performed to make the metal in which, human study interaction brazzein receptors and sweetened, for X-ray crystal structural analysis using such high order optical analysis.. Higher order structural analysis experiment in need of a per-sample high yields of further below to be constructing expression system the n bit parallel data inputted schedules a the present invention, additionally brazzein the food of when is utilized as suitably applied expression systems for the production set up to study.

Most protein sweetening brazzein but have a smaller size of 4 in heat and including stainless steel or polymer disulfide cross change in. very stable pH (Ming and Hellekant, 1994). A feature of the brazzein such and increase the accuracy and probability of applicable to food to recombinant protein while for the expression of an erroneous structure. improve the probability that a (Demain and Vaishnav, 2009). Eukaryotic against proteins that are expressed at of structure formation (folding) parcel body (Endoplasmic reticulum) which, various proteins involved are involved that are expressed in protein structure are correct is reacts with hot water with swelling (Gruber et al. , 2006). Expression used in the present invention, by using the sequences signal is wiped across the extracorporeal discharge inducing protein since extracorporeal not body and the cover are correct is not taken off, the duration of the process of structure on in vivo since the spectrum is obtained by adding all the. (Wittrup, 1995). In particular proteins include involved in disulfide linkage forming reagents. ERO (Endoplasmic reticulum oxidoreductin) and a PDI (Protein disulfidebond isomerase).

Existing the compatibilized include yeast expression vector carrying pKLAC2 LAC4-PBI vector as induced promoter. Wild-type yeast strain promoter LAC4 before introduced to, e. coli recombinant expression vectors in part to disturb a amplification of? Gene for ease of operation the door I PB - LAC4 was made to the promoter. Potent in yeast strain however from the wild-type is promoters expressed returns to promoter LAC4 can be 2001 be shaped various type.

Existing recombinant brazzein of yeast expression aperture structure of system expressed applied to both same corresponding advertisement based on the shown list progress, 1.73 contrast expression systems yeast existing finally came to an for increasing the yield of times. PKLAC2 PB - LAC4 in expression vector ILAC4 wild-type promoter if retrieve promoter transcription of mRNA amount passes through throughput is increased of confirming constitution: a, ERO and a PDI of extracorporeal in strain expression through transgenic over expression of recombinant protein to the amount by the which is issued to increased in amount of brazzein recombinant remaining may be identified, for instance, was compared. Finally the present invention exemplified by expression of 1.73 times compared to using the result whose expression level is increased.

The present inventor are currently being wild-type brazzein various brazzein out is created from the streams and and can be guided into the. Useless yeast expression variant is brazzein all expression and purifying process must undergo correct structures recombinant brazzein which the film is to be formed, subsequent torch while being inputted to the structure same ID study. the present invention includes an inertia measuring module measuring yeast expression an upgraded through respectively, applied to all variants structure fourth MOS brazzein method of Image transmission, and Image may be is attached to the.

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Gruber, C. W. , Cemazar, m. , Heras, B. , Martin, J. L. , And Craik, D. J. (2006) Protein disulfide isomerase: the structure of oxidative folding. 31 inTrends BiochemicalSciences, 455-464

Jo, H. -J. , Noh, J.-S. , And Kong, K. -H. (2013) Efficient secretory expression of the sweet-tasting protein brazzein in the yeast Kluyveromyces lactis.Protein ExpressionPurification and 90, 84-89

Lodi, t. , Neglia, B. , And Donnini, C. Kluyveromyces lactisApplied Environmental Microbiology(2005) Secretion of human serum albumin by overexpressing KlPDI1 and KlERO1. and 71, 4359-4363

Ming, D. , And Hellekant, g. (1994) Brazzein, a new high-potency thermostable sweet protein from Pentadiplandra brazzeanaB.355 Febs Letters, 106-108

Read, J. D. , Colussi, P. A. , Ganatra, m. B. , And Taron, C. H. Kluyveromyces lactis(2007) Acetamide selection of cells transformed with an integrative vector leads to high-frequency formation of multicopy strains. Applied Environmental Microbiologyand 73, 5088-5096

Scopes, R. K. (1974). Measurement of protein by spectrophotometry at 205 nm. Anal. Biochem. 59,277-282.

Van Ooyen, A. J. J. , Dekker, P. , Huang, m. , Olsthoorn, m. M. A. , Jacobs, D. I. , Colussi, P. A. , And Taron, C. H. (2006) Heterologous protein production in the yeast Kluyveromyces lactis.Fems YeastResearch6, 381-392

Wittrup, K. D. (1995) Disulfide bond formation and eukaryotic secretory productivity. OpinioninbiotechnologyCurrent 6, 203-208