CN101148669B - Multi-copy beefy meaty peptide expression gene and carrier, and recombination Pichia pastoris construction - Google Patents

Abstract

The present invention provides a multicopy beefy meaty peptide (BMP) expression gene, vector and construction process of recombinant Pichia pastoris, and relates to a method for obtaining the BMP in the Pichia pastoris by using molecular biotechnology, which overcomes the big difficulty, high cost and low yield and so on in the extraction or chemical synthesis of BMP in the prior art. Based on the codon partiality expressed and translated with Pichia pastoris, a 4-copy-containing BMP expressing gene sequence and essential limiting cleavage sites, 6His and a termination codon are designed. Through in vitro operation, the copy number of the BMP is increased to 16, and the BMP is inserted to expression vector pPIC9, after linearization of pPIC9, the 16-copy-containing BMP expressing gene as a target segment is recombined onto the Pichia pastoris genome by means of electric transformation. BMP is obtained in high yield through fermentation and methanol induction of Pichia pastoris, and is separated and purified by 6His affiliated Ni column.

Description

The structure of multi-copy beefy meaty peptide expression gene and carrier and recombinant yeast pichia pastoris

Technical field

The present invention relates to a kind of Protocols in Molecular Biology and make up recombinant DNA in the pichia spp fermented liquid, to obtain beef-flavouring peptide (Beefy Meaty Peptide, method BMP).

Background technology

Beef-flavouring is strengthened peptide (BMP) and from the papain hydrolysis thing of beef, is separated to obtain, and its primary structure is: Lys-Gly-Asp-Glu-Glu-Ser-Leu-Ala.BMP and salt, MSG have the collaborative preferably effect that is flavor, and have good thermostability, are suitable for the heat treatment requirements in the foodstuffs industry production.Therefore, BMP is expected to replace MSG to become flavour enhanced dose of a new generation, has vast market prospect.

At present, the flavor peptides major part forms through proteinic enzymolysis, but can produce a large amount of by products; In addition, when the protein enzymic hydrolysis, can form bitter peptides, this has seriously limited the utilization of protein hydrolystate.Chemical synthesis also is the effective ways of synthesized micromolecule peptide, but itself has drawback: 1. racemization and side reaction; 2. need protect the gene in the peptide side chain, especially during solid phase synthesis; 3. need excessive link reagent and acyl carrier; 4. the toxic residue that links reagent influences goods and environment.

Along with the development of Protocols in Molecular Biology, utilize recombinant DNA technology to transform mikrobe and make its synthesis secretion desirable proteins become a kind of mode of production of economy.

The pichia pastoris phaff heterologous gene expression system is a kind of outstanding eukaryotic expression system of development in recent years, compares with the yeast saccharomyces cerevisiae expression system with traditional intestinal bacteria, and its many advantage that have makes it researching value and using value constantly embodies.Its maximum characteristics are that to keep metabolism low, can carry out high density fermentation.Since the seventies in 20th century; The fermentation process in high density that pichia pastoris phaff is produced bacterium as single cell protein is quite ripe; Dried cell weight can be up to 160g/L after fermentation; And there are some researches show that the thalline content of fermentative prodn and the expression level of foreign protein have certain proportional relation.Pichia spp is closely similar to the translation post-treatment (formation of glycosylation, disulfide linkage, folding) and the higher eucaryote of foreign protein; Make foreign protein can form correct space conformation; Not only make expressed proteins have good biological activity, and very favourable to proteic secretion.The integration of foreign gene is highly stable to be another characteristics of this expression system.The growth of pichia spp and can in the synthetic medium of cheapness, carry out to the expression of foreign protein, the secretory protein of pichia spp self seldom has data to show in addition, and it is proteic more than 30% that the excretory foreign protein can account for total secretion.Therefore, the purifying that very helps secretory protein.

Summary of the invention

One of the object of the invention is to overcome the deficiency of above-mentioned prior art, and a kind of recombinant plasmid vector and construction process that contains multi-copy beefy meaty peptide expression gene is provided.

Two of the object of the invention provides a kind of engineering strain that is made up of above-mentioned expression vector importing pichia spp.

Three of the object of the invention provides the application of above-mentioned engineering strain in the beef-flavouring peptide of the many tandem copies of preparation.

In order to realize the object of the invention; At first design the expressing gene fragment that contains 4 copy BMP according to the codon-bias of pichia spp accurate translation; This fragment synthetic two strands is inserted on the common carrier, is built into through external connection to contain multiple copied BMP expressing gene, is integrated into suitable expression (pPIC9) through respective limits property restriction enzyme site; Import pichia spp again and constitute recombinant DNA, make it express the BMP of many tandem copies.

The acquisition of the object of the invention product B MP expressing gene is that the codon-bias according to the pichia spp accurate translation designs voluntarily and is responsible for synthetic by the precious biotech firm in Dalian.Wherein, and 4 copy beef-flavouring peptides (Beefy Meaty Peptide, expressing gene fragment BMP) is shown in sequence table sequence 1; The expressing gene fragment of 16 copy beef-flavouring peptides is shown in sequence table sequence 2.

Wherein, the expressing gene method of design of 4 copy BMP is following:

The first, design the expressing gene fragment that contains 4 copy BMP according to the codon-bias of pichia spp accurate translation, the BMP expressing gene of 4 copies is from beginning to end, and each is expressed between the fragment and does not contain other sequences;

The second, the upstream position at last step 4 copy BMP expressing genes adds Xho I restriction enzyme site, and downstream position adds Sal I restriction enzyme site;

Three, the upstream position at the Xho I restriction enzyme site of the gene fragment in last step adds EcoR I restriction enzyme site; The downstream position of Sal I restriction enzyme site adds 6His encoding sox, Not I restriction enzyme site and TAA terminator codon in proper order, finally obtains shown in sequence table sequence 1, containing the expressing gene fragment of 4 copy BMP.

The expressing gene construction process of 16 copy BMP is:

The first, with Xho I or Sal I the carrier enzyme of 4 copy BMP expressing genes of above synthetic structure is cut the back linearizing, produce sticky end;

The second, comprise the small segment that 4 copy BMP, two ends contain Xho I and Sal I sticky end with reclaiming behind Xho I and Sal I the carrier double digestion with 4 copy BMP expressing genes of above synthetic structure;

Three, the first step and second gene fragment that obtain of step are connected through the T4 dna ligase, connect that to obtain to contain 8 copies, 12 copies, 16 copies or copy number be 4 arbitrary integer BMP expressing gene carrier doubly;

Four, because ligation does not have forward and reverse selection; Need to contain the transformant that forward inserts through choosing artificially with Xho I and Sal I Restriction Enzyme blanking method; And through the definite quantity that connects small segment in its agarose gel electrophoresis position; The final selection of the present invention contains the transformant (also can select other copy numbers) of the BMP of 16 copies, promptly obtains the BMP expression vector of 16 copies.

The expression vector commonly used that the pPIC9 that the present invention adopts expresses for the pichia spp secreted protein has the AOX1 promotor.In the expression vector that makes up, the polyclone restriction enzyme site of pPIC9 is positioned at after the alpha factor secretion signal gene; Adopt molecular cloning method that the BMP expressing gene is inserted between EcoR I and the Not I, concrete construction process is following:

The first, above-mentioned 16 copy BMP expressing genes, one fragment that builds is passed through EcoR I and Not I double digestion, reclaim the small segment that contains the BMP expressing gene;

The expression vector pPIC9 that the second, will be used for pichia spp is with EcoR I and Not I double digestion;

Three, the recovery small segment with the above-mentioned the first step and the second step gained is connected through the T4 dna ligase with the big fragment of carrier, makes that to contain copy number be that 16 BMP expressing gene fragment is inserted on the pPIC9 and obtains.

The construction process of the object of the invention two described engineering strains is following:

The first, be that 16 BMP expression vector pPIC9 is with the linearizing of Sac I digestion with restriction enzyme with the above-mentioned copy number that builds;

The second, the method that transforms through electric shock will go up linearizing expression vector pPIC9 of step and import pichia spp, recombinate;

Three, identify through Histidine auxotrophy plate screening and PCR method, select positive transformant, promptly obtain the recombinant yeast pichia pastoris engineering strain.

Engineering strain called after pichia pastoris provided by the invention (Pichia pastoris) GS115-16BMP bacterial strain; Be preserved on July 26th, 2007 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center " (Datun Road, Chaoyang District, Beijing City 100101; Institute of Microorganism, Academia Sinica), its preserving number is CGMCC No.2136.

The application of the object of the invention three described engineering strains is following:

This recombinant yeast pichia pastoris engineering strain is through methanol induction, produces that to contain copy number be 16 BMP polypeptide, and has a 6His mark, and this mark is used for qualitative, the quantitative analysis that Western Blot carries out product, or affine with the Ni post, carries out separation and purification.

Advantage of the present invention and positively effect:

The present invention makes up recombinant DNA with this multiple copied resultant string linked method; Improved the expression amount of BMP in recombinant yeast pichia pastoris; This method uses isocaudarner for connecting bridge, can reach high-caliber different copies series connection number through the external connection of cheapness, makes up the expressing gene that multi-copy gene can construct different copy numbers with this method; Thereby, optimized the selectivity of experiment greatly for selecting the optimum expression amount that the gradient selection is provided; And this method has adopted the method for fusion rotein, behind the BMP of 16 copies, has added the 6His tail, can greatly reduce the difficulty that target protein reclaims through the Ni affinity column with the BMP purifying and recovering, has improved the purifying rate.Choosing pichia spp is engineering bacteria, and this bacterium is induced simply, and output is big; And can high-density growth; And foreign gene is integrated in pichia spp in the host DNA, can be stable go down to posterity and do not lose foreign gene, its stability is much higher than the additive type plasmid of yeast saccharomyces cerevisiae.Thereby established theoretical basis for a large amount of industrial production, had important use and be worth.

Embodiment

The structure of embodiment 1:16 copy BMP

With the plasmid vector that contains 4 copy BMP expressing genes (precious biotech firm is responsible for synthetic by Dalian) that designs voluntarily with Xho I and Sal I double digestion after, the enzyme system of cutting is carried out agarose gel electrophoresis, the recovery size is the small segment of 106bp.The plasmid vector that contains 4 copy BMP expressing genes that will design again with Xho I single endonuclease digestion after, oneself connects to prevent plasmid vector with the SEAP dephosphorylation, the 106bp small segment with recovery is connected with this linearized vector again.Connect product and change the competence intestinal bacteria over to, bacterium liquid is coated on the LB solid plate that contains penbritin through electric shock.Cultivate the positive transformant that picking is grown after 16 hours for 37 ℃ on flat board, extract plasmid, use the checking of Xho I and Sal I double digestion, select and to cut out the big or small segmental transformant that 208bp, 310bp, 412bp or equal proportion increase.

Embodiment 2: the structure of expression vector pPIC9

The forward that to choose inserts the transformant plasmid of 16 copy BMP expressing genes and uses EcoR I and Not I double digestion again, reclaims the small segment that downcuts subsequent use.Expression vector pPIC9 is connected with above-mentioned small segment with behind EcoR I and the Not I double digestion, connects product and change the competence intestinal bacteria over to through electric shock, bacterium liquid is coated on the LB solid plate that contains penbritin through connecting product.Cultivate the positive transformant that picking is grown after 16 hours for 37 ℃ on flat board, extract plasmid, verify through PCR.

Embodiment 3: the structure of pichia pastoris engineered strain

Electricity changed pichia spp GS115 over to after the positive transformant plasmid that checking is good (pPIC9 that contains 16 copy BMP genes) used Sac I linearization for enzyme restriction, and bacterium liquid is coated the MD flat board.Cultivate picking positive transformant after 50 hours for 28 ℃, extract its host DNA, carry out the PCR checking.

Embodiment 5: the application of pichia pastoris engineered strain in the beef-flavouring peptide of the many tandem copies of preparation

At BMGY (1.34%YNB, 0.00004% vitamin H, 1% glycerine; 1% yeast extract powder, 2% polyprotein peptone, 0.1mol/L phosphoric acid buffer pH6.1) in the substratum 30 ℃; The 200rpm shaking table was cultivated after 40 hours; Add 1% methyl alcohol/sky, induce 70 hours after, can produce a large amount of target peptide-16copy BMP.

Use the pichia spp novel strain of the described utilization molecule manipulation of the inventive method technique construction expressed BMP; Overcome and produced shortcomings such as the existing appointed condition requirement of BMP is high, technical difficulty is big, cost is high, yield poorly in the past; Make the synthesis secretion that BMP can be a large amount of, cost is lower, and behind the BMP expression product, is connected with the 6His tail; Can carry out separation and purification through the Ni post, therefore have the certain economic exploitation and be worth.

Sequence table

< 110>University Of Science and Technology Of Tianjin of Tianjin Chunfa Food Additives Co., Ltd

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caccaccacc?accaccacta?agcggccgc?449。

Claims (8)

1. fragment that contains 4 copy beef-flavouring peptide expressing genes, its sequence is shown in sequence table sequence 1.

2. fragment that contains 16 copy beef-flavouring peptide expressing genes, its sequence is shown in sequence table sequence 2.

3. an expression vector is characterized in that containing the described segmental expression vector pPIC9 of claim 2.

4. engineering strain, it is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, bacterial classification called after pichia pastoris (Pichia pastoris) GS115-16BMP, deposit number is CGMCC No.2136.

5. described segmental construction process of claim 1 is characterized in that the step of this construction process is following:

The first, design the fragment that contains 4 copy beef-flavouring peptide expressing genes according to the codon-bias of pichia spp accurate translation, 4 copy beef-flavouring peptide expressing genes are from beginning to end, do not contain other sequences between each expressing gene;

The second, the upstream position at last step 4 copy beef-flavouring peptide expressing genes adds Xho I restriction enzyme site, and downstream position adds Sal I restriction enzyme site;

Three, the upstream position at the Xho I restriction enzyme site in second step adds EcoR I restriction enzyme site; The downstream position of Sal I restriction enzyme site adds 6His encoding sox, Not I restriction enzyme site and TAA terminator codon in proper order, finally obtains the fragment that contains 4 copy beef-flavouring peptide expressing genes shown in sequence table sequence 1.

6. described segmental construction process of claim 2 is characterized in that the step of this construction process is following:

The first, will contain the segmental carrier enzyme that the said method of claim 5 makes up with Xho I or Sal I and cut the back linearizing, produce sticky end;

The second, will contain to reclaim behind the segmental carrier double digestion that the said method of claim 5 makes up with Xho I and Sal I and comprise the small segment that 4 copy beef-flavouring peptide expressing genes, two ends contain Xho I and Sal I sticky end;

Three, the gene fragment that the linearized vector that the first step is obtained and second step obtain is connected through the T4DNA ligase enzyme, connects to obtain to contain the carrier that 8 copies, 12 copies, 16 copies or copy number are 4 arbitrary integer beef-flavouring peptide expressing gene doubly;

Four, contain the transformant that forward inserts through choosing with Xho I and Sal I Restriction Enzyme blanking method; And through the definite quantity that connects small segment in its agarose gel electrophoresis position; Final selection contains the transformant of 16 copy beef-flavouring peptide expressing genes, promptly obtains containing the carrier of 16 copy beef-flavouring peptide expressing genes.

7. the construction process of the described expression vector of claim 3 is characterized in that the step of this construction process is following:

The first, will use that the said method of claim 6 makes up contain copy number be the fragment of 16 beef-flavouring peptide expressing gene through EcoR I and Not I double digestion, reclaim the small segment that contains beef-flavouring peptide expressing gene;

The expression vector pPIC9 that the second, will be used for pichia spp obtains the big fragment of carrier with EcoR I and Not I double digestion;

Three, the small segment with the above-mentioned the first step and the second step gained is connected through the T4DNA ligase enzyme with the big fragment of carrier, makes that to contain copy number be that the fragment of 16 beef-flavouring peptide expressing gene is inserted on the pPIC9 and obtains.

8. the application of the described engineering strain of claim 4 in the beef-flavouring peptide of the many tandem copies of preparation; It is characterized in that: this recombinant yeast pichia pastoris engineering strain passes through methanol induction; Generation contains the polypeptide that copy number is 16 beef-flavouring peptide, and has a 6His mark, and this mark is used for qualitative, the quantitative analysis that Western Blot carries out product; Or affine with the Ni post, carry out separation and purification.