CN103484488B - Optimized cattle chymosin proto-gene and secretory expression method and application thereof - Google Patents
Optimized cattle chymosin proto-gene and secretory expression method and application thereof Download PDFInfo
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Abstract
The invention discloses an optimized cattle chymosin proto-gene and a secretory expression method and the application thereof. According to the optimized cattle chymosin proto-gene, influence factors of difference between protein interpret rates and the like due to use frequency of codon, adjustment of GC content, deletion of unstable sequences, and different distribution of secondary mRNA structure and the codon are comprehensively considered, and optimized cattle chymosin proto-gene bodies respectively shown by SEQ ID NO.1, 2 and 3 are obtained by modifying cattle chymosin proto-gene bodies according to the preference codon of pichia pastoris. The invention further provides a production method of recombining the cattle chymosin proto-gene. The production method comprises the steps of converting recombinant expression carriers containing the optimized cattle chymosin proto-gene bodies into host cells to obtain recombination bacterial strains, cultivating the recombination bacterial strains, inducing expression of the recombination cattle chymosin proto-gene bodies, and recycling and purifying expressed products. Compared with original cattle chymosin proto-gene, the secretory expression amount and the enzyme activity of the optimized cattle chymosin proto-gene in the pichia pastoris are remarkably promoted.
Description
Technical field
The present invention relates to the prochymosin gene of optimization, particularly relate to and carry out codon optimized rear obtained prochymosin gene by deriving from ox prochymosin gene, the prochymosin gene that the invention still further relates to optimization, producing the application in rennin, belongs to the preparation field of recombinant chymosin.
Background technology
Cheese is nutritious, delicious flavour, and storage period is long and of a great variety, is the important component part in World of Food, have the title of " kings of milk-product ".In the production process of cheese, curdled milk is the critical process affecting cheese productive rate and quality.Rennin (chymosin, EC3.4.23.4) plays the key enzyme of curdled milk effect in the curdled milk process manufacturing cheese, and it is the one in aspartate protease, can Phe in specificity cracking k-casein
105-met
106peptide bond, causes milk-protein to condense, in milk-product particularly cheese processing, have vital role, and rennin plays very important effect to the matter structure of cheese and the formation of peculiar taste simultaneously.
Traditional rennin mainly extracts from its abomasum by slaughtering non-weaner calf, rennin synthesizes with the form of gathering peptide-renninogen precursor containing 381 amino acid whose precursors in calf abomasum, in secretion process, can remove 16 signal peptides to be formed containing 365 amino acid whose renninogens, size is 40.8kDa.Renninogen non-activity, in acid condition can autocatalytically, and remove N and hold 42 amino acid to form activated rennin, this enzyme contains 323 amino acid, and size is 35.6kDa.Along with the sharp increase of cheese industry, the rennin that calf abomasum extracts cannot satisfy the demands.The stomach en-of the more substitute of current use mainly sheep, pig and chicken, but these enzymes all have stronger proteolysis ability, the cheese made separately all can exist there is bitter taste, the curdled milk time is long, quality is softer, the problems such as yield rate is low.In recent years, some investigators have also extracted rennin from hydrocoles, there is as found in turtle gastric mucus the aspartic protease of milk-clotting activity, it is the proteolytic enzyme having fine milk-clotting activity within the scope of 6.0-6.8 in pH value that the people such as Kristinsson extract a kind of from the stomach and gastric mucus of sea dog, the cheesy flavour produced with this proteolytic enzyme is normal, being applicable to commercially producing, is the potentiality substitute of ox ferment rennet.
Although researchist has found some zoogenous ox chymosin substitutes, Animal resources have still been enriched not as plant and microorganism, and therefore, goal in research is turned to plant and microbial source rennin by scholars one after another.Many plant proteases all have the function of curdled milk, are mainly distributed in the positions such as the fruit of plant, leaf, stem and root.In world's rennin, this kind of enzyme dosage accounts for about 1%.The plant origin rennin reported at present is mainly papoid, bromeline, ficin, acacia proteolytic enzyme etc.Due to animal and plant source rennin limitation, microbial rennet gradually pay close attention to by people and study.The rennin of microbial source has wide material sources, growth cycle is short, and output is large, be convenient to extract, be beneficial to the advantages such as industrialization scale operation.Do not stop so far, existing 40 multiple-microorganisms can produce the proteolytic enzyme with certain condenser water level, mainly bacterium, actinomycetes and fungi.The maximum microbe-derived rennin of current application is produced by Mucor pusillus (Mucor pusillus), and the proteolysis energy force rate ox rennin of this enzyme is strong, but more weak than other proteolytic enzyme of originating.Other common microorganism for producing lab ferment has Rhizopus oryzae (Rhizopus oryzae), parasitic interior seat shell bacterium (Endothaparasitisa), Rhizomucor (Rhizomucor) etc.Compared with plant origin rennin, the quality of the cheese that microbe-derived rennin makes and quality all increase, but under normal pasteurize condition, because of the thermotolerance that it is higher, protease activity in whey can be caused higher, affect later stage work.After comparing with ox rennin, its milk-curdling activity and protease activity ratio (c/p) still on the low side, cheese is second-rate, and ripened cheese local flavor also changes to some extent, also can with bitter taste.
Along with engineered develop rapidly, utilize DNA recombinant technology to realize animal rennet and express to become gradually in microorganism host and produce the new effective way of of rennin.Genetically engineered rennin is mainly expressed in the rennin channel genes microbial host cell of animality, thus alleviates crude rennet situation in short supply, meets the production of cheese industry.At present, existing many animals source rennin is expressed in different Microbial Expression Systems.1980, Uchiyama etc. first in vitro successful translation go out ox rennin mRNA and attempt in intestinal bacteria, produce rennin.Nishimori etc. construct the expression plasmid pCR301 for expressing ox renninogen cDNA afterwards, and successful expression is in the cytoplasm of host cell.The people such as Beppu utilize renninogen cDNA at expression in escherichia coli, expression product finally with the form of inclusion body at thin intracellular accumulation.Although produce rennin with prokaryotic expression system achieved many progress, expression product exists mainly with the form of inclusion body, brings difficulty to follow-up work, also can cause the problems such as cytolemma fragility, cellular respiration ability and fecundity forfeiture simultaneously.Eukaryotic expression system has advantages such as not producing intracellular toxin, can carry out posttranslational modification, downstream extraction is convenient and be easily esthetically acceptable to the consumers, is more and more applied in the middle of the production of foreign protein.Conventional eukaryotic expression system mainly yeast expression system and filamentous fungus expression system.Ward etc. utilize Aspergillus awamori as expressive host, and construct the expression vector that an ox prochymosin gene and glucose amylase gene (glaA) merge, recombinant plasmid is transferred in aspergillus, obtains a large amount of rennin and is secreted into outside born of the same parents.2004, the caprine prochymosin gene of the successful clone such as Vega-Hemandez MC was in yeast and express, and proved to obtain activated rennin through dairy products agglutination test.2008, Vallejo etc. constructed ox rennin precursor, ox renninogen and ox rennin three kinds of expression vectors and are converted into pichia spp, measured, only have the expression vector of renninogen form can produce activated ox rennin by induction.Feng Zhen etc. successfully construct renninogen yeast secretion type expression carrier, and obtain secreting, expressing in Kluyveromyces lactis GG799.2009, ox prochymosin gene is inserted into Yeast expression carrier pPICZaA α by Zhang Li etc., after the expression plasmid linearizing built, electricity transforms Pichia pastoris GS115 bacterial strain, and under methanol induction, carry out the expression of rennin, in medium supernatant, the activity of rennin is 12.2SU/mL.This research is domestic first Application Pichia anomala expression ox rennin.2010, the people such as Yuan Wei proceed in Kluyveromyces lactis GG799 bacterial strain after carrying out codon optimized transformation to ox renninogen, after cultivating 96h, detect that condenser water level can reach 99.67SU/mL from supernatant, for obtaining expression (Yuan Wei etc. to through codon optimized ox prochymosin gene in Kluyveromyces lactis first both at home and abroad, the synthesis of ox prochymosin gene and the expression in Kluyveromyces lactis thereof. biotechnology journal, September 25 in 2010; 26 (9): 1281-1286).
In sum, all there is low, the expressed recombinant bovine rennin enzyme enzyme of secreting, expressing amount and to live the defect such as lower in the expression of ox prochymosin gene in pichia spp optimized disclosed in ox prochymosin gene and even document, haves much room for improvement.
Summary of the invention
Main purpose of the present invention is that secreting, expressing amount existing when expressing in yeast strain for the ox prochymosin gene optimized disclosed in original ox prochymosin gene or document is lower, enzyme lives not high problem, there is provided a kind of new codon through transformation and the ox prochymosin gene of the good optimization of mRNA secondary structural stability, this ox renninogen optimized gene secreting, expressing amount in yeast strain is high, and expressed recombinant bovine rennin enzyme is lived remarkable lifting;
Two of the object of the invention is to provide the recombinant expression vector of the ox prochymosin gene containing this optimization and the recombinant host cell containing this recombinant expression vector;
Three of the object of the invention is ox prochymosin gene Restruction ox renninogens of the optimization described in utilization;
Four of object of the present invention is to provide a kind of method improving ox prochymosin gene secreting, expressing amount in pichia spp of original ox prochymosin gene or optimization.
For achieving the above object, first ox renninogen original gene carries out codon optimized etc. modifying the ox prochymosin gene that transformation obtains three different optimizations by the present invention, called after pcm respectively, pcm-1, and pcm-2, wherein, the nucleotides sequence of pcm is classified as shown in SEQ ID No.1, the nucleotides sequence of pcm-1 is classified as shown in SEQ ID No.2, the nucleotides sequence of pcm-2 is classified as shown in SEQ ID No.3.
The present invention is for overcoming the problem of the low expression level brought due to ox renninogen original gene and the pichia spp difference on codon usage frequency, according to the preferences of pichia spp codon to ox renninogen original gene (pcw) (shown in SEQ ID NO.4) under the prerequisite not changing its protein amino acid sequence, consider codon usage frequency, the adjustment of GC content, the deletion of unstable sequence, the influence factors such as mRNA secondary structure, first modification transformation is carried out to ox renninogen original gene (pcw) according to the preference codon of pichia pastoris phaff, and then consider in mRNA translation process, codon usage frequency is higher, its translation rate is faster, otherwise it is then slower.But the expression amount of activated protein except with the overall frequency of utilization of codon mutually outside the Pass, also relevant to the distribution situation of codon.In codon optimisation process, all sections all can not be transform as the codon of very fast translation rate, but need to carry out reasonably combined, such guarantee translation rate and protein folding speed are to coupling.Therefore, devise identical but three the sequence (pcm of distribution situation difference (protein translation speed is different) of three codon usage frequencies, pcm-1, pcm-2), article three, the ox prochymosin gene after the optimization shown in sequence is respectively shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, these three sequences optimized, compared with original gene, all change 218 bases, account for 19.8% of base sum; Relate to 187 amino acid, account for 51.2% of amino acid sum.The GC content of the gene after optimization reduces to 47.3% by 57.4; In addition, compare original gene, after optimizing, the mRNA secondary structure free energy of ox prochymosin gene have also been obtained raising by a relatively large margin, reduces and transcribes energy barrier, improve transcriptional efficiency, finally effectively improve the secreting, expressing amount in pichia spp and enzyme work.
Another object of the present invention is to provide the recombinant expression vector of the ox prochymosin gene containing described optimization and the recombinant host cell containing this recombinant expression vector;
Preferably, described recombinant expression vector is recombinant eukaryon expression vector, is more preferably recombinant yeast expression vector, most preferably is recombinant yeast pichia pastoris (Pichia pastoris) expression vector.
Described recombinant host cell is preferably recombinant yeast cell, is more preferably recombinant yeast pichia pastoris (Pichia pastoris) cell.
Exercisable for the ox prochymosin gene of optimization to be connected with the expression regulation sequence of yeast just can be obtained the recombinant yeast expression vector of secreting, expressing ox renninogen in yeast cell; In order to reach better secreting, expressing effect, exercisable for the ox prochymosin gene of optimization to be connected with the expression regulation sequence of pichia spp is obtained the restructured Pichia pastoris in expression carrier of secreting, expressing recomposited ox prorennin in Pichia pastoris by the present invention.
Yeast cell of the present invention comprises any one yeast cell in each primary yeast of the ox prochymosin gene can expressing optimization.Selecting for expressing the suitable yeast of recomposited ox prorennin is within the limit of power of those of ordinary skill in the field.Selecting in the yeast host of expressing, suitable host can comprise and has active, the good soluble protein of such as good secretion capacity, low proteolytic and produce and the yeast of the overall characteristic such as firm.These yeast include but not limited to ascosporogenous yeast (Endomycetale (Endomycetales), sporidium yeast and belong to the yeast of imperfect fungi (gemma guiding principle (Blastomycetes)) class.Described ascosporogenous yeast is divided into two sections, that is: Spermophthoraceae (Spermophthoraceae) and Saccharomycetaceae (Saccharomycetaceae).The latter comprises four subfamilies, and Schizosaccharomycoideae(is Schizosaccharomyces (Schizosaccharomyces) such as), Nadsonioideae, Lipomycoideae and Saccharomycoideae(such as Pichia (Pichia), genus kluyveromyces (Kluyveromyces) and yeast belong (Saccharomyces).Sporidium yeast comprises Leucosporidium genus, Rhodosporidium (Rhodosporidium), yeast belong (Sporidiobolus) thrown by lock, Filobasidium belongs to and Filobasidiella (Filobasidiella).
Preferably, yeast of the present invention is pichia spp (Pichia pastoris).
Be well-known for the expression regulation sequence of Yeast expression carrier for those of ordinary skill in the field, include but not limited to the promoter region from such as following gene:
Alcoholdehydrogenase (ADH), Hydratase, phosphoenolpyruvate, glucokinase, GPI, glyceraldehyde-3-phosphate dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phoshoglyceric acid mutase and pyruvate kinase (PYK).Other Suitable promoter sequences for yeast host comprises for glycerol 3-phosphate acid kinase (Hitzeman et al., J.Biol.Chem. (1980) 255:2073) and sucroclastic promotor (Holland et al., Biochemistry (1978) 17:4900 of other such as pyruvic carboxylase, triosephosphate isomerase and phosphoglucose isomerase; Hess et al., J.Adv.ENZYMEREG. (1968) 7:149).Yeast enhancers also can use together with Yeast promoter.In addition, synthetic promoter also can play the effect of Yeast promoter.Yeast promoter can comprise the promotor of the non-yeast sources of the natural generation of the ability with combining yeast RNA polymerase and initiation transcription, other controlling elements that can comprise partial yeast expression vector comprises the terminator (Holland et al., J.Biol.Chem. (1981) 256:1385) such as from GAPDH or enolase gene.Be the trp1 gene be present in yeast plasmid for the Suitable selection genes in yeast, described trp1 gene is provided for the selective marker of the yeast mutant lacking the ability grown in tryptophane.
Further, the invention provides a kind of method of Restruction ox rennin, comprising: the optimization shown in SEQID NO.1, SEQ ID NO.2 or SEQ ID NO.3 obtained recombinant expression vector being connected with expression vector of ox prochymosin gene operability; By described recombinant expression vector transformed host cell, obtain recombinant bacterial strain; Cultivate recombinant bacterial strain, the expression of induction recombinant bovine rennin, reclaims the recombinant bovine rennin also expressed by purifying, to obtain final product.
In the method for above-mentioned Restruction ox rennin, described recombinant expression vector is preferably recombinant eukaryon expression vector, more preferably recombinant yeast expression vector, is more preferably restructured Pichia pastoris in expression carrier; Described host cell is preferably yeast cell, is more preferably pichia spp (Pichia pastoris) cell.
The method be transformed into by the ox prochymosin gene of optimization in yeast host is well-known to those of ordinary skill in the field.For example, yeast conversion can be carried out according to the method described in Publication about Document: Hsial et al., P.Natl.Acad.SCI.USA (1979) 76:3829 and Van Solingen et al., J.Bact. (1977) 130:946, also can according to usually at SAMBROOK et al., method described in Molecular Cloning:A Lab Manual (2001) transforms, such as by modes such as core injection, electroporation or protoplast fusions, ox renninogen optimized gene is incorporated in yeast cell, as long as construct recombinant host cell bacterial strain (introducing the recombination yeast host cell being also separated in yeast cell and there is Suitable expression vectors by recombinant yeast expression vector), then be suitable for cultivating recombinant host cell bacterial strain under the condition producing recomposited ox prorennin, the method of cultivating recombinant host cell bacterial strain depends on the character of expression carrier used thereof and the characteristic of host cell, this belongs to conventional technique means concerning those of ordinary skill in the field, it includes but not limited to fermentor tank, shake flasks, fluidized bed aerosol generator, hollow fiber bio-reactor, the methods such as spinner culture system and steel basin bioreactor system, each of these methods can adopt in batches, the method such as feed supplement or continuous mode is carried out, simultaneously gather cell with pattern in batches or continuously or gather culture supernatants.Recombinant host cell is cultivated in the assimilable source containing carbon, nitrogen and inorganic salt and the liquid nutrient medium optionally containing VITAMIN, amino acid, somatomedin and other well-known albumen cultivation fill-in.For cultivate host cell liquid nutrient medium can optionally containing microbiotic or anti-mycotic agent to prevent undesirable microorganism growth and/or containing including but not limited to that antibiotic compound is to select the host cell containing expression vector.
Well-known for other method of expressing heterologous albumen in yeast host cell to those of ordinary skill in the field.Can use those of ordinary skill in the field's well-known standard fed-batch fermentation method (standard feed batch fermentation method) that yeast host bacterial strain is grown in fermentor tank in the amplification stage.Described fermentation method can the difference that utilizes path or express in master mode of the carbon through adjusting to solve specific yeast host.For example, the fermentation of Saccharomyces cerevisiae host may need single glucose, compound nitrogen source (such as caseic hydrolysate) and multivitamin to supplement.The growth limitation nutrient substance being generally carbon can add in fermentor tank in the amplification stage to allow maximum growth.In addition, the usual Application Design of fermentation method is the fermention medium of carbon containing q.s, nitrogen, basic salt, phosphorus and other secondary nutrient substance (such as VITAMIN, trace mineral and salt etc.).
Unless otherwise defined, otherwise all technology used herein and scientific terminology all have with those skilled in the art usually understand identical implication.
Term " yeast host " or " yeast host cell " comprise the yeast of the receptor that can be used as or be used as recombinant vectors or other transfer DNAs.Described term comprises the offspring of the original yeast host cell receiving recombinant vectors or other transfer DNA.Due to accidentally or the sudden change had a mind to, the offspring of single parental cell can morphologically or with the genome of original parent complementation or STb gene on completely the same.
Term " recombinant host cell " or " host cell " mean the cell comprising Exogenous polynucleotide, and no matter use which kind of method to carry out inserting to produce recombinant host cell, such as directly absorb, transduce, known additive method in f pairing or affiliated field.Exogenous polynucleotide can remain the non-integrated vector of such as plasmid or can be integrated in host genome.
Term " polynucleotide " means the deoxyribonucleotide of sub-thread or bifilar form, dezyribonucleoside, ribonucleoside or ribonucleotide and polymkeric substance thereof.Except nonspecific restriction, otherwise the nucleic acid of the known analogue containing natural nucleotide contained in described term, and described analogue has the binding characteristic that is similar to reference nucleic acid and carries out metabolism in the mode of the Nucleotide being similar to natural generation.Unless other specific restriction, otherwise described term also means oligonucleotide analogs, and it comprises PNA(peptide nucleic acid(PNA)), DNA analogue (thiophosphatephosphorothioate, phosphamide acid esters etc.) used in antisense technology.Unless otherwise, otherwise the specific nucleic acid sequence sequence that also impliedly contains its conservative varient (including, but is not limited to degenerate codon replace) of modifying and complementary sequence and clearly specify.Particularly; the 3rd sequence replaced through mixing base and/or deoxyinosine residue by producing (or all) codons selected by one of them or more than replaces (Batzer et al., Nucleic Acid Res.19:5081 (1991) to realize degenerate codon; Ohtsuka et al., J.Biol.Chem.260:2605-2608 (1985); Cassol et al., (1992); Rossolini et al., Mol Cell.Probes8:91-98 (1994)).
Term " polypeptide ", " peptide " and " albumen " exchange in this article and use with the polymkeric substance meaning amino-acid residue.That is, be equally applicable to describe peptide for the description of polypeptide and describe albumen and vice versa.It is the aminoacid polymers of non-naturally encoded amino acids that described term is applicable to natural generation aminoacid polymers and one of them or more than one amino-acid residue.As used herein, the amino acid chain of any length contained in described term, and it comprises full-length proteins (i.e. antigen), and wherein amino-acid residue connects via covalent peptide bonds.
Term " expression " refers to foreign gene transcribing and/or translating in host cell.
Term " conversion " refers to the method be incorporated into by foreign gene in host cell.
Term " foreign gene " refers to specific host cell, this gene order belongs to external source, or from identical source but its original series carried out modifying or transformation.
Accompanying drawing explanation
The codon usage frequency of Fig. 1 ox renninogen original gene (pcw).
The codon usage frequency of the ox prochymosin gene (pcm, pcm-1, pcm-2) that Fig. 2 optimizes.
The comparison of Fig. 3 ox renninogen original gene (pcw) and optimized gene (pcm, pcm-1, pcm-2) translation rate.
The structure schematic diagram of Fig. 4 yeast recombinant expression plasmid pPIC9-pcw (pcm, pcm-1, pcm-2).
Fig. 5 turns the multiple sieve rennin enzyme slip-knot fruit of optimized gene pcm-1 yeast recon.
Fig. 6 turns the multiple sieve rennin enzyme slip-knot fruit of optimized gene pcm-2 yeast recon.
Fig. 7 turns the multiple sieve rennin enzyme slip-knot fruit of optimized gene pcm yeast recon.
Fig. 8 turns pcw (pcw-105
#), turn pcm-1(pcm1-220
#), turn pcm-2 (pcm2-116
#), turn pcm (pcm-4
#) yeast strain 3 liters of fermentor tanks in ox rennin protoenzyme slip-knot fruit.
Fig. 9 pcm-4
#the SDS-PAGE of yeast strain ox renninogen expression amount in methanol induction different time in 3 liters of fermentor tanks; M: molecular weight of albumen marker.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Illustrate: in following specific embodiment, used genetic recombination learns a skill the routine techniques be in this area.The technology do not described in detail in the examples below, all carry out according to the related Sections in following laboratory manual or document or part, comprise: Sambrook et al, Molecular Cloning, ALaboratory Manual (the 3rd edition .2001); Kriegler, Gene Transfer and Expression:ALaboratory Manual (1990); Current Protocols in Molecular Biology (Ausubel etal, 1994).
The optimization of embodiment 1 rennin original gene
The optimization of 1.1 renninogen original genes
Select the prochymosin gene pcw(GenBank Accession No.FJ768675.1 of Niu Laiyuan) be original gene sequence.Renninogen original gene (pcw) total length 1098bp(SEQ IDNO.4), encode 365 amino acid and a terminator codon.By the analysis to ox renninogen original gene sequence feature, according to the principle not changing protein amino acid sequence, consider codon usage frequency, GC content, conventional restriction enzyme site, the unstable sequence A TTTA of yeast, AACCAA, AATAA, the various factors such as Translation initiator mRNA secondary structure, tentatively transform ox prochymosin gene sequence according to the preferences of pichia pastoris phaff codon.Change 218 bases altogether, account for 19.8% of base sum; Relate to 187 amino acid, account for 51.2% of amino acid sum.Before and after transformation, codon usage frequency is as Fig. 1 and Fig. 2.Obtain the ox prochymosin gene optimized after modifying transformation and be respectively pcm, pcm-1, pcm-2, its nucleotide sequence is respectively shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and its GC content reduces to 47.3% (table 1) by 57.4; Gene (pcm, pcm-1, pcm-2) after optimization is by software prediction (RNA structure4.5), its mRNA secondary structure free energy is compared pcw (SEQ ID NO.4) and is obtained raising by a relatively large margin, be conducive to reduction like this and transcribe energy barrier, improve transcriptional efficiency, and then improve expressing quantity (table 2).Meanwhile, before and after optimizing, the secondary structure of the mRNA of gene also there occurs very large change, and the mRNA secondary structure of optimized gene decreases hairpin structure, more simply, is conducive to transcribing of gene.In addition, although the frequency of utilization of 3 optimized gene codons identical due to position in the sequence variant, so cause variant between protein translation speed (Fig. 3).Be connected on universal support after these three majorizing sequence synthetic, build plasmid pGH-pcm-1, pGH-pcm-2, pGH-pcm.
Based composition before and after the optimization of table 1 prochymosin gene is compared
MRNA secondary structure free energy before and after the optimization of table 2 prochymosin gene compares
Note: mRNA secondary structure free energy uses RNA structure4.5 software to calculate.
Recombinant expressed in pichia spp of embodiment 2 Ns of renninogens
2.1 experiment material
2.1.1 bacterial strain and plasmid
Pichia pastoris GS115 bacterial strain and carrier pPIC9 are purchased from Invitrogen; Intestinal bacteria Trans1-T1 competence is purchased from Beijing Quanshijin Biotechnology Co., Ltd; PGH-pcm, pGH-pcm-1, pGH-pcm-2 plasmid is the prosperous bio tech ltd synthesis of Beijing AudioCodes.
2.1.2 substratum and related solution preparation
1, LB substratum: 10g peptone, 5g yeast extract, 10g sodium-chlor are dissolved in beaker, is settled to 1000ml(solid containing 15g agar with deionized water), 121 DEG C of moist heat sterilizations, 20min.
2, MD solid medium: 2g glucose, 2g agarose are dissolved in beaker, are settled to 100ml with deionized water, 108 DEG C of moist heat sterilizations, 30min.
3, BM mother liquor: 10g yeast extract, 20g peptone are in 100ml1mol/l phosphate buffered saline buffer (pH6.0), and deionized water is settled to 900ml.
4, BMGY:900ml BM mother liquor, adds 10ml100% glycerine (v/v), 121 DEG C of moist heat sterilizations, and 20min treats that temperature is down to less than 60 DEG C, adds the sterilized 10ml10 × YNB of filter and 200 μ l500 × vitamin H.
5, BMMY:900ml BM mother liquor, 121 DEG C of moist heat sterilizations, 20min, treats that temperature is down to less than 60 DEG C, adds the aseptic methyl alcohol of 10ml100%, filters sterilized 10ml10 × YNB and 200 μ l500 × vitamin H.
6, YPD substratum: 10g yeast extract, 20g peptone and 20g glucose are dissolved in beaker, are settled to 1L with deionized water, 108 DEG C of moist heat sterilizations, 30min.
7,10 × YNB: take 13.4g YNB and be dissolved in a small amount of distilled water, be settled to 100ml, 0.22 μm of membrane filtration is degerming.
8,500 × vitamin H: take 0.02g vitamin H and be dissolved in 100ml water, 0.22 μm of membrane filtration is degerming.
9, Amp: take 500mg penbritin and be dissolved in 5ml distilled water, is mixed with the stock solution that concentration is 100mg/ml, and 0.22 μm of membrane filtration is degerming, in-20 DEG C of preservations.
10, X-gal: take 0.2g X-gal and be dissolved in 10ml dimethyl formamide solution, be made into the stock solution of 20mg/ml, in-20 DEG C of preservations.
11, IPTG: take 1.2g IPTG and be dissolved in 10mL distilled water, 0.22 μm of membrane filtration is degerming.In-20 DEG C of preservations.
12,10% skimming milk solution: take 10g skim-milk and be dissolved in 100ml0.01mol/LCaCl2 solution.
13, CaCl
2+ MgCl
2solution: take the anhydrous CaCl of 0.89g
2, 0.19g MgCl
2* 6H
2o adds after a small amount of distilled water dissolves and adds 20ml100% glycerine, is settled to 100ml.
14,1M sorbyl alcohol: take after 18.22g sorbyl alcohol is dissolved in a small amount of distilled water and be settled to 100ml.
2.1.3 toolenzyme and main agents box
T
4dNA ligase and restriction enzyme are Fermentas Products; TransStart
tMfast Pfu archaeal dna polymerase and carrier T pEASY-T1Simple are Beijing Quanshijin Biotechnology Co., Ltd's product; DNA sepharose recovery test kit and plasmid extraction kit are purchased from TIANGEN Biotech (Beijing) Co., Ltd.; Yeast extract paste and peptone are OXOID Products.
2.2 the structure of the ox prochymosin gene yeast expression vector of ox rennin original gene and optimization
2.2.1 the structure of expression vector
Extract pGH-pcm respectively, pGH-pcm-1, pGH-pcm-2 and pPIC9 plasmid, and with EcoRI and NotI, double digestion process is carried out to these two plasmids respectively, and the ox prochymosin gene (pcm, pcm-1, pcm-2) (SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3) optimized is carried out reclaiming and being connected with expression vector pPIC9 digestion products, cut by enzyme and check order and positive colony identified, construct yeast recombinant expression vector pPIC9-pcm, pPIC9-pcm-1, pPIC9-pcm-2 (Fig. 4) thus.
With reference to the construction process of yeast recombinant expression vector pPIC9-pcm, renninogen original gene (pcw) (SEQ ID NO.4) is carried out reclaiming and being connected with expression vector pPIC9 digestion products, cut by enzyme and check order and positive colony identified, build and obtain yeast recombinant expression vector pPIC9-pcw(Fig. 4).
2.2.2 the expression of the prochymosin gene of renninogen original gene and optimization in pichia spp and screening
2.2.2.1 pichia spp is transformed
With BglII respectively enzyme cut recombinant expression plasmid PIC9-pcm, pPIC9-pcm-1, pPIC9-pcm-2 and pPIC9-pcw, make plasmid linearization, transformed pichia spp Host Strains GS115 respectively according to Pichia anomala expression handbook.Coat on MD flat board with every plate 200 μ l bacterium liquid measure, 28 DEG C are cultured to and grow transformant.
2.2.2.2 producing lab ferment recombinant yeast pichia pastoris is in the screening of shaking table level
With sterile toothpick, transformant longer on MD flat board is copied on the numbered MM flat board of tool, be replicated in simultaneously and have on the MD flat board of identical numbering, MM replica plate and MD replica plate are placed in 28 DEG C of incubators to cultivate, grow well until bacterium colony single on MM flat board, be inoculated in 48 hole flat boards according to numbering, containing 500 μ l BMGY in each hole, 28 DEG C, 200r/min cultivates 2d; The centrifugal 5min of 4000r/min, abandons supernatant, and add 500 μ l BMMY, 28 DEG C, 200r/min cultivates 3d; Every 12h adds a methyl alcohol (methyl alcohol final concentration is 1%).After induction terminates, the centrifugal 5min of 4000r/min, collects supernatant, measures the activity of rennin in supernatant respectively.Choose primary dcreening operation enzyme higher bacterial strain of living and carry out multiple sieve, by the inoculation that activate in 10ml BMGY substratum, after 200r/min cultivation 48h, replace medium to 5ml BMMY, every 12h adds the methyl alcohol of one time 100% according to the addition of 1%.After induction 72h, collected by centrifugation enzyme liquid, for measuring rennin vigor.
The mensuration of milk-curdling activity
Activation of zymogen: first the enzyme liquid of collection be adjusted to pH2.0 with 1mol/L HCl, under room temperature, (25 DEG C) place 2h, then with 2mol/L NaOH, its pH are adjusted to pH5.5.
The method of Arima K is adopted to carry out the mensuration of rennin vigor.Use 0.01mol/L CaCl
2solution preparation 10% skimming milk, uses after at room temperature placing 40min after this solution preparation.Get 1mL10% skimming milk in 32 DEG C of insulation 10min, add the enzyme liquid (unactivated enzyme liquid in contrast) of appropriate dilution, shaken well also starts timing, observes on tube wall and starts to occur that curds granules is terminal, records curdled milk time t(second, s).Under these conditions, the enzyme amount that 40min condenses needed for 1mL10% skimming milk is defined as a Soxhlet unit (SU).Enzyme activity calculation formula is as follows:
Rennin vigor (SU)=experiment breast amount/curdled milk enzyme amount × d × 2400/t
Note: d is the multiple of enzyme liquid dilution
Screened each 800 strains of yeast transformant turning renninogen original gene (pcw) and turn optimized gene (pcm, pcm-1, pcm-2) respectively, the positive rate of each transformation event is all between 28%-35%.Wherein picking out the enzyme turning pcw the highest bacterial strain alive is pcw-105
#, its milk-curdling activity is 125SU/ml; And turn in 261 strain positive transformants of optimized gene (pcm-1) and have the milk-curdling activity of nearly 67% higher than pcw-105
#, therefrom filter out enzyme live higher bacterial strain 20 strain carry out multiple sieve, result as Fig. 5, wherein, pcm1-220
#rennin vigor is the highest, reaches 309.98SU/ml in multiple sieve level; Turn in the 255 strain positive transformants of optimized gene pcm-2 and have the milk-curdling activity of nearly 70% higher than pcw-105
#, therefrom filter out enzyme live higher bacterial strain 20 strain carry out multiple sieve, result as Fig. 6, wherein, pcm2-116
#rennin vigor is the highest, reaches 378.06SU/ml in multiple sieve level; Turn in the 269 strain positive transformants of optimized gene pcm and have the milk-curdling activity of nearly 78% higher than pcw-105
#, therefrom filter out enzyme live higher bacterial strain 20 strain carry out multiple sieve, result as Fig. 7, wherein, pcm-4
#rennin vigor is the highest, reaches 558.14SU/ml in multiple sieve level.
The fermentation of embodiment 3 rennin recombinant yeast pichia pastoris
Respectively with the recombinant yeast pichia pastoris bacterium pcw-105 that the expressing rennin amount turning renninogen original gene (pcw) is the highest
#with through living in multiple sieve experiment the highest pcm-4 than control strain enzyme prochymosin gene (pcm, pcm-1, pcm-2) enzyme optimized that turns significantly improved of living
#, pcm1-220
#, pcm2-116
#carry out the fermentation tank level induction of 3 liters, laboratory and produce enzymic fermentation.
Respectively above-mentioned 4 strain yeast transformant sterile toothpick are chosen in 40ml YPD substratum, after (28 DEG C, 200rpm) 48h cultivated by shaking table, be transferred in 200ml YPD substratum, shaking table cultivates (28 DEG C, 200rpm) 24h as ferment-seeded bacterium liquid, is inoculated in 3 liters of fermentor tanks.Fermentor tank optimum configurations is pH5.5, temperature 30 DEG C, stir speed (S.S.) is 1000rpm, air flow is 200, initial inoculum is 200ml bacterium liquid, when dissolved oxygen drops to minimum, when then starting again to rise to 100%, start to mend sugar (the 40% glucose+12mlPTM salt of 300ml), after mending sugared 4-5h, start mixed feeding (the 40% glucose+1mlPTM salt+7ml methyl alcohol of 80ml), start stream after mixed feeding 2-4h and add methyl alcohol (500ml methyl alcohol+12mlPTM salt) induction product enzyme, after starting induction, get fermented sample every 12h to measure thalline weight in wet base and sample enzyme and live and keep sample, enzyme work increases along with the prolongation of induction time, when enzyme work starts to decline, stop fermentation, lower tank, by centrifugal for fermented liquid (10000rpm, 10min, 4 DEG C), clear enzyme solution in collection.
From 3 liters of lab scale level fermentations result after methanol induction 132h, turn the yeast transformant pcw-105 of renninogen original gene pcw
#the activity of rennin is 721.8SU/ml; Turn the yeast transformant pcm1-220 of renninogen optimized gene pcm-1
#enzyme is lived as 1231.44SU/ml, and the work of its enzyme is pcw-105
#1.7 times of transformant, turn the yeast transformant pcm2-116 of pcm-2
#enzyme is lived as 1658.2SU/ml, is pcw-105
#2.3 times; And turn the yeast transformant pcm-4 of pcm
#enzyme is lived as 2987SU/ml, is pcw-105
#4.1 times (Fig. 8, Fig. 9).
As can be seen here, by considering GC content, codon preference, after the factors such as mRNA secondary structure are optimized renninogen original gene, the expression level of this gene in pichia spp is significantly improved.Even if under the condition that codon usage frequency is all identical, also can cause the difference between foreign protein translation rate in the distribution difference of the different positions codon of sequence, thus cause the greatest differences between final expression level.Study with domestic and international part and compare, the former final expression amount of recombinant chymosin of the present invention reaches higher expression level, for further industrialization expanding production is laid a good foundation.
Claims (4)
1. the ox prochymosin gene optimized, is characterized in that: its nucleotides sequence is classified as shown in SEQ ID No.1.
2. the restructured Pichia pastoris in expression carrier containing the ox prochymosin gene optimized described in claim 1.
3. the recombinant yeast pichia pastoris host cell containing restructured Pichia pastoris in expression carrier described in claim 2.
4. a production method for recombinant bovine rennin, comprising: being connected with expression vector of ox prochymosin gene operability of optimization according to claim 1 is obtained recombinant expression vector; By described recombinant expression vector transformed host cell, obtain recombinant bacterial strain; Cultivate recombinant bacterial strain, the expression of induction recombinant bovine rennin, reclaims the recombinant bovine rennin also expressed by purifying, to obtain final product; Described expression vector is yeast expression vector; Described host cell is pichia spp (Pichia pastoris) cell.
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| FJ768675.1;Zhang,L. 等;《GenBank》;20090323;序列表 * |
| 牛凝乳酶原基因的合成及其在乳酸克鲁维酵母中的表达;袁伟 等;《生物工程学报》;20100925;第26卷(第9期);1281-1286 * |
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