重组纳豆激酶在毕赤酵母中表达及大规模发酵研究
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摘要
纳豆激酶(Nattokinase, NK)是一种碱性丝氨酸蛋白酶,能显著地溶解体内外的血栓,它由纳豆枯草杆菌(Bacillus subtilis var.natto)产生,最早从纳豆中分离得到。国内外大量研究证实了纳豆激酶的医用价值,但由于获取成本过高,所以应用基因工程途径获取纳豆激酶是近年来的研究热点。
本研究从纳豆芽孢杆菌中,利用PCR技术扩增并获取了纳豆激酶基因,构建了表达载体pPICZaC-NK,建立了重组NK毕赤酵母真核表达系统。通过对产物进行SDS-PAGE分析和四肽底物法活性分析,筛选获得了高表达纳豆激酶的毕赤酵母菌株,并对其最佳发酵条件进行初步探索,明确了发酵时间以及不同pH等对其发酵的影响,进行了大规模发酵纯化工艺研究,为重组纳豆激酶的工业化生产奠定了基础。
Nattokinase is one kind of serine protein generated by Bacillus subtils natto. In 1987, Japanese scholar Dr. Hiroyuki Sumi discovered it in natto food and named it nattokinase. In the initial study, the enzyme extract added to fibrin plate showed obvious thrombolysis function, this effect identified the fibrinolytic activity of the kinase. Compared to existing thrombolytic agents, NK has a longer half-life in vivo (2-8 h), less antigenicity and side effects, widely source of raw material and remains active in the gastrointestinal pH environment, etc. It can directly act on the main constituents of thrombus-fibrin, and indirectly activate the internal plasminogen to dissolve fibrin. Therefore, nattokinase becomes a potential new thrombolytic medicine, and attracts the attention of scholars. The current production methods of nattokinase mainly rely on extraction and purification from Bacillus natto fermentation broth. But since the high cost of raw materials of this method, and the unsatisfactory activity, the mass production of nattokinase is limited.
In recent years, scholars hope to produce recombinant nattokinase through genetic engineering techniques, and have made some progresses. The major evolution is cloning the gene of nattokinase, using the E.coli or pichia pastoris expression system and obtaining nattokinase via purification methods. But the large-scale fermentation of recombined nattokinase are rarely reported. In this experiment we extracted the genomic DNA from Bacillus natto and cloned the sequences encoding the mature peptide, then linked the fragment to the expression vector pPICZaC, named as pPICZaC-NK. The vector was imported into pichia pastoris X-33 by electroporation in the following procedure. Then the positive clones were selected and used for identification and analysis. Finally the highly expressing clone were selected and applied in a 30L fermenter for large-scale fermentation study, by which we established the large-scale fermentation process for nattokinase and could provide an experimental basis for its industrialization..
1. Cloning and identification of NK
Genomic DNA was extracted from Bacillus natto, the sequence of nattokinase coding region was cloned from genomic DNA by PCR and linked to pMD18-T vector, so the recombinant cloning vector pMD18-T-NK was established. After the digestion by restriction enzyme, according to the reports of other literatures, an specific band appeared at the position of 1220 kb. The above steps have made the foundation of the expression of recombinant Nattokinase.
2. Construction of recombinant expression vector pPICZaC-NK
The cloning vector pMD18-T-NK was used as template, with the method of PCR, a restriction site Xho I and a signal recognition site Kex2 were inducted in the 5'terminal of the nattokinase mature peptide gene, besides, the other restriction site EcoR I was inducted in the 3' terminal. The production of PCR was digested by Xho I and EcoR I, linked to the plasmid pPICZaC, which had been digested by the same two restriction enzymes, so the recombinant expression vector pPICZα-NK was made. Restriction enzyme digestion and DNA sequencing showed that the construction of vector pPICZa-NK achieved the desired effect.
3. Expression of recombinant NK gene in Pichia pastoris
Recombinant expression vectors pPICZa-NK were digested by restriction enzyme SacⅠextracted by phenol and chloroform and precipitated by ethanol. The vectors were transformed to the competent cells of Pichia pastoris X-33 by electrotransformation, the positive strains were screened by PCR, and induced to express NK by methanol. Experiments showed that the best pH for expression is 5.5, the best time for inductive expression is 72 hours.
4. Research on large-scale fermentation of recombinant NK in Pichia pastoris
When recombinant NK was produced by the large-scale fermentation, plasmin activity could be detected 6 h after the induction of methanol and the maximum of plasmin activity would be reached at 36 h, this maximum is more than twice the maximum of expression by shake flask. This phenomenon showed that under the large-scale fermentation condition, both the living environment and expression status of the yeast are better than that in shake flask. The production of expression was purified from the fermenting liquor and analyzed by 12% SDS-PAGE, a limpid band appeared at the position of 27 kD and no band of hybridprotein appeared, indicating that the whole system of the expression, large-scale fermentation and purification of NK in Pichia pastoris had been basically established. This research has provided an experimental basis for the industrial production of NK.
本研究从纳豆芽孢杆菌中,利用PCR技术扩增并获取了纳豆激酶基因,构建了表达载体pPICZaC-NK,建立了重组NK毕赤酵母真核表达系统。通过对产物进行SDS-PAGE分析和四肽底物法活性分析,筛选获得了高表达纳豆激酶的毕赤酵母菌株,并对其最佳发酵条件进行初步探索,明确了发酵时间以及不同pH等对其发酵的影响,进行了大规模发酵纯化工艺研究,为重组纳豆激酶的工业化生产奠定了基础。
Nattokinase is one kind of serine protein generated by Bacillus subtils natto. In 1987, Japanese scholar Dr. Hiroyuki Sumi discovered it in natto food and named it nattokinase. In the initial study, the enzyme extract added to fibrin plate showed obvious thrombolysis function, this effect identified the fibrinolytic activity of the kinase. Compared to existing thrombolytic agents, NK has a longer half-life in vivo (2-8 h), less antigenicity and side effects, widely source of raw material and remains active in the gastrointestinal pH environment, etc. It can directly act on the main constituents of thrombus-fibrin, and indirectly activate the internal plasminogen to dissolve fibrin. Therefore, nattokinase becomes a potential new thrombolytic medicine, and attracts the attention of scholars. The current production methods of nattokinase mainly rely on extraction and purification from Bacillus natto fermentation broth. But since the high cost of raw materials of this method, and the unsatisfactory activity, the mass production of nattokinase is limited.
In recent years, scholars hope to produce recombinant nattokinase through genetic engineering techniques, and have made some progresses. The major evolution is cloning the gene of nattokinase, using the E.coli or pichia pastoris expression system and obtaining nattokinase via purification methods. But the large-scale fermentation of recombined nattokinase are rarely reported. In this experiment we extracted the genomic DNA from Bacillus natto and cloned the sequences encoding the mature peptide, then linked the fragment to the expression vector pPICZaC, named as pPICZaC-NK. The vector was imported into pichia pastoris X-33 by electroporation in the following procedure. Then the positive clones were selected and used for identification and analysis. Finally the highly expressing clone were selected and applied in a 30L fermenter for large-scale fermentation study, by which we established the large-scale fermentation process for nattokinase and could provide an experimental basis for its industrialization..
1. Cloning and identification of NK
Genomic DNA was extracted from Bacillus natto, the sequence of nattokinase coding region was cloned from genomic DNA by PCR and linked to pMD18-T vector, so the recombinant cloning vector pMD18-T-NK was established. After the digestion by restriction enzyme, according to the reports of other literatures, an specific band appeared at the position of 1220 kb. The above steps have made the foundation of the expression of recombinant Nattokinase.
2. Construction of recombinant expression vector pPICZaC-NK
The cloning vector pMD18-T-NK was used as template, with the method of PCR, a restriction site Xho I and a signal recognition site Kex2 were inducted in the 5'terminal of the nattokinase mature peptide gene, besides, the other restriction site EcoR I was inducted in the 3' terminal. The production of PCR was digested by Xho I and EcoR I, linked to the plasmid pPICZaC, which had been digested by the same two restriction enzymes, so the recombinant expression vector pPICZα-NK was made. Restriction enzyme digestion and DNA sequencing showed that the construction of vector pPICZa-NK achieved the desired effect.
3. Expression of recombinant NK gene in Pichia pastoris
Recombinant expression vectors pPICZa-NK were digested by restriction enzyme SacⅠextracted by phenol and chloroform and precipitated by ethanol. The vectors were transformed to the competent cells of Pichia pastoris X-33 by electrotransformation, the positive strains were screened by PCR, and induced to express NK by methanol. Experiments showed that the best pH for expression is 5.5, the best time for inductive expression is 72 hours.
4. Research on large-scale fermentation of recombinant NK in Pichia pastoris
When recombinant NK was produced by the large-scale fermentation, plasmin activity could be detected 6 h after the induction of methanol and the maximum of plasmin activity would be reached at 36 h, this maximum is more than twice the maximum of expression by shake flask. This phenomenon showed that under the large-scale fermentation condition, both the living environment and expression status of the yeast are better than that in shake flask. The production of expression was purified from the fermenting liquor and analyzed by 12% SDS-PAGE, a limpid band appeared at the position of 27 kD and no band of hybridprotein appeared, indicating that the whole system of the expression, large-scale fermentation and purification of NK in Pichia pastoris had been basically established. This research has provided an experimental basis for the industrial production of NK.
引文
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