萝卜硫素抗肿瘤活性研究及其关键酶myrosinase基因的克隆表达
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摘要
萝卜硫素(1-异硫氰酸-4-甲磺酰基丁烷)(SFN)是迄今为止蔬菜中所发现的抗痛效果最好的天然活性物之一,美国、日本等国家从20世纪50年代就开始研究SFN的性质、制备方法、肿瘤抑制机制及生理功能等,由于其具有多种抑瘤机制且具有分子量小、治疗指数好等优点,SFN现已成为重点研究的抗肿瘤药候选药物。我国关于SFN的研究较少,关于其抑瘤活性的研究几无报道。本论文利用MTT法、荧光显微术和流式细胞术等方法研究了SFN对人胶质母细胞廇细胞A172、非小细胞肺癌细胞株H460、肝癌细胞株HepG2、人肺癌细胞株A549生长的影响。对人胶质瘤细胞A172的实验结果表明,较低浓度的萝卜硫素能够诱导A172发生凋亡和调控细胞周期,并主要以后者为主。而萝卜硫素对H460增殖抑制其所需浓度较高,并且主要是通过诱导凋亡通路来实现。可见,SFN对于不同瘤细胞株其增殖抑制机制不同。此外，SFN对A549和HepG2也有增殖抑制作用,结合本课题组前期MTT试验结果：SFN对瘤细胞株HeLa、HL-60、CNE、PC3及P388细胞等均有显著的体外增殖抑制活性且IC50值较低,说明SFN具有很好的新药开发潜力。
硫代葡萄糖苷酶(myrosinase, EC 3.2.3.1,简称硫苷酶)又称黑芥子酶,是黑芥子苷(硫代葡萄糖苷的一种)水解生成SFN过程的关键酶。在生命进化历程中,硫苷酶基因往往以多个拷贝的形式存在于物种基因组中并在遗传过程中被各物种保留,最终形成了一个相当复杂的基因家族。在甘蓝型油菜中,该基因的研究已非常广泛,而在同为十字花科植物的西兰花中其研究却少见报道。因此,对西兰花硫苷酶基因进行克隆并对其异源表达进行研究对进一步研究该植物的防卫机理和硫苷活性水解产物的获得具有重要的学术意义和实际应用价值。
硫苷酶在十字花科植物中的重要功能对硫苷酶基因结构保守性的要求与硫苷酶编码基因的多基因存在及在进化中被保留的现象说明硫苷酶编码基因序列存在相当保守的序列模体和变异相当活跃的区域。因此,利用现有基因数据库中不同物种的硫苷酶编码序列进行同源性比较,探询硫苷酶编码基因的保守序列,设计扩增西兰花硫苷酶编码基因保守序列的引物,结合利用RACE技术,在西兰花中获得硫苷酶基因的全长序列是可行的。本实验以西兰花幼苗为材料,根据同源序列法PCR扩增硫苷酶特异cDNA片段,进一步用3'RACE和5'RACE分别扩增cDNA 3'端序列和5’端序列,经序列拼接获得西兰花硫苷酶全长cDNA序列。利用在线蛋白质分析系统对其编码的氨基酸序列进行结构特征分析,NCBI CDD数据库中进行保守结构域查找,在SWISS-MODEL进行在线蛋白同源模建,MEG A3.1构建NJ系统树。结果得到一全长1830bp硫苷酶cDNA,含有一个1641bp的ORF及31bp的5’端非翻译区和158bp的3’端非翻译区,命名为BoMyr2(GenBank登录号为EU004075)。编码氨基酸序列含有20氨基酸长度的信号肽并含有9个天冬酰胺N末端糖基化位点,编码的蛋白分子量(Mw)约为62.2kD,等电点(pI)为8.71；BoMyr2推测氨基酸序列含有糖基水解酶家族1特有的糖基水解酶结构域,其编码蛋白与PDB库中白芥硫苷酶有相似的三维结构,具有明显的β/α(TIM)桶结构；BoMyr2应归为硫苷酶基因家族的MB亚族。
由于硫苷酶是一类非组织型表达的蛋白,通常在植物体内含量较低,并且由MB亚族基因编码的硫苷酶在植物体内往往与硫苷酶结合蛋白形成复合物,因此,从植物源出发通过分离纯化获得大量的硫苷酶很难实现。西兰花硫甘酶的原核表达现无资料公开报道。根据RACE结果设计引物扩增硫苷酶成熟蛋白编码区序列,以pGEX-4T-1为载体,在大肠杆菌BL21中进行GST融合蛋白表达。经SDS-PAGE检测,发现在90kD左右处有明显的蛋白条带。预测的硫苷酶成熟蛋白分子量为60.17kDa,而融合蛋白N端融合标签蛋白分子量约为26kDa,表达产物与与预期大小一致。说明硫苷酶成熟蛋白在BL21中融合表达成功。不同时间点GST融合蛋白表达比较发现,融合蛋白的诱导表达具有累积作用,在诱导4h后,蛋白表达量已较高,到6小时达最大。融合蛋白活性测定发现,表达的融合蛋白基本没有催化底物sinigrin的能力,即融合蛋白缺乏硫苷酶活性,有必要进行进一步的凝血酶酶切加工以及蛋白变性、复性等方面的研究。
蛋白数据库(PDB)中三维结构已闸明的硫苷酶均存在糖基化现象,利用不同模型对GenBank中已登录的硫苷酶全长cDNA序列进行信号肽预测发现,其编码基因均含有一段典型信号肽编码序列,说明硫苷酶蛋白在黑芥子酶细胞ER膜上合成后转运到黑芥子酶颗粒的过程中发生了糖基化反应。毕赤酵母(Pichia pastoris)具有对外源蛋白进行适度糖基化修饰的作用。因此,进一步将西兰花BoMyr2成熟蛋白编码序列重组到Pichia pastoris分泌型表达载体pPIC9K上,得到重组酵母表达载体pPIC9K-BoMyr2,经SacⅠ线性化后电转导入Pichia pastoris KM71。菌落PCR以及重组酵母的SDS-PAGE分析发现,硫苷酶在Pichia pastoris中进行分泌表达,表达蛋白分子量约为65kDa。酶活测定显示,表达较好的6株菌株(13#、15#、5#、21#、41#、29#、36#),13#、15#重组菌株其发酵液上清硫苷酶酶活可达1.7～1.9U／ml,而36#菌株其活性为0.75U／ml。硫苷酶比活力分析发现,同样是13#、15#重组菌株比活力较高,相关分析发现两者相关性达95%,说明发酵上清的蛋白量可基本反映外源蛋白的表达情况和硫苷酶活性。
将酶活相对较高的13#菌株,进一步进行摇瓶发酵研究。结果表明,在BMMY培养基中,pH 6.0、1.0%甲醇浓度可以获得较好的表达水平；0.05%的油酸和0.1%的表面活性剂吐温20可以少量提高硫苷酶的表达。诱导培养36小时后发酵上清中硫苷酶总酶活较好,适于发酵液的回收。重组毕赤酵母在合适条件下其发酵上清酶活可达到3U／ml左右
MA基因亚族编码的自由态硫苷酶已得到广泛深入的研究,而关于MB基因亚族编码的硫苷酶因在植物体内的表达情况、存在形式等因素其生化特性至今很少被触及。BoMyr2基因的克隆及其在毕赤酵母基因组中整合、分泌表达不但为大量获得纯硫苷酶提供了一条可行的新途径,而且必将有助于研究该酶的结构、功能,研究该酶与其结合蛋白、相关蛋白等相互作用并形成复合物的过程及各类蛋白对蛋白复合体活性影响等方面均具有重要的意义。
Sulforaphane (SFN), which has been identified in broccoli as a product of enzymatic or acid hydrolysis of the corresponding glucoraphanin(one of glucosinolates), is viewed as a conceptually promising agent in cancer prevention and/or therapy. SFN was prepared by author laboratory and its purity was about 85% determined by HPLC and GC/MS. The effect of SFN on the proliferation of human glioma cell line A172, human intestinal adenocarcinoma cell lines A549 and H460, human hepatoma carcinoma cell HepG2 was investigated in vitro by MTT assay、Hoechst fluorescent staining、Annexin V /PI fluorescent staining and FCS Technique. The results show that SFN can induce A172 cells apoptosis and SFN-mediated apoptosis depend on dose and time. In vitro experiments with SFN Simultaneously indicated a pronounced role for cell cycle arrest in its anti-cancer properties; G2/M arrest is the predominant stage of cell cycle arrest induced by SFN which indicated that SFN could inhibit cell microtube formation. Apoptosis and cell cycle arrest both contribute to the cell proliferation inhibition and the latter is the dominance mechanism for cell line A172. to contrast with cell line A172, sulforaphene concentration for cell H460 proliferation inhibition are higher than it's concentration for cell line A172, and the chief mechanism for proliferation inhibition is due to apoptosis. So the mechanisms of the proliferation inhibition are different with regard to different tumor cell lines. And it's essential to approach the mechanisms for tumor cell line when we do rearch for drug anti-tumor experiment.The proliferation inhibition evoked by SFN was also observed in tumor cell lines A549 and HepG2. Combined with the results about proliferation inhibition of SFN on tumor cell lines HeLa, HL-60, CNE, PC3 and P388 which were got by author laboratory by MTT method, the conclusion was that SFN had anti-tumor activity and can act via several mechanisms to suppress cancer progression, such as modulate cell growth and cell death signals. In conclution, SFN has a promising potential as a new anti-tumor drug.
Myrosinases, thioglucoside glucohydrolases (EC 3.2.3.1) are isoenzymes present in most organs of Brassicaceae species. They catalyse the degradation of glucosinolates, a group of low molecular weight compounds to form an aglucone and D-glucose. The aglucone is unstable and spontaneously decomposes into nitriles, thiocyanates, isothiocyanates or indoles depending on the nature of the side chain and the reaction conditions. In some Brassica vegetables such as cabbage, cauliflower, brussels sprouts and broccoli, glucosinolate degradation products, especially isothiocyanates have been shown to have anticarcinogenic properties. Myrosinase-glucosinolate system in plant is also an important prevention mechanism for responding to the attacks by herbivores, entomologica, pathogenic microorganisms et al. All plant myrosinases characterized to date are glycosylated and are probably transported via the secretory pathway to the myrosin grains present in idioblasts called myrosin cells. Only when the plant tissues are damaged by physical or biological factors and the physical separation is disappeared, glucosinolates can contact with myrosinases and be hydrolyzed by myrosinases.
Myrosinases have been well characterized in biochemical and molecular biological studies. Myrosinase genes have been cloned fr om oilseed rape (B. napus), turnip (B. campestris), leaf mustard (B. juncea), white mustard (Sinapis alba), and thale cress (Arabidopsis thaliana) and classified into four subtypes, MA, MB, MC and TGG, on the basis of amino acid sequences. Studies of myrosinases in B. napus and Sinapis alba showed that they are encoded by large gene families, with up to 15-20 genes in B.napus. MB showed dominant expression in seeds, seedlings, young leaves, and other organs in the mature plant, whereas MA and MC were expressed only in developing seeds. Few reports were about the Myrosinase genes from Broccoli (Brassica oleracea var. italica). In this paper, a full-length cDNA encoding myrosinase from Broccoli are cloned and characterized. And the gene is further fusion expressed in E.coli BL21 and secretory expressed in Pichia pastoris. Moreover, fermentation research of the recombinant yeast for myrosinase secretion was carried on in the last chapter.
Based on the reported plant myrosinase genes in GenBank, software Primer Premier 5 was used to design degenerate primers MyrF and MyrR for amplification gene consensus regions. And then, using 3'RACE and the corresponding 5'RACE technique, the full-length cDNA of Broccoli homologous gene of Brassicaceae myrosinase (BoMyr2, GenBank number: EU004075) was obtained. The 1830bp cDNA contained an ORF of 1641bp, which coded for a polypeptide of 548 amino acids. The deduced polypeptide had a predicted molecular weight of 62.2kD, a pI of 8.71. And The predicted amino acid sequence include a signal peptide, nine N-glycosylation sites and a characteristic domain of the glycosyl hydrolases family 1 which include N-terminal signature(FiFGvAsSAYQiEgG) and active site(IYITENGFS). Homology model of the myrosinase from Broccoli was obtained with MOE on the base of the crystal structure of myrosinase from S. alba as template. Putative three-dimensional structures of the myrosinase showed a (β/α)8 barrel structure common to other glucosidases. Neighbor-joining tree constructed by MEGA 3.1 showed that BoMyr2 was belonged to myrosinase MB subfamily.
The codon region of BoMyr2 was amplified using designed primers based on the RACE results and inserted into E.coli expression vector pGEX-4T-1.The recombinant plasmid was transformed into E.coli BL12 to produce expression strain which would express GST-myrosinase fusion protein. SDS-PAGE showed that the recombinant protein was highly expressed at 90KDa which is coincided with the designed protein. The protein was accumulated in the E.coli BL12 cells by IPTG induced. The product of fusion protein was fairly well after four hours IPTG induced and reached the maxium after six hours IPTG induced. Enzyme activity analysis found that the fusion protein was lack of myrosinase activity. This phenomenon maybe due to the fusion protein was deficient in modification or had a GST addition. The fusion protein presented mostly as inclusion body would also lead to enzyme deficient. So the further investigation, such as protein degeneration and regeneration, enzyme cleavage, is necessary for the aim to get enzymatic protein.
Primary myrosinase has a signal peptide using different SignalP prediction models predicted. And all mature myrosinase which three dimension structure are mentioned and published in PDB database are glycosylated. Pichia pastoris expression system has glycosylation and other modification after protein translated. So it was also used to express recombinant myrosinase BoMyr2 in this paper. The interested BoMyr2 gene was got by digesting the pMD-18-BoMyr2 vector using restriction endonuclease, and then it was inserted into the secretory pPIC9K Pichia pastoris expression vector and transformed into E.coli. Positive recombinant plasmids were selected、sequenced and named pPIC9K-BoMyr2 and was linearized by Sac I, then the linear DNA transferred into Pichia pastoris KM71 by electroporation. The recombinant expression vector pPIC9K-BoMyr2 integrated into KM71 via homologous recombination between the transforming DNA and regions of homology within yeast genome. The positive transformants were screened by MD His- plates and colony PCR. The recombinants were induced expression with methanol and the expression products were tested by SDS-PAGE and enzymatic activity. The results showed that the BoMyr2 gene had been expressed successfully in Pichia pastoris and secreted in the culture medium. The expression product is about a 65 kDa protein and can degrade sinigrin in suitable condition. Six strains were selected in the experiment. Myrosinase activities in the culture supernatant of number 13 # and 15# reached 1.7-1.9U/ml. Determination of the special activity of myrosinase reflected that protein expression levels and enzyme activity had good correlation in culture supernatant. This is the first report on successful heterologous expression of a myrosinase in Pichia pastoris KM71 and provides an important tool for, e.g., further characterization of myrosinase by site-directed mutagenesis and for studying the interaction between myrosinase and myrosinase-binding proteins, myrosinase-associated proteins, and epithiospecifier proteins.
Further fermentation research on number 13# strain were carried out using single factor analysis. The result show that the optimum pH and methanol concentration were respectively as 6.0 and 1.0% for myrosinase expression in BMMY medium.0.05% oleic acid or 0.1% tween-20 can also enhance myrosinase expression in small scale. Opportunity for recovering the fermentation culture was 36 hours after methanol inducing began. And myrosinase activity in the supernatant would come up to 3U/ml or so.
The free, soluble form of myrosinase of the MA gene family has so far been intensively analyzed. In contrast, only limited information is available about the biochemical properties of the myrosinases encoded by the MB gene family for the reason of its low-level exression and complex form. the successful heterologous expression of recombinant BoMyr2 myrosinase from Broccoli in the yeast Pichia pastoris provides a new feasible procedure to produce free myrosinase homogeneity, facilitates studies on structure and function of the enzyme and will be a key for determining the consequences of complex formation with, e.g., myrosinase-binding proteins and myrosinaseassociated proteins, including their effection on myrosinase complex activity.
硫代葡萄糖苷酶(myrosinase, EC 3.2.3.1,简称硫苷酶)又称黑芥子酶,是黑芥子苷(硫代葡萄糖苷的一种)水解生成SFN过程的关键酶。在生命进化历程中,硫苷酶基因往往以多个拷贝的形式存在于物种基因组中并在遗传过程中被各物种保留,最终形成了一个相当复杂的基因家族。在甘蓝型油菜中,该基因的研究已非常广泛,而在同为十字花科植物的西兰花中其研究却少见报道。因此,对西兰花硫苷酶基因进行克隆并对其异源表达进行研究对进一步研究该植物的防卫机理和硫苷活性水解产物的获得具有重要的学术意义和实际应用价值。
硫苷酶在十字花科植物中的重要功能对硫苷酶基因结构保守性的要求与硫苷酶编码基因的多基因存在及在进化中被保留的现象说明硫苷酶编码基因序列存在相当保守的序列模体和变异相当活跃的区域。因此,利用现有基因数据库中不同物种的硫苷酶编码序列进行同源性比较,探询硫苷酶编码基因的保守序列,设计扩增西兰花硫苷酶编码基因保守序列的引物,结合利用RACE技术,在西兰花中获得硫苷酶基因的全长序列是可行的。本实验以西兰花幼苗为材料,根据同源序列法PCR扩增硫苷酶特异cDNA片段,进一步用3'RACE和5'RACE分别扩增cDNA 3'端序列和5’端序列,经序列拼接获得西兰花硫苷酶全长cDNA序列。利用在线蛋白质分析系统对其编码的氨基酸序列进行结构特征分析,NCBI CDD数据库中进行保守结构域查找,在SWISS-MODEL进行在线蛋白同源模建,MEG A3.1构建NJ系统树。结果得到一全长1830bp硫苷酶cDNA,含有一个1641bp的ORF及31bp的5’端非翻译区和158bp的3’端非翻译区,命名为BoMyr2(GenBank登录号为EU004075)。编码氨基酸序列含有20氨基酸长度的信号肽并含有9个天冬酰胺N末端糖基化位点,编码的蛋白分子量(Mw)约为62.2kD,等电点(pI)为8.71；BoMyr2推测氨基酸序列含有糖基水解酶家族1特有的糖基水解酶结构域,其编码蛋白与PDB库中白芥硫苷酶有相似的三维结构,具有明显的β/α(TIM)桶结构；BoMyr2应归为硫苷酶基因家族的MB亚族。
由于硫苷酶是一类非组织型表达的蛋白,通常在植物体内含量较低,并且由MB亚族基因编码的硫苷酶在植物体内往往与硫苷酶结合蛋白形成复合物,因此,从植物源出发通过分离纯化获得大量的硫苷酶很难实现。西兰花硫甘酶的原核表达现无资料公开报道。根据RACE结果设计引物扩增硫苷酶成熟蛋白编码区序列,以pGEX-4T-1为载体,在大肠杆菌BL21中进行GST融合蛋白表达。经SDS-PAGE检测,发现在90kD左右处有明显的蛋白条带。预测的硫苷酶成熟蛋白分子量为60.17kDa,而融合蛋白N端融合标签蛋白分子量约为26kDa,表达产物与与预期大小一致。说明硫苷酶成熟蛋白在BL21中融合表达成功。不同时间点GST融合蛋白表达比较发现,融合蛋白的诱导表达具有累积作用,在诱导4h后,蛋白表达量已较高,到6小时达最大。融合蛋白活性测定发现,表达的融合蛋白基本没有催化底物sinigrin的能力,即融合蛋白缺乏硫苷酶活性,有必要进行进一步的凝血酶酶切加工以及蛋白变性、复性等方面的研究。
蛋白数据库(PDB)中三维结构已闸明的硫苷酶均存在糖基化现象,利用不同模型对GenBank中已登录的硫苷酶全长cDNA序列进行信号肽预测发现,其编码基因均含有一段典型信号肽编码序列,说明硫苷酶蛋白在黑芥子酶细胞ER膜上合成后转运到黑芥子酶颗粒的过程中发生了糖基化反应。毕赤酵母(Pichia pastoris)具有对外源蛋白进行适度糖基化修饰的作用。因此,进一步将西兰花BoMyr2成熟蛋白编码序列重组到Pichia pastoris分泌型表达载体pPIC9K上,得到重组酵母表达载体pPIC9K-BoMyr2,经SacⅠ线性化后电转导入Pichia pastoris KM71。菌落PCR以及重组酵母的SDS-PAGE分析发现,硫苷酶在Pichia pastoris中进行分泌表达,表达蛋白分子量约为65kDa。酶活测定显示,表达较好的6株菌株(13#、15#、5#、21#、41#、29#、36#),13#、15#重组菌株其发酵液上清硫苷酶酶活可达1.7～1.9U／ml,而36#菌株其活性为0.75U／ml。硫苷酶比活力分析发现,同样是13#、15#重组菌株比活力较高,相关分析发现两者相关性达95%,说明发酵上清的蛋白量可基本反映外源蛋白的表达情况和硫苷酶活性。
将酶活相对较高的13#菌株,进一步进行摇瓶发酵研究。结果表明,在BMMY培养基中,pH 6.0、1.0%甲醇浓度可以获得较好的表达水平；0.05%的油酸和0.1%的表面活性剂吐温20可以少量提高硫苷酶的表达。诱导培养36小时后发酵上清中硫苷酶总酶活较好,适于发酵液的回收。重组毕赤酵母在合适条件下其发酵上清酶活可达到3U／ml左右
MA基因亚族编码的自由态硫苷酶已得到广泛深入的研究,而关于MB基因亚族编码的硫苷酶因在植物体内的表达情况、存在形式等因素其生化特性至今很少被触及。BoMyr2基因的克隆及其在毕赤酵母基因组中整合、分泌表达不但为大量获得纯硫苷酶提供了一条可行的新途径,而且必将有助于研究该酶的结构、功能,研究该酶与其结合蛋白、相关蛋白等相互作用并形成复合物的过程及各类蛋白对蛋白复合体活性影响等方面均具有重要的意义。
Sulforaphane (SFN), which has been identified in broccoli as a product of enzymatic or acid hydrolysis of the corresponding glucoraphanin(one of glucosinolates), is viewed as a conceptually promising agent in cancer prevention and/or therapy. SFN was prepared by author laboratory and its purity was about 85% determined by HPLC and GC/MS. The effect of SFN on the proliferation of human glioma cell line A172, human intestinal adenocarcinoma cell lines A549 and H460, human hepatoma carcinoma cell HepG2 was investigated in vitro by MTT assay、Hoechst fluorescent staining、Annexin V /PI fluorescent staining and FCS Technique. The results show that SFN can induce A172 cells apoptosis and SFN-mediated apoptosis depend on dose and time. In vitro experiments with SFN Simultaneously indicated a pronounced role for cell cycle arrest in its anti-cancer properties; G2/M arrest is the predominant stage of cell cycle arrest induced by SFN which indicated that SFN could inhibit cell microtube formation. Apoptosis and cell cycle arrest both contribute to the cell proliferation inhibition and the latter is the dominance mechanism for cell line A172. to contrast with cell line A172, sulforaphene concentration for cell H460 proliferation inhibition are higher than it's concentration for cell line A172, and the chief mechanism for proliferation inhibition is due to apoptosis. So the mechanisms of the proliferation inhibition are different with regard to different tumor cell lines. And it's essential to approach the mechanisms for tumor cell line when we do rearch for drug anti-tumor experiment.The proliferation inhibition evoked by SFN was also observed in tumor cell lines A549 and HepG2. Combined with the results about proliferation inhibition of SFN on tumor cell lines HeLa, HL-60, CNE, PC3 and P388 which were got by author laboratory by MTT method, the conclusion was that SFN had anti-tumor activity and can act via several mechanisms to suppress cancer progression, such as modulate cell growth and cell death signals. In conclution, SFN has a promising potential as a new anti-tumor drug.
Myrosinases, thioglucoside glucohydrolases (EC 3.2.3.1) are isoenzymes present in most organs of Brassicaceae species. They catalyse the degradation of glucosinolates, a group of low molecular weight compounds to form an aglucone and D-glucose. The aglucone is unstable and spontaneously decomposes into nitriles, thiocyanates, isothiocyanates or indoles depending on the nature of the side chain and the reaction conditions. In some Brassica vegetables such as cabbage, cauliflower, brussels sprouts and broccoli, glucosinolate degradation products, especially isothiocyanates have been shown to have anticarcinogenic properties. Myrosinase-glucosinolate system in plant is also an important prevention mechanism for responding to the attacks by herbivores, entomologica, pathogenic microorganisms et al. All plant myrosinases characterized to date are glycosylated and are probably transported via the secretory pathway to the myrosin grains present in idioblasts called myrosin cells. Only when the plant tissues are damaged by physical or biological factors and the physical separation is disappeared, glucosinolates can contact with myrosinases and be hydrolyzed by myrosinases.
Myrosinases have been well characterized in biochemical and molecular biological studies. Myrosinase genes have been cloned fr om oilseed rape (B. napus), turnip (B. campestris), leaf mustard (B. juncea), white mustard (Sinapis alba), and thale cress (Arabidopsis thaliana) and classified into four subtypes, MA, MB, MC and TGG, on the basis of amino acid sequences. Studies of myrosinases in B. napus and Sinapis alba showed that they are encoded by large gene families, with up to 15-20 genes in B.napus. MB showed dominant expression in seeds, seedlings, young leaves, and other organs in the mature plant, whereas MA and MC were expressed only in developing seeds. Few reports were about the Myrosinase genes from Broccoli (Brassica oleracea var. italica). In this paper, a full-length cDNA encoding myrosinase from Broccoli are cloned and characterized. And the gene is further fusion expressed in E.coli BL21 and secretory expressed in Pichia pastoris. Moreover, fermentation research of the recombinant yeast for myrosinase secretion was carried on in the last chapter.
Based on the reported plant myrosinase genes in GenBank, software Primer Premier 5 was used to design degenerate primers MyrF and MyrR for amplification gene consensus regions. And then, using 3'RACE and the corresponding 5'RACE technique, the full-length cDNA of Broccoli homologous gene of Brassicaceae myrosinase (BoMyr2, GenBank number: EU004075) was obtained. The 1830bp cDNA contained an ORF of 1641bp, which coded for a polypeptide of 548 amino acids. The deduced polypeptide had a predicted molecular weight of 62.2kD, a pI of 8.71. And The predicted amino acid sequence include a signal peptide, nine N-glycosylation sites and a characteristic domain of the glycosyl hydrolases family 1 which include N-terminal signature(FiFGvAsSAYQiEgG) and active site(IYITENGFS). Homology model of the myrosinase from Broccoli was obtained with MOE on the base of the crystal structure of myrosinase from S. alba as template. Putative three-dimensional structures of the myrosinase showed a (β/α)8 barrel structure common to other glucosidases. Neighbor-joining tree constructed by MEGA 3.1 showed that BoMyr2 was belonged to myrosinase MB subfamily.
The codon region of BoMyr2 was amplified using designed primers based on the RACE results and inserted into E.coli expression vector pGEX-4T-1.The recombinant plasmid was transformed into E.coli BL12 to produce expression strain which would express GST-myrosinase fusion protein. SDS-PAGE showed that the recombinant protein was highly expressed at 90KDa which is coincided with the designed protein. The protein was accumulated in the E.coli BL12 cells by IPTG induced. The product of fusion protein was fairly well after four hours IPTG induced and reached the maxium after six hours IPTG induced. Enzyme activity analysis found that the fusion protein was lack of myrosinase activity. This phenomenon maybe due to the fusion protein was deficient in modification or had a GST addition. The fusion protein presented mostly as inclusion body would also lead to enzyme deficient. So the further investigation, such as protein degeneration and regeneration, enzyme cleavage, is necessary for the aim to get enzymatic protein.
Primary myrosinase has a signal peptide using different SignalP prediction models predicted. And all mature myrosinase which three dimension structure are mentioned and published in PDB database are glycosylated. Pichia pastoris expression system has glycosylation and other modification after protein translated. So it was also used to express recombinant myrosinase BoMyr2 in this paper. The interested BoMyr2 gene was got by digesting the pMD-18-BoMyr2 vector using restriction endonuclease, and then it was inserted into the secretory pPIC9K Pichia pastoris expression vector and transformed into E.coli. Positive recombinant plasmids were selected、sequenced and named pPIC9K-BoMyr2 and was linearized by Sac I, then the linear DNA transferred into Pichia pastoris KM71 by electroporation. The recombinant expression vector pPIC9K-BoMyr2 integrated into KM71 via homologous recombination between the transforming DNA and regions of homology within yeast genome. The positive transformants were screened by MD His- plates and colony PCR. The recombinants were induced expression with methanol and the expression products were tested by SDS-PAGE and enzymatic activity. The results showed that the BoMyr2 gene had been expressed successfully in Pichia pastoris and secreted in the culture medium. The expression product is about a 65 kDa protein and can degrade sinigrin in suitable condition. Six strains were selected in the experiment. Myrosinase activities in the culture supernatant of number 13 # and 15# reached 1.7-1.9U/ml. Determination of the special activity of myrosinase reflected that protein expression levels and enzyme activity had good correlation in culture supernatant. This is the first report on successful heterologous expression of a myrosinase in Pichia pastoris KM71 and provides an important tool for, e.g., further characterization of myrosinase by site-directed mutagenesis and for studying the interaction between myrosinase and myrosinase-binding proteins, myrosinase-associated proteins, and epithiospecifier proteins.
Further fermentation research on number 13# strain were carried out using single factor analysis. The result show that the optimum pH and methanol concentration were respectively as 6.0 and 1.0% for myrosinase expression in BMMY medium.0.05% oleic acid or 0.1% tween-20 can also enhance myrosinase expression in small scale. Opportunity for recovering the fermentation culture was 36 hours after methanol inducing began. And myrosinase activity in the supernatant would come up to 3U/ml or so.
The free, soluble form of myrosinase of the MA gene family has so far been intensively analyzed. In contrast, only limited information is available about the biochemical properties of the myrosinases encoded by the MB gene family for the reason of its low-level exression and complex form. the successful heterologous expression of recombinant BoMyr2 myrosinase from Broccoli in the yeast Pichia pastoris provides a new feasible procedure to produce free myrosinase homogeneity, facilitates studies on structure and function of the enzyme and will be a key for determining the consequences of complex formation with, e.g., myrosinase-binding proteins and myrosinaseassociated proteins, including their effection on myrosinase complex activity.
引文
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