DSPAα1缺失突变体在毕赤酵母中的表达及活性研究

吸血蝙蝠唾液纤溶酶原激活剂(Desmodus salivary plasminogen activators,DSPAs)共有4种:DSPAα1、α2、β及γ。其中,DSPAα含有指形区(F)、表皮生长因子区(E)、kringle区(K)和丝氨酸蛋白酶区(P),DSPAβ含E、K、P区,DSPAγ含K和P区。以DSPAα1为基础,研究其结构对纤溶活性的影响。采用重叠延伸PCR技术(splicing overlap extension PCR,SOE-PCR)获得缺失E区的DSPAα1突变体(m DSPAα1),分别构建m DSPAα1/p PIC9K、DSPAβ/p PIC9K和DSPAγ/p PIC9K重组质粒并转化到巴斯德毕赤酵母(Pichia pastoris)菌株GS115中表达,纤维平板法测活。结果显示未检测到DSPAγ的表达,DSPAα1活性为2.64×105 U/mg,DSPAβ的活性为1.32×104 U/mg,m DSPAα1活性为151.52 U/mg;同时使用DSPAα1、m DSPAα1、DSPAβ时,总活性几乎不受影响,单独使用任意两者时,发现活性均降低2-3倍。m DSPAα1纤溶活性几乎没有,同时DSPAβ与野生型DSPAα1相比保留了相当的催化活性。DSPAα1的N端区域可以影响其溶栓活性,而缺失E区后几乎丧失活性,说明E区在DSPAα1溶栓过程中起着至关重要的作用。     

表达食血蝙蝠唾液纤溶酶原激活剂的毕赤酵母菌株的构建

食血蝙蝠(Desmodus rotundus)在吸食大型动物血液时能保持创口处血流的畅通。本研究根据食血蝙蝠唾液中发现的纤溶酶原激活剂(Bat-PA,另称DSPAα1)的全长基因序列(GenBank Accession No.J05082),首次在体外人工合成Bat-PA全长基因并亚克隆到巴斯德毕赤酵母(Pichia pastoris)表达载体pPIC9K上;转化毕赤酵母菌株GS115后,经甲醇诱导获得DSPAα1的分泌型表达,表达条带为47kD;通过抗G418浓度梯度筛选、毕赤酵母中小量表达后SDS-PAGE电泳检测以及纤维平板法测活,筛选出高表达的DSPAα1稳定菌株,对电泳中的目的蛋白条带进行密度扫描测定,产量达到约30mg/L。目前,DSPAα1主要来自哺乳动物细胞,如中国仓鼠卵巢细胞(CHO)、胚胎卵巢肾细胞(BHK)、非洲绿猴肾细胞(COS),代价高昂。在毕赤酵母中生产DSPAα1可以降低成本,增加产率,为探索Bat-PA作为新一代溶栓剂奠定基础。     

鸡α干扰素在毕赤酵母中的表达及糖基化对其表达的影响

目的:实现鸡α干扰素(ChIFNα)在毕赤酵母GS115中的表达,并考察糖基化对其表达的影响。方法:人工合成按照毕赤酵母偏好密码子优化的ChIFNα基因,克隆至分泌表达载体pPIC9,电转到毕赤酵母GS115中进行表达。利用定点突变对ChIFNα基因序列中4个糖基化位点进行缺失,SDS-PAGE分析N-糖基化对毕赤酵母表达ChIFNα的影响。结果:ChIFNα在GS115中获得了表达,在摇瓶发酵条件下,表达量为35.2 mg/L;构建了缺失1~4个糖基化位点的4个突变体,在GS115中实现了表达,分析表达结果显示,与未突变的ChIFNα对照相比,突变体的糖基化程度和表达量都有大幅度下降。结论:N-糖基化对于毕赤酵母表达ChIFNα具有重要影响,无糖基化的ChIFNα在毕赤酵母中表达量极低。     

短小芽孢杆菌木聚糖酶基因在毕赤酶母中的分泌表达及酶学性质研究

将短小芽孢杆菌HB030的内切-1,4-木聚糖酶基因克隆到毕赤酵母表达载体pPIC9K,得到重组质粒pH-BM220,将pHBM220经酶切后分别转化三株毕赤酵母KM71、GS115、SMD1168,该木聚糖酶基因在三株毕赤酵母中均实现了分泌表达,将重组毕赤酵母KM71（pHBM220）,GS115（pHBM220),GS115(pHBM220),SMD1168（pHBM220）分别诱导产酶,对重组酶进行相关的酶学性质分析表明,三的最适反应pH值约为5.5,最适反应温度约为60℃,在其最适反应条件下测得三粗酶液酶活分别为10.80IU/mL,11.63IU/mL,9.68IU/mL,重组毕赤酵母KM71（pHBM220）所产酶的热稳定性较好,而在pH稳定性方面三没有太大的差异。     

短小芽孢杆菌木聚糖酶基因在毕赤酵母中的分泌表达及酶学性质研究

将短小芽孢杆菌HB030的内切-1,4-木聚糖酶基因克隆到毕赤酵母表达载体pPIC9k上,得到重组质粒pHBM220,将pHBM220经酶切后分别转化三株毕赤酵母KM71、GS115、SMD1168,该木聚糖酶基因在三株毕赤酵母中均实现了分泌表达。将重组毕赤酵母KM71(pHBM220)、GS115(pHBM220)、SMD1168(pHBM220)分别诱导产酶,对重组酶进行相关的酶学性质分析表明,三者的最适反应pH值约为5.5,最适反应温度约为60℃。在其最适反应条件下测得三者粗酶液酶活分别为10.80IU/mL,11.63IU/mL,9.68IU/mL。重组毕赤酵母KM71(pHBM220)所产酶的热稳定性较好,而在pH稳定性方面三者没有太大的差异。     

乙醛脱氢酶2基因在毕赤酵母中的表达和分离纯化

将乙醛脱氢酶2（ALDH2）基因整合到质粒pPIC9K上,构建重组表达载体pPIC9K-coALDH2,用电转导将表达质粒pPIC9K-coALDH2转化至毕赤酵母GS115中,在毕赤酵母中表达经密码子改造的ALDH2。结果表明：重组基因工程菌GS115（pPIC9K-coALDH2）发酵液中蛋白质量浓度为8.40 mg/L,1 mL发酵液中酶活为11.35 mU。     

毕赤酵母菌高效表达乙肝表面抗原的研究

目的:筛选高效表达HBsAg的毕赤酵母茵,制备目的蛋白.方法:从已确诊的乙肝病人血清中提取DNA,PCR扩增HBVS基因,将其分别克隆入毕赤酵母胞内表达栽体pPICZA中.构建重组质粒pPICZA-S和pPICZA-SH,经Sac I线性化后,LiCI化学法转化入酵母茵株GS115、X-33、KM71H和SMD1168.结果:诱导表达后的GS115工程茼单位体积的培养基所得的抗原含量最高,诱导培养基中加入0.1%酪蛋氨基酸后,可抑制目的蛋白的水解,有利于目的蛋白的表达,粗略估算表达量为15.3mg/L,最佳收获时间为72 h.结论:经SDS-PAGE和Westcrn-blot分析表明,所得产物为乙肝表面抗原S蛋白.     

水稻白叶枯病抗性基因Xα21和稻瘟病抗性基因Pi-d2激酶蛋白质在毕赤酵母中的表达及条件优化

[目的]白叶枯病和稻瘟病是最主要的水稻病害,Xα21是水稻白叶枯病抗性基因,Pi-d2是稻瘟病抗性基因,二者都编码类受体激酶蛋白质.本研究旨在毕赤酵母系统中表达XA21和PI-D2激酶蛋白质.[方法]用Xα21和Pi-d2的激酶区PCR产物,构建了pPICZαA-Xα21K、pPICZαA-Pi-d2K重组质粒,酶切及测序验证后,将重组质粒线性化,转化到毕赤酵母菌株中,系统地比较了不同酵母菌株(KM71、GS115、X33),不同甲醇浓度(1%、2%、3%),不同pH(pH5、pH6、pH7、pH8)值,不同诱导时间(24 h、48 h、72 h)条件下激酶蛋白质的表达情况.[结果]XA21和PI-D2激酶蛋白质可以在毕赤酵母中表达,但表达的蛋白质不能分泌到培养基上清中,而只能在菌体中检测到,对表达条件的系统比较发现,毕赤酵母菌株KM71和X33、2%的甲醇诱导浓度、pH5和48 h以上的诱导时间有利于激酶蛋白质的表达,最后我们在酵母裂解物上清中获得了纯化的考染可见的激酶蛋白质.[结论]在毕赤酵母中表达了XA21和PI-D2激酶蛋白质,为下一步生化特性研究奠定了基础.     

水稻白叶枯病抗性基因Xa21和稻瘟病抗性基因Pi-d2激酶蛋白质在毕赤酵母中的表达及条件优化

【目的】白叶枯病和稻瘟病是最主要的水稻病害,Xa21是水稻白叶枯病抗性基因,Pi-d2是稻瘟病抗性基因,二者都编码类受体激酶蛋白质。本研究旨在毕赤酵母系统中表达XA21和PI-D2激酶蛋白质。【方法】用Xa21和Pi-d2的激酶区PCR产物,构建了pPICZαA-Xa21K、pPICZαA-Pi-d2K重组质粒,酶切及测序验证后,将重组质粒线性化,转化到毕赤酵母菌株中,系统地比较了不同酵母菌株(KM71、GS115、X33),不同甲醇浓度(1%、2%、3%),不同pH(pH5、pH6、pH7、pH8)值,不同诱导时间(24h、48h、72h)条件下激酶蛋白质的表达情况。【结果】XA21和PI-D2激酶蛋白质可以在毕赤酵母中表达,但表达的蛋白质不能分泌到培养基上清中,而只能在菌体中检测到,对表达条件的系统比较发现,毕赤酵母菌株KM71和X33、2%的甲醇诱导浓度、pH5和48h以上的诱导时间有利于激酶蛋白质的表达,最后我们在酵母裂解物上清中获得了纯化的考染可见的激酶蛋白质。【结论】在毕赤酵母中表达了XA21和PI-D2激酶蛋白质,为下一步生化特性研究奠定了基础。     

玫瑰微球菌中treZ基因在毕赤酵母中的表达研究

首次将玫瑰微球菌中麦芽寡糖基海藻糖水解酶(MTHase)treZ基因序列连接到表达载体pPICZαA中,通过电转化法将构建好的表达载体分别转入巴斯德毕赤酵母GS115和KM71菌株中.利用含有Zeocin的YPD平板筛选到阳性转化子,并经PCR、SDS-PAGE电泳以及Western blot最终验证海藻糖水解酶基因treZ已经整合到巴斯德毕赤酵母的基因组上,并且得到了预期的表达.     

Secretion and N-linked glycosylation are required for prostatic acid phosphatase catalytic and antinociceptive activity

Secretory human prostatic acid phosphatase (hPAP) is glycosylated at three asparagine residues (N62, N188, N301) and has potent antinociceptive effects when administered to mice. Currently, it is unknown if these N-linked residues are required for hPAP protein stability and activity in vitro or in animal models of chronic pain. Here, we expressed wild-type hPAP and a series of Asn to Gln point mutations in the yeast Pichia pastoris X33 then analyzed protein levels and enzyme activity in cell lysates and in conditioned media. Pichia secreted wild-type recombinant (r)-hPAP into the media (6-7 mg protein/L). This protein was as active as native hPAP in biochemical assays and in mouse models of inflammatory pain and neuropathic pain. In contrast, the N62Q and N188Q single mutants and the N62Q, N188Q double mutant were expressed at lower levels and were less active than wild-type r-hPAP. The purified N62Q, N188Q double mutant protein was also 1.9 fold less active in vivo. The N301Q mutant was not expressed, suggesting a critical role for this residue in protein stability. To explicitly test the importance of secretion, a construct lacking the signal peptide of hPAP was expressed in Pichia and assayed. This "cellular" construct was not expressed at levels detectable by western blotting. Taken together, these data indicate that secretion and post-translational carbohydrate modifications are required for PAP protein stability and catalytic activity. Moreover, our findings indicate that recombinant hPAP can be produced in Pichia--a yeast strain that is used to generate biologics for therapeutic purposes.     

Site-specific glycosylation at Asn-292 in ovalbumin is essential to efficient secretion in yeast

Chicken ovalbumin (OVA) exists as mono-N-glycosylated form with a carbohydrate chain on Asn-292 in egg white, despite the possession of two potential N-glycosylation sites. To investigate the roles of N-glycosylation of OVA, we constructed a series of N-glycosylation mutants deleted N-glycosylation site and compared the secretion level of the mutants in Pichia pastoris. N292Q and N292/311Q mutants resulted in greater lowering of the secretion level as compared with wild-type, whereas N311Q mutant was secreted in approximately equal amounts to wild-type. However, secretion of wild-type and N311Q mutant was inhibited completely by tunicamycin treatment. All the N-glycosylation mutants have been expressed in the cells, as well as wild-type. Circular dichroism and fluorescence spectra of secreted N311Q mutant were almost identical to those of wild-type, while those of N292Q and N292/311Q mutants were different from wild-type; and, N292Q and N292/311Q mutants showed considerably lower denaturation temperature than wild-type. The results indicate that N-glycosylation at Asn-292 of OVA is required for the folding and secretion.     

Heterologous expression of wild type and deglycosylated human sex steroid-binding protein (SBP or SHBG) in the yeast, Pichia pastoris. Characterization of the recombinant proteins.

Wild type, partially and fully-deglycosylated human sex steroid-binding protein (SBP or SHBG) cDNAs lacking the native cucaryotic signal sequence were cloned into a yeast expression vector containing the Saccharomyces cerevisiae alpha-factor for extracellular secretion. Following transformation into Pichia pastoris, the wild type and all constructed mutants were successfully expressed. The levels were lower for the deglycosylated mutants indicating that oligosaccharide side chains may play a role in SBP secretion. Under fermentation conditions, the wild type protein was expressed at a level of 4 mg/l while the fully-deglycosylated mutant T7A/N351Q/N367Q was expressed at about 1.5 mg/l. The latter was purified from several fermentation runs and was found to be completely deglycosylated, electrophoretically homogeneous and fully active. The aminoterminus was found to have the sequence NH2QSAHDPPAV- indicating that cleavage of the alpha-factor occurred at the A(+7)-Q(+8) peptide bond. The molecular mass of the subunit was determined to be 39,717.8 Da, which is in complete agreement with the amino acid sequence of the T7A/N351Q/N367/Q mutant. The equilibrium constants for the dissociation of 5alpha-dihydrotestosterone and steroid binding specificity were found to be identical to that of the human plasma protein indicating that the missing N-terminal segment NH2-LRPVLPT and the removal of oligosaccharide side chains do not affect the stability and active conformation of the protein. In conclusion, the data presented reveal that the SBP mutant T7A/N351Q/N367/Q is the protein of choice for solving the three-dimensional structure.     

Identification,purification, and pharmacological activity analysis of Desmodus rotundus salivary plasminogen activator alpha1 (DSPAα1) expressed in transgenic rabbit mammary glands

Desmodus rotundus plasminogen activator alpha 1(DSPAα1) is a thrombolytic protein with advantages, such as a long half-life, high accuracy and specificity for thrombolysis, wide therapeutic window, and no neurotoxicity. To date, DSPAα1 has only been expressed in the Chinese hamster ovary, insect cells, transgenic tobacco plants, and Pichia pastoris. To the best of our knowledge, we are the first to report the expression of DSPAα1 in transgenic rabbit mammary glands, extract the product, and analyze its pharmacology activity. An efficient mammary gland-specific expression vector pCL25/DSPAα1 was transferred to prokaryotic zygotes in rabbits by microinjection to generate six DSPAα1 transgenic rabbits. The recombinant DSPAα1 (rDSPAα1) expression in transgenic rabbit milk was 1.19?±?0.26 mg/mL. The rDSPAα1 purification protocol included pretreatment, ammonium sulfate precipitation, benzamidine affinity chromatography, cation exchange chromatography, and Cibacron blue affinity chromatography; approximately 98% purity was achieved using gel electrophoresis. According to sequencing results, the primary structure of rDSPAα1 was consistent with the theoretical design sequence, and its molecular weight was consistent with that of the natural protein. N-terminal sequencing results indicated rDSPAα1 to be a mature protein, as the goat signal peptide sequence of the expression vector was no longer detected. The fibrinolytic activity of rDSPAα1 was estimated to be 773,333 IU/mg. Fibrin-agarose plate assay and in vitro rat blood clot degradation assay showed that rDSPAα1 had strong thrombolytic activity. In conclusion, we report recombinant DSPAα1 with high thrombolytic activity expressed in transgenic rabbit mammary glands.

     

重组人干扰素ω在对数生长期毕赤酵母中的高效表达特性研究

用不同比生长速率μ的毕赤酵母探讨其表达外源重组蛋白的差异性,通过起始pH值、甲醇诱导浓度和周期、菌体浓度、装液量等实验,优化具有较高μ的对数生长期毕赤酵母表达rhIFNω的摇瓶条件。结果表明,μ对毕赤酵母表达rhIFNω有显著影响。μ为0.1612h-1的毕赤酵母表达rhIFNω最高为558mg/L,较μ为0.1321、0.0505和0.0052h-1的毕赤酵母分别提高50%、68%和99%。对数生长期的毕赤酵母表达rhIFNω的最适摇瓶表达条件为:250mL摇瓶装入30mL BMMY,控制菌体浓度达到200~300g/L(WCW),起始pH值自然,每24h添加甲醇15g/L一次,诱导表达周期为4d。通过表达条件的优化,rhIFNω的表达量达到1070mg/L,较优化前提高149%。     

人白细胞介素12基因在巴斯德毕赤酵母细胞中的表达(英文)

人白细胞介素 12 (hIL 12 )是人体内具有多种生物学活性的免疫调节因子 ,由p4 0和p35两个亚基经多对二硫键连接而成 .根据hIL 12的结构特点 ,采用LiCl二次转化将hIL 12的p4 0和p35两亚基基因导入巴斯德毕赤酵母X33细胞中 ,并经同源交换分别插入酵母基因组AOX1区域 ,构建成含hIL 12双亚基基因的酵母工程菌PichiapastorisX33 p4 0 p35 .经 0 .5 %甲醇诱导 ,p4 0和p35两亚基在同一酵母细胞中得到了表达 ,并组装成具有生物学活性的hIL 12p70分子     

Importance of N-glycosylation positioning for secretion and folding of ovalbumin

To investigate the role of the carbohydrate chain of hen egg ovalbumin (OVA), potential N-glycosylation site-deletion OVA mutants were expressed in yeast. The secretion level of the N292Q and N292/311Q mutants was greatly reduced compared with the wild-type OVA. Furthermore, secretion of the mutants without a carbohydrate chain on Asn-292 could hardly be detected in the culture medium, even if an additional N-glycosylation site was introduced to the OVA molecule. The reduction in secretion level seems to be due to incorrectly folded protein. Moreover, the secretion levels of the wild-type and N311Q mutant reduced in a similar extent as those of the mutants without a carbohydrate chain on Asn-292 in calnexin-disrupted yeast. These results indicate that the carbohydrate chain attached to Asn-292 of OVA has an important role for the secretion and folding in the cells.     

黑曲霉Aspergillus niger 963植酸酶基因phyA2 N-糖基化突变体的构建与表达分析

为研究N-糖基化对黑曲霉Aspergillus niger963植酸酶蛋白酶学性质的影响,利用Megaprimer PCR介导基因定点突变的技术,构建了植酸酶phyA2基因两个N-糖基化突变体,即将该基因编码蛋白质N87位和N102位的天冬酰胺密码子置换为编码与其具有相似结构的谷氨酰胺密码子,两个突变体分别命名为N87Q、N102Q,经测序结果比对和图谱分析,表明在核酸水平上成功实现了点突变,构建了酵母表达载体pPIC9-N87Q,pPIC9-N102Q,转化毕赤酵母GS115,经发酵罐水平诱导表达后,获得了N-糖基化缺失突变蛋白,对突变体蛋白在60℃进行处理发现,突变体N87Q处理1h后剩余50%的酶活,N102Q处理10min后酶活完全丧失,在37℃,不同的pH缓冲体系(pH1~10)处理1h,N87Q剩余约大于70%的活性,而N102Q在pH8的环境下,没有检测到酶活。     

人胰岛素原在甲醇酵母(Pichia pastoris)中的高效表达

甲醇营养型酵母Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ在近十几年已被人们广泛用作外源基因表达的系统。表达的是可溶性蛋白,且胞外分泌。本研究系将外源基因（胰岛素原基因）连接到穿梭质粒ＰＨＩＬ－Ｓ１上,再通过同源重组到酵母染色体,筛选表达株。用１ 升发酵罐在甲醇诱导下可获得０．３ｇ／Ｌ胰岛素原的产率。