猪生产激素基因在巴斯德毕赤酵母中的高效分泌表达及产物的N—糖基化分析

利用含有强启动子PAOX1和α-MF信号肽序列的巴斯德毕赤酵母载体质粒pPICZαA构建出含PST基因的重复组质粒pPICZαA－pST。通过电击将经SacI酶后线化的pPICZαA－pST质粒转化到巴斯德毕赤酵母X－33菌中,并筛选Mut^ 表型的重型的生组菌。表达产物的SDS－PAGE和Western blot结果表明,分泌于胞外的PST蛋白分子量比天然PST分子量稍大,而胞内的PST蛋白分子量与天然PST大小相同,将经SacI酶切后线性化的pPICZαA－pST再次转化重组酵母细胞X－33／pPICZαA－pST(Mut^ ）,所得表达产物的SDS－PAGE和Western blot结果显示,PST基因的表达水平明显提高,且表达产生的蛋白均可发生正确的抗原－抗体结合反应,表达量达956mg/L。将发酵液上清进行N－糖基化分析,显示rPST无N－糖基化加工修饰。     

猪生长激素基因在巴斯德毕赤酵母中的高效分泌表达及产物的N-糖基化分析

利用含有强启动子P_AOX1 和α-MF信号肽序列的巴斯德毕赤酵母载体质粒pPICZαA构建出含PST基因的重组质粒pPICZαA pST。通过电击将经SacⅠ酶切后线性化的pPICZαA pST质粒转化到巴斯德毕赤酵母X 33菌中 ,并筛选Mut+ 表型的重组菌。表达产物的SDS PAGE和Westernblot结果表明 ,分泌于胞外的PST蛋白分子量比天然PST分子量稍大 ,而胞内的PST蛋白分子量与天然PST大小相同。将经SacⅠ酶切后线性化的pPICZαA pST再次转化重组酵母细胞X 33 pPICZαA pST(Mut⁺) ,所得表达产物的SDS PAGE和Westernblot结果显示 ,PST基因的表达水平明显提高 ,且表达产生的蛋白均可发生正确的抗原 抗体结合反应 ,表达量达 95 6mg L。将发酵液上清进行N 糖基化分析 ,显示rPST无N 糖基化加工修饰     

米曲霉木聚糖酶基因的克隆及其在毕赤酵母中的表达

目的:构建米曲霉木聚糖酶基因的真核表达载体,并转化巴斯德毕赤酵母,进行分泌表达。方法:以米曲霉总RNA为模板,根据已知的米曲霉木聚糖酶基因序列设计引物,采用RT-PCR技术克隆木聚糖酶基因cDNA序列,将其与pPIC9K质粒连接构建表达载体后转化毕赤酵母,经MM/MD快慢斑筛选,得到Muts型重组子,进行甲醇诱导表达。结果:克隆得到的cDNA序列全长666 bp,连续编码221个氨基酸;阳性克隆子在诱导培养数天后,将菌液点于RBB-木聚糖平板上,产生了明显的透明圈,表明重组木聚糖酶在毕赤酵母中获得表达。结论:木聚糖酶基因的真核表达载体构建成功,并能够在毕赤酵母中表达。     

嗜热毛壳菌CT2纤维二糖水解酶Ⅰ在毕赤酵母中的高效表达

嗜热毛壳菌ChaetomiumthermophilumCT2可产生具有重要工业生产价值的纤维素酶类。RT-PCR扩增cbh1成熟蛋白的编码基因,利用基因重组的方法构建可在毕赤酵母分泌表达系统中表达纤维二糖水解酶的重组表达载体,并转化毕赤酵母得到重组子。在毕赤酵母醇氧化酶AOX1基因启动子的作用下,重组蛋白得到高效表达,小规模发酵量达1.42mg/mL。表达蛋白分泌到培养基中,分子量约80kD;以脱脂棉为底物测得酶活为21U/mL。表达蛋白在60℃稳定,70℃保温60分钟仍保持90%的酶活力,具有较高的热稳定性。     

GAPDH基因克隆及其作为毕赤酵母内标物的可靠性研究

为了测定GAPDH在巴斯德毕赤酵母中作为内参蛋白的有效性,利用PCR鉴定巴斯德毕赤酵母中GAPDH基因,分别在含葡萄糖、甲醇、甘油培养基及不同温度下培养巴斯德毕赤酵母,采用SDS-PAGE、Western blotting和催化活性检测。测序结果与已报道的巴斯德毕赤酵母GAPDH基因完全一致。SDS-PAGE结果显示,表达蛋白分子量为35.4 kD,与预期分子量相符。Western blotting和催化活性测定显示,在葡萄糖诱导下GAPDH表达最高,甲醇次之,甘油最低。37℃下GAPDH表达略高于28℃。在同一诱导物或温度下,GAPDH表达不会随浓度或培养时间发生明显变化。结果表明,在同一种诱导方式下,GAPDH可以作为异源蛋白表达的内参对照。     

巴斯德毕赤酵母表达系统研究进展

巴斯德毕赤酵母表达系统现在已经发展成为一种高效的外源蛋白基因优秀表达系统,该系统具有高表达、高稳定、高分泌、容易放大和成本低等优点,目前已有多种外源蛋白基因在该系统中实现高效表达,对巴斯德毕赤酵母表达系统的进一步研究将会促进其大规模的工业化应用。     

信号肽序列对毕赤酵母表达外源蛋白质的影响

乙醇氧化酶启动子被分离、克隆 ,并建立了转化方法后 ,毕赤酵母已被发展成为一种高效的外源蛋白表达宿主。为了进一步提高外源蛋白质的分泌表达 ,对信号肽序列进行了研究。首先按毕赤酵母的偏爱密码合成了酿酒酵母的α因子信号肽序列MF4I,随后在MF4I信号肽序列的N端分别引入 1～ 10个毕赤酵母Aox1蛋白质的N端氨基酸 ,构成 10种不同的分泌信号肽序列 ,10种不同的分泌信号肽序列被用于植酸酶基因的毕赤酵母分泌表达。以上新的信号肽序列都可使植酸酶的分泌表达量增加 ,而以N端增加A、I、P三个氨基酸的信号肽序列引起的提高最大 ;和野生型的酿酒酵母α因子信号肽序列相比 ,使植酸酶分泌表达量增加 5倍 ,摇瓶中植酸酶的分泌表达量为 90mg/L。此外在MF4I信号肽的引导序列和内切蛋白酶间增加了EEAEAEAEP和K共 10个氨基酸 ,进一步提高信号肽的分泌效率 ,使表达又提高约 35 % ,使得摇瓶中酸性植酸酶的表达量达到 12 0mg/L ,是pPCI9K表达量的 8倍。     

mNUDC基因在巴斯德毕赤酵母中表达载体的构建及表达

目的:探索小鼠核迁移蛋白C(mNUDC)在毕赤酵母分泌表达的方法.方法:应用PCR扩增本实验室所构建的重组质粒PET28b-his-mNUDC中的mNUDC基因,使用基因重组方法构建毕赤酵母真核表达栽体pPICZaA-his-mNUDC,电击转化酵母茵株KM71后,经摇瓶发酵和甲醇诱导,SDS-PAGE和Westernblot分析鉴定上清中重组mNUDC蛋白表达量.结果:经过PCR方法,有效扩增了mNUDC基因,构建了pPICZα A-his-mNUDC酵母表达质粒,序列分析表明所构建的含mNUDC基因的质粒与设计相同,mNUDC蛋白得到正确表达.使用SDS-PAGE和Western blot方法可以检测到mNUDC的稳定、高效地分泌表达.结论:成功地构建了mNUDC基因的毕赤酵母表达载体pPICZα A-his-mNUDC,并在毕赤酵母中实现分泌型离表达,为进一步研究mNUDC蛋白对小鼠的生物活性奠定了实验基础.     

改良毕赤酵母分泌表达外源蛋白能力的研究进展

巴斯德毕赤酵母(Pichia pastoris)由于能高效表达正确折叠加工的外源蛋白而成为目前最具应用前景的表达宿主.但随着对大量不同外源蛋白在毕赤酵母中分泌表达的研究发现,并不是所有蛋白均能高效分泌表达,这严重限制了毕赤酵母这一表达系统的推广应用.相关研究发现,外源蛋白在内质网中的聚集是限制酵母分泌表达外源蛋白的主要因素,因此近年来开始尝试通过基因操作改良毕赤酵母表达外源蛋白的能 力.本文综述了这一领域的研究进展.     

猪瘟病毒E2蛋白A/D抗原区基因在酵母中的分泌表达与鉴定

基于猪瘟病毒主要保护性抗原E2囊膜糖蛋白有两个相对独立的抗原结构单位-B/C抗原区和A/D抗原区,设计一对特异性的引物扩增猪瘟病毒E2蛋白的A/D抗原区基因,并将PCR产物克隆入含有强启动子PAox1和α-MF信号肽序列的巴斯德毕赤酵母表达载体pPICZαC中,构建成重组质粒pPICZα-AD,酶切线性化后电穿孔导入巴斯德毕赤酵母X33菌中,经ZeocinTM筛选得到5株高拷贝转化子,甲醇诱导表达.SDS-PAGE和Westernblot试验表明酵母培养上清液中含有具有良好反应原性的E2蛋白,蛋白表达量达175.8μg/mL.N-糖基化分析显示该表达蛋白在分泌过程中发生糖基化.该研究为研制防治猪瘟的亚单位疫苗与诊断试剂盒奠定基础.     

Molecular cloning and heterologous expression of an alkaline xylanase from Bacillus pumilus HBP8 in Pichia pastoris

The xynHB gene, encoding alkaline xylanase was cloned from Bacillus pumilus by a shot-gun method. The gene was cloned into vector pHBM905A, and expressed in Pichia pastoris GS115. Xylanase-secreting transformants were selected on plates containing RBB-xylan. Enzymatic activity in the culture supernatants was up to 644 U mL^-1 and the optimal secretion time was 4 days at 25°C. SDS-PAGE showed two bands, of 32.2 kDa and 29.6 kDa, both larger than the predicted mass of 22.4 kDa based on its amino acid sequence. Zymogram analysis demonstrated that the enzyme in both bands could hydrolyze xylan. Deglycosylation by endoglycosidase H revealed that both were derived from the same protein but contain different extents of glycosylation (30 and 25%). The optimal pH and temperature of the enzyme was pH6-9 and 50°C, respectively.     

Expression of a Bacillus subtilis pectate lyase gene in Pichia pastoris

The methylotrophic yeast Pichia pastoris is an attractive heterologous protein expression host, mainly for genes from higher eukaryotes. However, no successful examples for the expression of bacterial gene encoding pectate lyase in P. pastoris have been reported. The present study reports for the first time the cloning and functional expression of the bacterial Bacillus subtilis gene encoding alkaline pectate lyase in P. pastoris. A molecular weight of 43,644 Da was calculated from the deduced amino acid sequence. A pectate lyase activity as high as 100 U/ml was attained in the fermentation broth of P. pastoris GS 115, which was about 10 times higher than when the gene is expressed in Escherichia coli. The recombinant pectate lyase was purified to homogeneity and maximal activity of the enzyme was observed at 65 °C, and pH 9.4. The recombinant enzyme showed a wider pH and thermal stability spectrum than the purified pectate lyase from B. subtilis WSHB04-02. Pectate lyase activity slightly increased in the presence of Mg²⁺ (ion) but decreased in the presence of other metal ions. Analysis of polygalacturonic acid degradation products by electrospray ionization-mass spectrometry revealed that the degradation products were unsaturated trigalacturonic acid and unsaturated bigalacturonic acid, which confirms that the enzyme catalyzes a trans-elimination reaction.     

毕赤酵母底盘芳香族氨基酸合成途径改造生产肉桂酸及对香豆酸*

目的:改造毕赤酵母使其异源合成类黄酮生物合成途径的重要中间体肉桂酸、对香豆酸,并优化前体芳香族氨基酸生物合成途径以提高毕赤酵母的生产能力。方法:在毕赤酵母GS115中利用乙醇诱导型人工转录系统表达Rhodotorula glutinis来源的苯丙氨酸解氨酶,并在该重组菌株中分别过表达胞内芳香族氨基酸生物合成途径中的关键酶或其突变体以进行优化。结果:异源表达苯丙氨酸解氨酶可使毕赤酵母将自身产生的L-苯丙氨酸、L-酪氨酸转化为肉桂酸(38.8 mg/L)、对香豆酸(34.2 mg/L),而通过过表达相关酶进行优化,最终肉桂酸和对香豆酸的产量分别达到124.1 mg/L和302.0 mg/L。结论:利用新的异源宿主毕赤酵母成功合成了肉桂酸、对香豆酸,并对胞内的芳香族氨基酸生物合成途径进行了优化,表明毕赤酵母具有生产黄酮类化合物的应用潜力,也为其他芳香族氨基酸衍生物或植物化合物在毕赤酵母中的异源合成奠定了基础。     

Two-step ion-exchange chromatographic purification of recombinant hirudin-II and its C-terminal-truncated derivatives expressed in Pichia pastoris

The scene of the protein micro-heterogeneity of recombinant hirudin-II (HV2) expressed in Pichia pastoris was investigated. It was shown that three derivatives of HV2 were present in the fermentation broth of P. pastoris, which were intact HV2 and its two derivatives truncated the C-terminal amino acid residue Gln and Leu-Gln, respectively. To purify the minor degradation derivatives of HV2, a simple, biocompatible and scale-up-feasible purification process with two-step ion-exchange chromatography was established instead of usual reverse phase chromatography. The purities of end products were over 96% and the residual endotoxin less than 0.5 EU/ml.     

Inhibition of soil-borne plant pathogens by the treatment of sinigrin and myrosinases released from reconstructed Escherichia coli and Pichia pastoris

Myrosinases (thioglucoside glucohydrolase, EC 3.2.3.1) are able to hydrolyse glucosinolates in natural plant products. In Arabidopsis thaliana three different genes with different tissue-specific expressions and distribution patterns encode myrosinases. cDNAs of myrosinase genes (TGG1 and TGG2) were isolated from A. thaliana and expressed in Escherichia coli and Pichia pastoris. The enzyme activities of myrosinase TGG1 and TGG2 genes expressed in P. pastoris were higher than those expressed in E. coli. Among six glucosinolates tested for specificity to myrosinases TGG1 and TGG2, the suitable substrates for these two genes expressed in P. pastoris and E. coli were sinigrin, gluconapin, glucobrassicanapin and glucoraphanin. Treatment of sinigrin with myrosinases excreted from reconstructed E. coli and P. pastoris with TGG1 and TGG2 genes showed strong fungicidal effects on mycelial growth of Rhizoctonia solani AG-4, Sclerotium rolfsii, and Pythium aphanidermatum. This study suggests that the combination of glucosinolate with myrosinases excreted from the reconstructed microbes may be of potential for control of soil-borne diseases.     

重组猪胰蛋白酶及其R122位点突变体在毕赤酵母中的高效表达及其性质研究

目的:研究R122位点突变重组猪胰蛋白酶,与野生型酶相比较,该位点对重组猪胰蛋白酶(RPT)性质的影响。方法:以毕赤酵母GS115作为表达宿主,对RPT、突变体mRPT(R122H)和 mRPT(R122H/R73G/R130T)进行表达及纯化。并对其性质和稳定进行对比研究。结果:重组胰蛋白酶及其突变体在毕赤酵母中均获得了高效表达。相对于RPT,突变体mRPT(R122H)和 mRPT(R122H/R73G/R130T)在以N-苯甲酰-L-精氨酸乙酯 (BAEE)为底物时,具有更强底物结合力,三者的米氏常数分别为18.8μmol/L、9.0μmol/L和11.0μmol/L;两突变体耐高温耐碱能力增强;在Ca ²⁺存在及去除的条件下,突变体具有更强的抗自降解能力。 结论:可以利用毕赤酵母高效表达重组胰蛋白酶及其突变体。mRPT(R122H)和mRPT(R122H/R73G/R130T) 相对于野生型RPT,对高pH条件和高温的耐受性增强,该稳定性的提高主要归因于R122位点的突变。     

功能性新型冠状病毒RBD结构域在毕赤酵母表面的展示*

目的:设计并构建新型冠状病毒(SARS-CoV-2)受体结合结构域(receptor binding domain,RBD)在毕赤酵母表面的展示体系,并对表面展示的RBD进行功能性评价,从而为以RBD为靶点的高通量药物筛选平台奠定基础。方法:将四种锚定分子与新冠病毒RBD融合,电转化至毕赤酵母中;通过细胞免疫荧光分析,筛选能够成功展示RBD的锚定系统;进一步分析其与血管紧张素转化酶2(angiotensin-converting enzyme 2,ACE2)受体的亲和力,证明展示在细胞表面RBD分子的功能。结果:仅Sed1p锚定分子能够有效呈递RBD至毕赤酵母细胞表面,展示效率约为70%;亲和力分析结果表明,ACE2受体和表面展示RBD的亲和力(K_D = 30.42 nmol/L)与溶液中RBD的亲和力(K_D = 16.00 nmol/L)较为接近。结论:这一体系能够在毕赤酵母表面高效地展示具有生物学功能的RBD,可用于抗新冠病毒RBD药物的高通量筛选和评价。     

基于重组毕赤酵母的fusaruside生物合成

目的:构建产fusaruside的毕赤酵母菌株,解决天然小分子免疫抑制剂fusaruside的来源问题。方法:从禾谷镰刀菌Fusarium graminearum PH-1中扩增获得合成fusaruside的相关基因-3位去饱和酶[Δ3(E)-SD]和10位去饱和酶[Δ10(E)-SD]基因;并通过2A肽策略构建两种基因的共表达载体,转化到毕赤酵母GS115中进行双酶的诱导表达;对诱导后的毕赤酵母菌体进行甲醇和二氯甲烷的处理后,经高效液相色谱质谱联用仪(HPLC-MS)检测其中产物变化。结果:3位去饱和酶和10位去饱和酶在毕赤酵母中成功共表达,SDS-PAGE显示3位去饱和酶分子量约为48kDa,10位去饱和酶分子量约为65kDa; HPLC-MS显示重组酵母可以产生fusaruside。结论:与fusaruside原产菌株镰刀菌相比,该酵母菌的发酵时间更短、产量更高,为fusaruside的进一步开发与应用奠定基础。     

Production and purification of a novel thermostable phytase by Pichia pastoris FPHY34

A genetically engineered Pichia pastoris FPHY34 strain containing a 1.3 kb thermostable phytase gene (fphy) evolved by DNA shuffling was constructed and screened. Expression and purification conditions for the recombinant phytase were developed in this study. The effect of Pi on recombinant phytase expression and cell growth of P. pastoris FPHY34 was tested in shake flask culture. Optimization of carbon sources for cell growth and methanol feeding strategies for phytase expression in P. pastoris FPHY34 was carried out in a 50-L fermenter by fed-batch fermentation. The purification of phytase was investigated by micro-filtration and ultra-filtration followed by desalting, ion-exchange chromatography, and gel filtration in the ÄKTA system. It showed that the optimum inorganic phosphorus is 13.6 g L⁻¹ and that glucose can be used as a substrate for P. pastoris cell growth instead of glycerol; the biomass yield of glycerol (Y_X/S) is slightly higher than that of glucose. Different profiles of lag phase and respiratory quotient (RQ) displayed between glucose and glycerol as the sole carbon source. The maximum phytase activity in per millimetre reached 2508 U mL⁻¹ at a methanol feed rate of 3.0 mL L⁻¹ h⁻¹ after 80 h period of induction. A purification factor of 41.1 with a 32% yield was achieved after chromatographic purification. The specific enzyme activity was 80 U mg⁻¹ and 3281 U mg⁻¹ in that supernatant fraction and after gel filtration purification, respectively. The strain P. pastoris FPHY34 showed a promising application in phytase industrial production.     

Production and secretion of biologically active recombinant canine growth hormone by Pichia pastoris

Production of recombinant canine (Canis familiaris) growth hormone (rCFGH) by two expression systems, methanol utilization slow (Mut^s) and methanol utilization plus (Mut⁺) based on Pichia pastoris. Led by the Saccharomyces cerevisiae -mating type signal sequence (SS), the hormone was secreted into the culture medium in its mature and active form. The level of total proteins secreted into the medium achieved at 25 ml working volume using Erlenmeyer flasks was approximately 40 and 15 μg/ml for Mut^s and Mut⁺ constructs, respectively. As judged by densitometry of proteins resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), the hormone produced by the fermented Mut^s strain upon induction with methanol reached 24 μg/ml, representing around 60% of the total secreted proteins and being eight times more abundant than in its Mut⁺ counterpart. Finally, the recombinant hormone showed activity when tested in the Nb2 cell proliferation assay.