基因重组Echistatin发酵工艺的优化

目的:利用基因工程方法对一种蛇毒锯鳞蝰素蛋白的发酵纯化工艺进行优化,以提高目的蛋白的产量和纯度。方法:对工程菌进行发酵培养并诱导表达,研究不同的培养基、不同补料方式、溶解氧浓度、培养和诱导时间对工程菌产量和目的蛋白表达量的影响,利用几丁质亲和层析纯化Ecs融合蛋白,通过合适温度和pH裂解融合蛋白得到Ecs纯品,并鉴定和检测Ecs活性。结果:经过高密度发酵优化后,菌体湿重可达110g/L,目的蛋白表达量约占总蛋白的40%;亲和层析纯化后,得到Ecs单体,得率为68mg/L发酵液。生物学活性分析显示,重组Ecs能有效抑制血小板的聚集,其活性与天然Ecs相似。结论:通过发酵和纯化工艺优化,大大提高了目的蛋白产量,为进一步规模化研究和生产奠定了基础。     

新型集成干扰素-人血清白蛋白融合蛋白在毕赤酵母中的分泌表达和纯化

将含编码HSA天然信号肽、HSA和新型集成干扰素(NIFN)的融合基因,插入毕赤酵母表达载体pPIC3.5,转染毕赤酵母GS115,用于分泌表达融合蛋白HSA-NIFN的酵母工程菌。诱导表达后,产物经SDS-PAGE、Western blot和MALDI-TOF-MS分析表明该蛋白分子量为86369Da,且能被抗HSA单抗特异性结合;表达的HSA-NIFN经超滤、Blue亲和层析和CM阳离子交换层析纯化后,HSA-NIFN的纯度大于95%。用细胞病变抑制法测定其比活性为7.75±0.39×106IU/mg。     

人胰高血糖素样肽-1与人血清白蛋白融合蛋白在毕赤酵母中的表达

目的：在巴斯德毕赤酵母中表达有降糖活性的人胰高血糖素样肽-1（hGLP-1）突变体（2Gly-hGLP-1）与人血清白蛋白（HSA）的融合蛋白。方法：为将GLP-1氨基酸序列第2位的丙氨酸（Ala）定点突变为甘氨酸（Gly）,根据毕赤酵母偏爱密码子合成编码2Gly-hGLP-1的基因;采用重叠PCR法拼接2Gly-hGLP-1和HSA的基因,使得2Gly-hGLP-1的C端与HSA的N端通过甘氨酸五肽接头连接;将该融合基因插入表达载体pPIC9构建为重组载体pPIC9/2Gly-hGLP-1-HSA,电击转化至毕赤酵母GS115细胞,通过表型筛选和诱导表达实验获得高效表达菌株;工程菌在5L发酵罐中培养后,对发酵产物进行分离纯化和生物学活性分析。结果：融合蛋白在5L发酵罐中的表达量约为200mg/L,经纯化后纯度可达95%以上;小鼠糖耐量实验表明该融合蛋白具有明显的控血糖活性。结论：在毕赤酵母中分泌表达的融合蛋白2Gly-hGLP-1-HSA具有降血糖活性。     

重组猪卵透明带抗原pZP3α在毕赤酵母中的分泌表达

为制备rpZP3α蛋白供发展避孕疫苗研究,将编码天然提取pZP3α上的DNA序列(446~1423)插入至毕赤酵母分泌型表达载体pPICZαA上,重组质粒pPICZαA-pZP3α线性化后通过电穿孔转入毕赤酵母GS115,经抗生素Zeocin筛选获得工程菌。在2L发酵罐中,用甲醇诱导工程菌进行高密度发酵生产rpZP3α。分离浓缩发酵上清液,通过螯合铜离子的亲和柱纯化rpZP3α,用SDS-PAGE和Western blot进行鉴定,以Quantity One软件对rpZP3α进行定量分析并计算纯度和回收率。用rpZP3α免疫家兔,以ELISA法和间接免疫荧光法检测抗血清对rpZP3α和猪卵透明带的抗体反应。获得了分泌表达rpZP3α的工程菌,其高密度发酵产物经分离纯化后获得能与抗pZP3抗体反应的46kD成分,命名为rpZP3α,平均产量为8mg/L,纯度达92%,回收率为63%。用其免疫家兔获得抗rpZP3α抗血清,ELISA测定显示能与rpZP3α和天然提取pZP3反应。间接免疫荧光法分析显示抗rpZP3α抗血清能与猪卵透明带反应,产生亮绿荧光。用酵母表达系统成功表达了rpZP3α,该蛋白保留有天然pZP3的免疫活性。     

人肝再生增强因子在毕赤酵母中的表达、纯化和生物学活性的研究

肝再生增强因子（ALR）是一类胞源性肝细胞生长因子。为在毕赤酵母中分泌表达人肝再生增强因子（rhALR）,以色谱法分离纯化后进行体外活性研究,构建表达载体pPICZαA- ALR,经电穿孔转入毕赤酵母中,用0.5％甲醇诱导表达;重组酵母培养上清经SDS-PAGE电泳和western blot鉴定后表明, rhALR以分子量为30kD的二聚体为主;定量分析结果表明,重组酵母培养上清中rhALR约占总蛋白的66％,表达量约为40mg/L;经DEAE柱和G75柱纯化后,获得的rhALR纯度大于95％,得率为52％;体外生物学活性实验表明,rhALR能明显促进HepG2、SMMC-7721和NIH-3T3细胞的增殖。     

重组猪卵透明带抗原pZP3a在毕赤酵母中的分泌表达

为制备rpZP3a蛋白供发展避孕疫苗研究,将编码天然提取pZP3a上的DNA序列（446—1423）插入至毕赤酵母分泌型表达载体pPICZaA上,重组质粒pPICZaA—pZP3a线性化后通过电穿孔转入毕赤酵母GS115,经抗生素Zeoein筛选获得工程菌。在2L发酵罐中,用甲醇诱导工程菌进行高密度发酵生产rpZP3a。分离浓缩发酵上清液,通过螯合铜离子的亲和柱纯化rpZP3a,用SDS-PAGE和Westernblot进行鉴定,以Quantity One软件对rpZP3a进行定量分析并计算纯度和回收率。用rpZP3a免疫家兔,以ELISA法和间接免疫荧光法检测抗血清对rpZP3a和猪卵透明带的抗体反应。获得了分泌表达rpZP3a的工程菌,其高密度发酵产物经分离纯化后获得能与抗pZP3抗体反应的46kD成分,命名为rpZP3a,平均产量为8mg／L,纯度达92％。回收率为63％。用其免疫家兔获得抗rpZP3a抗血清,ELISA测定显示能与rpZP3a和天然提取pZP3反应。间接免疫荧光法分析显示抗rpZP3a抗血清能与猪卵透明带反应,产生亮绿荧光。用酵母表达系统成功表达了rpZP3a,该蛋白保留有天然pZP3的免疫活性。     

甲醇酵母分泌表达重组人干扰素α1b纯化工艺的建立

建立适合大规模、低成本生产重组人干扰素α1b(Recombinanthumaninterferon α1b ,rhIFN α1b)的纯化工艺。采用高效分泌表达rhIFN α1b的甲醇酵母工程菌发酵 ,收集离心后的上清液 ,超滤脱盐 ,经离子交换柱和分子筛柱层析纯化。纯化的rhIFN α1b的纯度为 98%以上 ,比活性 2 .4× 10 7IU/mg ,活性回收率 14 % ,相对分子质量 1980 0和等电点 5 .0。经检测 ,rhIFN α1b蛋白N 端 15个氨基酸序列与正常对照完全符合。该纯化工艺简便 ,时程短 ,重复性好 ,适合于大规模生产     

人源性抗HBsAg Fab抗体的发酵生产研究

为了适应工业生产的需要,利用fed—batch方法,重组人源性抗HBsAg Fab抗体酵母工程菌在30L发酵罐中进行了高密度发酵,发酵最适温度30℃,pH值范围5．0～5．3,溶氧范围20％～30％。发酵液OD600值达到300时开始诱导,甲醇最佳诱导浓度为10mL／L。重组人源性抗HBsAg Fab抗体经离子交换层析纯化,纯化产品经SDS-PAGE、Western blot进行分析和ELISA方法进行活性测定。结果显示,重组Fab抗体在Fed-batch发酵系统中可高效表达,经过192h的发酵生产,重组人源性抗HBsAg Fab抗体的表达量可达412mg／L。发酵上清经过离子交换层析纯化,获得纯度为95％的重组Fab抗体,该Fab抗体经ELISA分析具有较高的HBsAg抗原亲和力和特异性。结果证实可以通过高密度发酵毕赤酵母工程菌来高效生产重组人源性抗HBsAg Fab抗体,为后续的工业化生产应用奠定了基础。     

纤维连接蛋白C端肝素结合域多肽在毕赤酵母中的表达、纯化及鉴定

为在毕赤酵母中表达纤维连接蛋白C端肝素结合域(Fibronectin C-terminal heparin-binding domainFNCHBD)多肽并研究其功能,通过PCR技术扩增FNCHBD目的基因,将目的基因与T载体连接,经测序正确后,插入pAo815SM酵母表达载体增加基因拷贝数,然后酶切克隆入酵母表达载pPIC9K;将重组质粒Sal I酶切线性化后转化毕赤酵母菌株,筛选工程菌,经甲醇诱导表达,用SDS-PAGE检测发酵上清液,表明有重组蛋白FNCHBD多肽的高表达,表达产物通过离心、超滤、离子交换层析纯化,纯化产物通过SDS-PAGE、Western blotting印迹、质谱及肝素亲和层沉析对表达产物进行鉴定。结果表明利用酵母工程菌成功表达和纯化了FNCHBD多肽,多肽的分子量接近32 kDa,纯化产物的纯度可达95%以上,能被FN多克隆抗体特异识别且具有多肽肝素结合活性,为后续结构及功能的研究奠定基础。     

人精氨酸酶在毕赤酵母的高效表达、纯化和活性研究

目的：人精氨酸酶（Arginase, Arg）的基因arg在毕赤酵母高效分泌表达,建立相应纯化工艺路线,研究重组人精氨酸酶的活性。方法：将人精氨酸酶基因arg按正确的阅读框架插入到毕赤酵母表达载体pPIC9α信号肽基因后,构建得到重组毕赤酵母表达质粒。转化毕赤酵母GS115筛选高表达菌株。结果：成功构建了酵母表达载体pPIC-Arg,转化毕赤酵母GS115后筛选到分泌表达目的蛋白Arg的菌株,目标蛋白可以分泌到培养基中。经过膜过滤和凝胶过滤层析对培养基上清进行纯化,即可获得纯度达到95%的活性产物。活性测定表明,纯化的Arg比活性为310 IU/mg。结论：成功构建了Arg的毕赤酵母高效表达菌种,建立了目标物质的分离纯化工艺。     

Expression of kringle 5 domain of human plasminogen in Pichia pastoris

The kringle 5 domain of plasminogen exhibits potent inhibitory effect on endothelial cell proliferation. It can also cause cell cycle arrest and apoptosis of endothelia cell specifically, and shows promise in antiangiogenic therapy. It has been prepared via both proteolysis of native plasminogen and recombinant DNA methodologies. When expressed in E. coli, recombinant, kringle 5 deposited mainly as inactive, insoluble inclusion bodies and the refolding yield was also low. In the present study, human kringle 5 encoding gene was cloned into secretory plasmid pPIC9K and then integrated into Pichia pastoris genome for expression. On methanol induction, biologically active recombinant kringle 5 was expressed and secreted into the culture medium by the integrated Pichia pastoris with the expression level around 30mg/L of yeast culture. After a simple and economical three-step purification protocol, namely precipitation, DEAE ion exchange chromatography, and gel filtration, the recombinant kringle 5 was purified to homogeneity, with the yield of 7.5 mg/liter yeast culture.     

促进大肠杆菌周质空间小分子抗体表达的菌种构建方法*

目的:构建一株表达TNF-α Fab'抗体的大肠杆菌工程菌,并设计一种高效实用的策略以促进大肠杆菌周质空间的可溶性Fab'抗体表达。方法:首先,通过更换不同表达载体,改变轻链和重链顺序,更换信号肽,共表达分子伴侣(Skp)、二硫键合成酶(Dsbc)、肽基辅氨酰顺反异构酶(PPIB)、二硫键异构酶(hPDI)、核酸酶(Nuclease),以评估对Fab'抗体表达量的改善。其次,纯化表达的Fab'抗体。通过周质提取、Q阴离子交换柱净化、苯基柱捕获、Protein L柱亲和三步纯化方案得到高纯度的Fab'抗体。最终将纯化后的Fab'抗体进行亲和力测定。结果:提高正确组装的Fab'抗体表达量的策略有——将目的蛋白构建至pET-30a载体;重链在前、轻链在后;轻、重链采用相异的信号肽;共表达hPDI。周质提取液中的Fab'抗体浓度达到588.0mg/L提取液,纯化后产量可达28.2mg/L发酵液,总回收率为32.0%,纯度为90.9%。Fab'抗体亲和力为(5.8±3.0)×10^-9mol/L,体外细胞学活性IC₅₀为(5.2±2.4)×10^-11mol/L。结论:通过大肠杆菌工程菌分子构建方式的优化,得到了一株高效表达可溶性Fab'抗体的工程菌株,为可溶性小分子抗体的规模化生产奠定了研究基础。     

Expression and purification of soluble B lymphocyte stimulator from recombinant Escherichia coli

In this work, the expression conditions of fusion protein thioredoxin (Trx)-soluble B lymphocyte stimulator (sBLyS) in shake flask and bioreactor from the recombinant Escherichia coli BL21 (DE3) with a pET system encoding the fusion protein gene of Trx-sBLyS and the purification method of the sBLyS were optimized to effectively obtain the bioactive protein sBLyS with a high purity. A yield of about 250 mg Trx-sBLyS/g DWC (1686 mg Trx-sBLyS/L) and expression level of about 38.5% in soluble Trx-sBLyS were obtained in a 30 1 bioreactor after optimization of the fermentation conditions. After the completion of the optimized purification procedure in order of affinity chromatography, enzymatic cleavage with enterokinase and DEAE ion exchange chromatography, about 200 mg sBLyS per liter fermentation broth was obtained with a purity of about 95% and a yield of near 30%, respectively. Furthermore, the molecular weight (MW) and the isoelectric point (pI) of the purified sBLyS were determined by 2-D gel electrophoresis and SDS-PAGE analysis and estimated to be over 16 kDa and about pH 4.15, respectively. In addition, the bioactivities of the soluble Trx-sBLyS in fermentation broth and the purified sBLyS were tested by two kinds of analytical methods of bioactivity. The good fermentation yield and the satisfied, purified sBLyS product with high purity, yield and bioactivity demonstrated the sBLyS production procedure was promising in industry.     

Optimized gene synthesis, expression and purification of active salivary plasminogen activator alpha2 (DSPAalpha2) of Desmodus rotundus in Pichia pastoris

Vampire bat salivary plasminogen activators (DSPAs) are thrombolytic agents that are under clinical investigation for the treatment of acute ischemic stroke. In this study, the synthetic active salivary plasminogen activator alpha2 (DSPAalpha2) gene optimized for the preferred codons of Pichia pastoris was assembled from 48 oligonucleotides, and cloned into the yeast expression vector pPIC9 with a strong enhancer from human cytomegalovirus (HCMV). This system achieved high expression of an active DSPAalpha2 in P. pastoris yeast GS115. Secreted active DSPAalpha2 recombinant protein was purified from broth supernatant by a simple one-step procedure on Sephadex chromatography and was confirmed by SDS-PAGE and Western blot analysis. ELISA showed that 2.5mg of recombinant protein could be obtained from 100-ml culture broth supernatant. The fibrinolytic activity of the recombinant DSPAalpha2 was 1.28 x 10(5)IU/mg.     

Expression and purification of soluble B lymphocyte stimulator from recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

In this work, the expression conditions of fusion protein thioredoxin (Trx)-soluble B lymphocyte stimulator (sBLyS) in a shake flask and bioreactor from the recombinant Escherichia coli BL21 (DE3) with a pET system encoding the fusion protein gene of Trx-sBLyS and the purification method of the sBLyS were optimized to effectively obtain the bioactive protein sBLyS with a high purity. A yield of about 250 mg Trx-sBLyS/g DWC (1686 mg Trx-sBLyS/L) and expression level of about 38.5% in soluble Trx-sBLyS were obtained in a 30-1 bioreactor after optimization of the fermentation conditions. After the completion of the optimized purification procedure in order of affinity chromatography, enzymatic cleavage with enterokinase and DEAE ion exchange chromatography, about 200 mg sBLyS per liter fermentation broth was obtained with a purity of about 95% and a yield of near 30%, respectively. Furthermore, the molecular weight (MW) and the isoelectric point (pl) of the purified sBLyS were determined by 2-D gel electrophoresis and SDS-PAGE analysis and estimated to be over 16 kDa and about pH 4.15, respectively. In addition, the bioactivities of the soluble Trx-sBLyS in fermentation broth and the purified sBLyS were tested by two kinds of analytical methods of bioactivity. The good fermentation yield and the satisified, purified sBLyS product with high purity, yield and bioactivity demonstrated the sBLyS production procedure was promising in industry. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2008, Vol. 44, No. 2, pp. 187–192. The text was submitted by the authors in English.     

High yield production of a mutant Nippostrongylus brasiliensis acetylcholinesterase in Pichia pastoris and its purification

The mutant M301A of the acetylcholinesterase B from Nippostrongylus brasiliensis (NbAChE) was produced in a high-cell-density fermentation of a recombinant methylotrophic yeast Pichia pastoris. Dissolved oxygen (DO) spikes were used as an indicator for feeding the carbon source. Wet cell weight (WCW) reached after 8 days a maximum value of 316 g/L and the OD600 at this time was 280. The acetylcholinesterase activity increased up to 6,600 U/mL corresponding to an expression rate of 2 g of NbAChE per liter supernatant. The specific activity of the mutant NbAChE was determined after purification as 3,300 U/mg. Active site titration with chlorpyrifos, a strong AChE inhibitor, yielded in a specific activity of 3,400 U/mg. The enzyme was secreted by Pichia pastoris. Therefore, it could be concentrated from culture broth by cross-flow-filtration (50 kDa cut-off membrane). It was further purified in one-step anion-exchange chromatography, using a XK 50/20 column filled with 125 mL Q Sepharose HP. Mutant NbAChE was purified 1.9-fold up to a purity of 97% and a yield of 87%. The isolated enzyme was nearly homogenous, as seen on the silver stained SDS-PAGE as well as by a single peak after gel filtration. This extraordinary high expression rate and the ease of purification is an important prerequisite for their practical application, for example in biosensors for the detection of neurotoxic insecticides.     

人67kD层粘连蛋白受体在毕赤酵母中的表达及活性分析

为实现人67kD层粘连蛋白受体(Human 67kD Laminin Receptor,67LR)蛋白的分泌表达,采用DNA重组技术将67LR cDNA片段插入分泌型酵母表达载体pPIC9K中,构建了相应的重组表达质粒pPIC9K-67LR并在GS115毕赤酵母菌株中表达,每升培养基经亲和层析可纯化目的蛋白12.56mg。纯化的目标蛋白能够与肺癌A549细胞竞争性结合其配体分子LN-1,具有相应的生物学活性,从而为深入研究人67LR的结构与功能奠定了基础。     

Heterologous expression of dammarenediol synthase gene in an engineered Saccharomyces cerevisiae

Aims: Dammarenediol production by an engineered yeast Saccharomyces cerevisiae was investigated. Methods and Results: A dammarenediol‐producing engineered yeast was constructed by heterologous expression of the dammarenediol synthase gene from Panax ginseng hairy roots through RT‐PCR. Fermentation was carried out in a 5‐L GRJY‐bioreactor with an inoculum size of 1% v/v at 30°C. Dammarenediol detection was performed with silica gel chromatography and HPLC. Determination of dammarenediol synthase activity subcellular distribution was carried out by surveying the enzyme activity in microsomes, lipid particles and total yeast homogenate. When cultured under aerobic conditions, the engineered yeast could produce dammarenediol up to 250 μg l^?1. However, when an anaerobic shift strategy was employed, dammarenediol accumulated at a level as twice as that under aerobic condition. The dammarenediol synthase and dammarenediol were mainly localized in lipid particles. Conclusions: Dammarenediol could be heterologously produced in engineered yeast. The heterologously expressed dammarenediol synthase is mainly localized in lipid particles. Anaerobic shift strategy could enhance the dammarenediol level in the engineered yeast. Significance and Impact of the Study: This study showed that the high‐value plant product dammarenediol could be produced by heterologous expression of the according gene in yeast. Furthermore, the anaerobic shift strategy could be potentially applied in oxidosqualene‐derived compounds production in yeast. Here, the information about subcellular distribution of heterologously expressed dammarenediol synthase in the engineered yeast was also provided.     

离子交换色谱法对大肠杆菌表达的人重组骨形态发生蛋白-7的纯化

带有BMP 7基因的大肠杆菌可以用来高量表达重组的人骨形态发生蛋白 7。升温诱导表达后 ,每升培养液大约可得到菌体湿重 3g ,其中目的蛋白约占菌体总蛋白量的 40 %。裂解离心 ,用低浓度变性剂洗涤初步纯化包涵体 ,上清中无目的蛋白损失 ,目的蛋白纯度提高到 60 %,将包涵体溶解于高浓度变性剂溶液中 ,然后在不同条件下用离子交换色谱法对变性状态下的蛋白质进行纯化 ,绝大部分杂蛋白被除去 ,目的蛋白纯度达 95 %以上 ,改变条件 ,可以减少rhBMP 7损失。并做Westernblot对目的蛋白进行特异性鉴定。     

Purification and characterization of human plasminogen activator inhibitor type I expressed in Saccharomyces cerevisiae

The rapidly acting inhibitor of plasminogen activators, PAI-1, was produced intracellularly in Saccharomyces cerevisiae by using the ADH2 promoter to drive the expression of the human PAI-1 cDNA. Approximately 8 mg of human PAI-1 was produced per liter of confluent yeast culture. A purification scheme which resulted in 20% recovery of isolated PAI-1 from the broken yeast cell homogenate was devised. Yeast-derived human PAI-1 differs from endothelial-type PAI-1 isolated from HT1080 fibrosarcoma cells in that the recombinant inhibitor does not contain carbohydrate side chains. Nevertheless, the activity and other functional attributes of yeast-derived PAI-1 are similar to those exhibited by HT1080 fibrosarcoma cell-derived PAI-1. Hence, this study demonstrates that expression of human PAI-1 in yeast is a viable strategy for the production of ample quantities of this key modulator of plasminogen activator-mediated proteolysis.