来源于柠檬酸杆菌的高比活植酸酶基因在毕赤酵母中的高效表达

柠檬酸杆菌(Citrobacterbraakii)来源的植酸酶是目前报道的比活最高的植酸酶。按照毕赤酵母(Pichiapastoris)对密码子的选择偏向性,对来源于柠檬酸杆菌的高比活植酸酶基因AppA进行了密码子优化改造。改造后的基因AppA(m)按正确的阅读框架融合到毕赤酵母表达载体pPIC9的α-因子信号肽编码序列3′端,通过电击转化得到重组转化子。通过PCR验证,AppA(m)已整合在酵母染色体上。SDS-PAGE分析和表达产物的研究表明,植酸酶得到了高效分泌表达,在5L发酵罐中植酸酶蛋白表达量达到3·2mg/mL发酵液,发酵效价达到每毫升发酵液1·4×107IU以上,高于目前报道的各种植酸酶基因工程菌株的发酵效价。     

Construction of intein-mediated hGMCSF expression vector and its purification in Pichia pastoris

As a novel attempt for the intracellular recombinant protein over expression and easy purification from Pichia pastoris, the therapeutic cytokine human granulocyte macrophage colony stimulating factor (hGMCSF) gene was fused to an intein-chitin-binding domain (gene from pTYB11 vector) fusion tag by overlap extension PCR and inserted into pPICZB vector, allowing for the purification of a native recombinant protein without the need for enzymatic cleavage. The fusion protein under the AOX1 promoter was integrated into the P. pastoris genome (SMD 1168) and the recombinant Pichia clones were screened for multicopy integrants. Expression of hGMCSF was done using glycerol and methanol based synthetic medium by three stage cultivation in a bioreactor. Purification of the expressed hGMCSF fusion protein was done after cell disruption and binding of the solubilized fusion protein to chitin affinity column, followed by DTT induced on column cleavage of hGMCSF from the intein tag. In this study, final biomass of 89 g dry cell weight/l and purified hGMCSF of 120 mg/l having a specific activity of 0.657 x 10(7) IU/mg was obtained. This strategy has an edge over the other--His or--GST based fusion protein purification where non-specific protein binding, expensive enzymatic cleavage and further purification of the enzyme is required. It distinguishes itself from all other purification systems by its ability to purify, in a single chromatographic step.     

重组牛肠激酶轻链基因在毕赤酵母中的表达与纯化

目的:构建重组牛肠激酶轻链的基因工程菌,并进行表达和纯化,以获得高纯度和高活性的重组牛肠激酶轻链蛋白。方法:以GenBank公共数据库中的牛肠激酶轻链基因序列(AccessionNo.NM174439)设计引物,利用RT-PCR合成牛肠激酶轻链基因片段,并克隆进pPIC9K载体,同时在基因N端插进6个组氨酸标签,转化毕赤酵母GS115,进行筛选和诱导表达。产物经镍离子螯和层析和Q-SepharoseFF柱纯化,并酶切融合蛋白检测其活性。结果:培养液中重组牛肠激酶轻链蛋白表达量为3.0mg/L。对含有肠激酶酶切位点的IL-11/MBP融合蛋白进行酶切,结果表明,酶解率可达到90%以上。结论:表达并获得了高纯度的重组肠激酶轻链蛋白,为大规模生产打下了基础。     

巴曲酶在毕赤酵母中的高效表达

目的研究巴曲酶在毕赤酵母菌中的表达。方法按Pichiapastoris偏好密码子人工合成巴曲酶全基因,克隆到酵母分泌型表达载体pPICZaA中,将重组载体酶切线性化后经电转化转入X-33。筛选鉴定转化子．经摇瓶发酵甲醇诱导,酵母菌分泌表达有凝血活性的巴曲酶。经SDS-PAGE电泳确定其分子量为33．0 kDa．免疫印迹证明重组巴曲酶具有天然巴曲酶的免疫活性。结果经发酵条件的优化．发酵罐的表达量达到25000Ku／L发酵液。从每升发酵液中可纯化出11．0mg重组巴曲酶。结论巴曲酶毕氏酵母菌成功的构建．为重组巴曲酶止血药的开发奠定了基础。     

重组巴曲酶在毕赤酵母中的高效表达

以毕赤酵母为表达系统,建立生产重组巴曲酶的技术工艺路线。通过递归式PCR的方法,人工合成了巴曲酶基因,将其插入pPIC9表达质粒中,转化至毕赤酵母GS115(his4),筛选出的表达株经甲醇诱导,表达了重组巴曲酶,并得以纯化。从每升发酵液中可纯化得到10mg重组巴曲酶,其比活为238NIHunits/mg,分子量为30.55kD。重组巴曲酶在体外可使纤维蛋白凝固,在体内缩短小鼠出血时间。为开发重组的蛇毒类凝血酶止血剂打下了基础。     

Production of enzymatically active recombinant full-length barley high pI alpha-glucosidase of glycoside family 31 by high cell-density fermentation of Pichia pastoris and affinity purification

Recombinant barley high pI alpha-glucosidase was produced by high cell-density fermentation of Pichia pastoris expressing the cloned full-length gene. The gene was amplified from a genomic clone and exons (coding regions) were assembled by overlap PCR. The resulting cDNA was expressed under control of the alcohol oxidase 1 promoter using methanol induction of P. pastoris fermentation in a Biostat B 5 L reactor. Forty-two milligrams alpha-glucosidase was purified from 3.5 L culture in four steps applying an N-terminal hexa-histidine tag. The apparent molecular mass of the recombinant alpha-glucosidase was 100 kDa compared to 92 kDa of the native barley enzyme. The secreted recombinant enzyme was highly stabile during the 5-day fermentation and had significantly superior specific activity of the enzyme purified previously from barley malt. The kinetic parameters Km, Vmax, and kcat were determined to 1.7 mM, 139 nM x s(-1), and 85 s(-1) using maltose as substrate. This work presents the first production of fully active recombinant alpha-glucosidase of glycoside hydrolase family 31 from higher plants.     

Expression, purification, and characterization of a novel methyl parathion hydrolase

The mpd gene coding for a novel methyl parathion hydrolase (MPH) was previously reported and its putative open reading frame was also identified. To further confirm its coding region, the intact region encoding MPH was obtained by PCR and expressed in Escherichia coli as a hexa-His C-terminal fusion protein. The fusion protein was purified to homogeneity by metal-affinity chromatography. The enzyme activity and zymogram assay showed that the fusion protein was functional in degrading methyl parathion. The amino terminal sequencing of the purified recombinant MPH indicated that a signal peptide of the first 35 amino acids was cleaved from its precursor to form active MPH. A rat polyclonal antiserum was raised against the purified mature fusion protein. The results of Western blot and zymogram demonstrated that mature MPH in native Plesiomonas sp. strain M6 was also processed from its precursor by cleavage of a putative signal peptide at the amino terminus. The production of active MPH in E. coli was greatly improved after the coding region for the signal peptide was deleted. HPLC gel filtration of the purified mature recombinant MPH revealed that the MPH was a monomer.     

猪IL-18在毕赤酵母中的表达及活性分析

采用PCR方法从pGEM-IL-18重组质粒中扩增出IL-18基因并构建真核融合表达载体pPIC9K-IL-18,电激法转化入毕赤酵母GS115,采用G418抗性梯度法筛选得到多拷贝重组菌株,甲醇诱导表达,应用SDS-PAGE分析重组蛋白的表达情况,并将表达蛋白用凝胶层析柱纯化后,用MTT法检测其生物学活性。实验结果表明重组的GS115酵母菌株可表达分泌pIL-18,其表达在72h时达高峰,分泌量可达160mg/L,纯化的重组pIL-18蛋白具有显著的促进淋巴细胞增殖的活性,说明本试验已在毕赤酵母中在国内首次成功表达了具有生物学活性的pIL-18。     

人甲状旁腺激素-血清白蛋白融合蛋白的构建及在毕赤酵母中的表达

利用重叠PCR技术拼接PTH和HSA基因,并将构建好的融合基因插入到载体pUC19测序后插入表达载体pPIC9K中,在启动子AOXⅠ和α交配因子信号肽的作用下,分泌表达融合蛋白PTH-HSA。重组质粒pPIC9K/PTH-HSA经SalⅠ线性化后,电击转化毕赤酵母KM71,经G418筛选得到的转化子。PCR鉴定后,用甲醇诱导表达,蛋白电泳分析表明融合基因得到表达; Western blot分析表明发酵液上清中表达的融合蛋白PTH-HSA具有HSA的抗原性:用酶标法测定发酵上清中融合蛋白的甲状旁腺激素活性为318IU/ml     

蛇毒类凝血酶calobin在毕赤酵母中的表达

蛇毒类凝血酶是临床上防治血栓栓塞性疾病的有效药物。参照朝鲜蝮蛇(Agkistrodon caliginosus,Korean Viper)类凝血酶calobin基因序列(GenBank AccessionNo.U32937.1),将人工合成的calobin基因克隆到酵母表达载体pPICZαA,于毕赤酵母中表达,得到了分子量约为32kD的重组calobin蛋白,经甲醇诱导培养,表达产物可获得3.5g/L的高表达量。重组蛋白经过阴离子交换柱Q-Sepharose Fast Flow和分子筛Sephacryl-S-100凝胶过滤层析等纯化步骤进行了初步纯化。纯化后的重组calobin可以在纤维蛋白原平板上形成水解圈,经SDS-PAGE实验显示,重组蛋白能水解纤维蛋白原的Aα链,产生一条约40kD左右的降解带。在实验中未能发现重组calobin对纤维蛋白原的凝固作用。     

高效表达N-乙酰高丝氨酸内酯酶-木聚糖酶融合蛋白及其酶学性质

摘要: 【目的】构建N-乙酰高丝氨酸内酯酶-木聚糖酶双酶活性表达毕氏酵母重组菌株,并对经纯化的重组蛋白SL2B 进行N-乙酰高丝氨酸内酯酶及木聚糖酶酶学性质的研究。【方法】利用PCR 拼接技术得到N-乙酰高丝氨酸内酯酶基因aiiA-B546 和木聚糖酶基因xynAS27cd 融合而成的基因Sl2b。构建重组表达载体pPIC9 / Sl2b 转化毕氏酵母,筛选得到同时具有木聚糖酶和N-乙酰高丝氨酸内酯酶活性的重组子,随后对经硫酸铵沉淀、分子筛纯化后得到的重组蛋白SL2B 进行N-乙酰高丝氨酸内酯酶及木聚糖酶     

人血清白蛋白和人干扰素α2b的融合蛋白在毕赤酵母中的表达

为了延长IFNα2b在血浆中的半衰期,构建了编码HSA和hIFNα2b的融合基因并在毕赤酵母中获得高效表达,工程菌经5L发酵罐培养后获得的含融合蛋白的培养液经超滤浓缩、蓝色葡聚糖凝胶层析、疏水柱层析以及阴离子柱层析,融合蛋白的纯度达到95%以上。该融合蛋白能与干扰素抗体和人血清白蛋白抗体结合,并表现出与重组干扰素α2b相似的抗病毒活性。以猕猴为动物模型,分别从静脉和皮下单剂量给药,给药浓度为90μg/kg时,在336h后血浆中仍可检测到融合蛋白。其静脉注射的血浆半衰期为101h,皮下注射的半衰期为68.2h。皮下注射的生物利用度为67.9%。IFNα2b与HSA融合后,明显的延长了血浆半衰期,显现了其良好的临床应用前景。     

增加植酸酶基因appA-m的拷贝提高其在巴斯德毕赤酵母的表达量

为了进一步提高植酸酶的发酵效价,降低植酸酶生产成本,对毕赤酵母表达载体pGAPZα-A进行了改造。将表达载体pPIC9的AOX1启动子序列引入pGAPZα-A,使之成为甲醇可诱导型表达载体pAOXZα,插入植酸酶基因appA-m后得到重组载体pAOXZα-appA-m。以染色体上带有一个拷贝的appA-m基因、发酵效价可达到7.5×106IU/mL发酵液的重组酵母菌株74#为受体菌进行转化,在该重组菌株的染色体上的另一位点整合含有植酸酶基因的表达盒,经筛选到高表达植酸酶的重组子。通过PCR进行验证,植酸酶基因被整合到重组酵母的染色体上,且受体菌中原有的植酸酶基因结构未改变。重组菌在5L发酵罐经甲醇诱导120h植酸酶蛋白表达量达到4mg/mL发酵液,酶活性(发酵效价)达到1.2×107IU/mL发酵液以上,较含单拷贝植酸酶基因的受体菌株表达量有较大程度提高。PCR检测及表达量分析证明改良的菌株具有很好的遗传稳定性和表达稳定性。     

High-level expression of active recombinant ubiquitin carboxyl-terminal hydrolase of Drosophila melanogaster in Pichia pastoris

Ubiquitin carboxyl-terminal hydrolases (UCHs) are implicated in the proteolytic processing of polymeric ubiquitin. The high specificity for the recognition site makes UCHs useful enzymes for in vitro cleavage of ubiquitin fusion proteins. In this work, an active C-terminal His-tagged UCH from Drosophila melanogaster (DmUCH) was produced as a secretory form in a recombinant strain of the methylotrophic yeast Pichia pastoris. The production of recombinant DmUCH by Mut(s) strain was much higher than that by Mut(+) strain, which was confirmed by Western blot analysis. When expression was induced at pH 6.0 in a BMMY/methanol medium, the concentration of recombinant DmUCH reached 210 mg l(-1). With the (His)(6)-tag, the recombinant DmUCH was easily purified by Ni-NTA chromatography and 18 mg pure active DmUCH were obtained from 100ml culture broth supernatant. Ubiquitin-magainin fusion protein was efficiently cleaved by DmUCH, yielding recombinant magainin with high antimicrobial activity. After removing the contaminants by Ni-NTA chromatography, recombinant magainin was purified to homogeneity easily by reversed-phase HPLC. Analysis of the recombinant magainin by ESI-MS showed that the molecular weight of the purified recombinant magainin was 2465 Da, which perfectly matches the mass calculated from the amino acid sequence. The result of mass spectrometry confirmed that the purified His-tagged DmUCH can recognize the ubiquitin-magainin fusion protein and cleave it at the carboxyl terminus of ubiquitin precisely. Our results showed that P. pastoris is a robust system to express the secreted form of DmUCH.     

米根霉糖化酶、类芽孢杆菌木聚糖酶融合蛋白的真核分泌表达

以米根霉(Rhizopus oryzae)3.866基因组DNA为模板,克隆得到糖化酶基因(glucoamylase gene, amyA),基因全长2 049 bp,编码604个氨基酸;以类芽孢杆菌(Paenibacillus sp.)H10-3基因组DNA为模板,克隆出基因木聚糖酶基因(xylanase A gene, xynA)的成熟肽编码序列,长636 bp,编码211个氨基酸。通过重叠延伸PCR(SOE-PCR)得到拼接片段amyA-l-xynA,并将其克隆到毕赤酵母表达载体pPIC9中,得到重组质粒pPIC9-amyA-l-xynA,重组质粒线性化后经电击转化到毕赤酵母(Pichia pastoris)GS115中,得到了表达成功的工程菌AX11。在AX11发酵上清液中同时检测到糖化酶活性(5.8 U/mL)和木聚糖酶活性(32.3 U/mL)。     

Expression and purification of a recombinant antibacterial peptide, cecropin, from Escherichia coli

Insect cecropins are small basic polypeptides synthesized in fat body and hemocytes in response to bacterial infections or hypodermic injuries. To explore a new approach for high expression of soluble cecropin in Escherichia coli cells, we fused the sequence encoding Musca domestica mature cecropin (named Mdmcec) in-frame to thioredoxin (TRX) gene to construct an expression vector pTRX-6His-Mdmcec. An enterokinase cleavage site was introduced between the 6xHis-tag and Mdmcec to facilitate final release of the recombinant Mdmcec. The fusion protein TRX-6His-Mdmcec was purified successfully by HisTrap HP affinity column and a high yield of 48.0mg purified fusion protein was obtained from 1L culture. Recombinant Mdmcec was readily obtained by enterokinase cleavage of the fusion protein followed by HPLC chromatography, and 11.2mg pure active recombinant Mdmcec was obtained from 1L E. coli culture. The molecular mass of recombinant Mdmcec determined by electrospray ionization-mass spectrometry (ESI-MS) is identical to that of native cecropin. Analysis of recombinant Mdmcec by circular dichroism (CD) indicated that recombinant Mdmcec contained predominantly alpha-helix with some random coil. Antimicrobial activity assays demonstrated that recombinant Mdmcec had a broad spectrum of activity against fungi, Gram-positive and negative bacteria. The procedure described in this study will provide a reliable and simple method for production of different cationic peptides for biological studies.     

PTD-NPY融合基因的克隆及其在毕赤酵母中的分泌表达

应用重叠延伸PCR方法扩增HIV-1 TAT蛋白转导结构域(PTD)与鼠源神经肽Y(NPY)的融合基因,克隆目的片段并插入酵母表达载体pPICZαA,构建成重组表达质粒pPICZα-PTD-NPY.PCR和酶切鉴定及测序正确后,经限制性内切酶Sac Ⅰ线性化重组表达质粒并通过电转化整合到巴斯德毕赤酵母菌GS115的染色体基因组中.阳性重组酵母菌用含1%甲醇的培养基诱导其分泌表达.经过120 h的诱导,取上清浓缩除盐后进行SDS-PAGE电泳,表明该系统成功表达了PTD-NPY融合蛋白,Western blotting实验证实表达产物具有特异性.获得真核表达的PTD-NPY融合蛋白,为下一步的应用研究提供了物质基础.     

Novel expression system for large-scale production and purification of recombinant class IIa bacteriocins and its application to piscicolin 126

Piscicolin 126 is a class IIa bacteriocin isolated from Carnobacterium piscicola JG126 that exhibits strong activity against Listeria monocytogenes. The gene encoding mature piscicolin 126 (m-pisA) was cloned into an Escherichia coli expression system and expressed as a thioredoxin-piscicolin 126 fusion protein that was purified by affinity chromatography. Purified recombinant piscicolin 126 was obtained after CNBr cleavage of the fusion protein followed by reversed-phase chromatography. Recombinant piscicolin 126 contained a single disulfide bond and had a mass identical to that of native piscicolin 126. This novel bacteriocin expression system generated approximately 26 mg of purified bacteriocin from 1 liter of E. coli culture. The purified recombinant piscicolin 126 acted by disruption of the bacterial cell membrane.     

庚型肝炎病毒E2区cDNA在毕赤酵母中的表达及抗原性鉴定

从含有庚型肝炎病毒(GBVC/HGV)包膜蛋白E2 cDNA(559bp)的质粒pGEX\|E2中,扩增得到能够编码日本血吸虫谷胱甘肽硫转移酶(GST)和GBVC/HGV包膜蛋白E2的融合基因片段。将此长度为1324bp的DNA片段插入到酵母表达载体pPIC9K中,使之位于α因子信号肽下游,且与之同框。通过电激转化将构建的重组表达质粒pPIC9K\|GST\|E2插入到Pichia pastoris GS115菌株染色体中。筛选His\++Mut\+s表型的转化子,震荡培养,用05%甲醇诱导表达5d后,在培养液中得到表达的GSTE2融合蛋白。经过表达条件的优化,GSTE2蛋白可占培养液中总蛋白的50%。通过谷胱甘肽亲和层析柱纯化,GSTE2融合蛋白的纯度可达95%左右。以庚型肝炎病人血清为探针,进行免疫印迹及ELISA实验,结果表明该融合蛋白具有能被庚型肝炎病人血清特异性识别的抗原性。     

Intein介导的重组昆虫毒素BmK IT在大肠杆菌中的可溶性表达、纯化及活性分析

为获得重组蝎昆虫毒素BmKIT,通过PCR方法在BmKIT基因的3′端融合了编码6个组氨酸残基的核苷酸序列,将其插入原核表达载体pTWIN1的内含肽Ssp DnaB Intein基因下游的多克隆位点(MCS)。将获得的表达质粒转化大肠杆菌BL21(DE3)中,用IPTG诱导融合蛋白表达。用Ni-NTA亲和层析柱从菌体裂解液中纯化了CBD-Intein-BmK IThis6融合蛋白,并在柱上诱导Intein自剪切,成功去除融合子CBD-Intein。通过Superdex75凝胶过滤层析获得了纯度达95%以上的BmK IThis6蛋白,该蛋白不仅具有正确的二级结构而且有生物活性。