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。     

毕赤酵母表达巴曲酶发酵条件的优化研究

目的对表达重组巴曲酶的巴斯德毕赤酵母的发酵条件进行优化,确定最佳的发酵控制条件以获得重组巴曲酶的最高表达量。方法通过多因素正交实验确定巴曲酶发酵培养的最适培养条件。结果表达温度在25℃,pH值为7．0,加入甲醇的量为10g／L时为最优发酵条件,诱导表达时间为84-96h。结论重组巴曲酶可以开发为止血药,代替临床应用的从蛇毒中提取的巴曲酶。     

植酸酶基因中稀有密码子的改造提高其在毕赤酵母中的表达量

对来源于黑曲霉N2 5(AspergillusnigerChinaStrain)的植酸酶基因phyA进行PCR介导的定点突变 ,不改变其所编码氨基酸 ,选用毕赤酵母偏爱的密码子对该基因保守序列中第 81位和第 85位的Arg密码子进行同义突变 .构建了含正确突变的克隆载体pUC18 phyAm 和酵母表达载体pPIC9k phyAm,电击转化毕赤酵母 ,经MM、MD平板筛选和产物的酶活性测定 ,筛选出突变与未突变高酶活酵母转化子各 2株 .这 4株转化子的Southern印迹结果表明 ,phyA基因以单交换方式单拷贝整合到酵母染色体DNA中 .表达产物的SDS PAGE分析表明 ,重组酵母中的植酸酶能有效分泌和表达 ,蛋白质分子大小为 70 15kD .转化子酶活测定结果表明 ,经密码子优化的突变重组酵母酶活力明显高于未进行优化的重组酵母转化子 .经密码子优化的突变重组酵母株PP NPm 8于麦芽汁培养基中诱导 36h后酶活力可达 4 76 0 0U/ml,其活力比未优化重组酵母株PP NP 2 (2 36 6 7U/ml)提高了约 1倍 ,且重组转化子遗传稳定性良好 .     

突变型人白细胞介素-2基因在巴斯德毕赤酵母中的表达

为了提高人重组白细胞介素 2的稳定性和活性以及减少毒副作用 ,有必要定向改造rhIL 2的分子结构 .用PCR法从白细胞介素 2 (IL 2 )cDNA全序列中扩增成熟的肽基因片段 ,并利用定点突变技术将人重组白细胞介素 2第 12 5位游离的半胱氨酸编码序列突变为丙氨酸序列 .编码 18位亮氨酸的序列突变为蛋氨酸序列 ;编码 19位亮氨酸的序列突变为丝氨酸序列 .突变型人白细胞介素 2 (MvIL 2 )基因与表达载体pPIC9K重组 ,酶切线性化后用Invitrogen转化毕赤酵母试剂盒导入酵母细胞进行整合 ,经筛选得到一高表达白介素 2的克隆 .SDS PAGE显示 ,表达量约占总量的4 5 7% .经Western印迹验证 ,重组人白介素 2有免疫活性 ;与野生型IL 2相比 ,所获得的突变型IL 2纯品的比活性为 4 0× 10 7IU mg蛋白 ,比天然型IL 2高 4～ 5倍     

地高辛标记探针检测重组定点突变巴曲酶蛋白宿主DNA残留量的研究

目的 建立检测重组定点突变巴曲酶原液中外源性DNA残留量的方法.方法 从重组定点突变巴曲酶酵母工程菌提取基因组DNA作为模板,制备地高辛标记探针.此探针与样品进行点样杂交,并进行显色反应.结果 3批重组定点突变巴曲酶原液中,每人份剂量的宿主DNA残留量均＜10 ng.结论 该方法检测灵敏度较好,特异性较强,可用于重组定点突变巴曲酶的检测.     

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。     

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。     

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     

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。     

One-step site-directed mutagenesis of the Kex2 protease oxyanion hole

BACKGROUND: Members of the subtilisin family of serine proteases usually have a conserved asparagine residue that stabilizes the oxyanion transition state of peptide-bond hydrolysis. Yeast Kex2 protease is a member of the subtilisin family that differs from the degradative subtilisin proteases in its high substrate specificity, it processes pro-alpha-factor, the precursor of the alpha-factor mating pheromone of yeast, and also removes the pro-peptide from its own precursor by an intramolecular cleavage reaction. Curiously, the mammalian protease PC2, a Kex2 homolog that is likely to be required for pro-insulin processing, has an aspartate in place of asparagine at the 'oxyanion hole'. RESULTS: We have tested the effect of making substitutions of the conserved oxyanion-hole asparagine (Asn 314) of the Kex2 protease. To do this, we have developed a rapid method of site-directed mutagenesis, involving homologous recombination of a polymerase chain reaction product in yeast. Using this method, we have substituted alanine or aspartate for Asn 314 in a form of Kex2 engineered for secretion. Transformants expressing the two mutant enzymes could be identified by failure either to produce mature alpha-factor or to mate. The Ala 314 enzyme was unstable but the Asp 314 enzyme accumulated to a high level, so that it could be purified and its activity towards various substrates tested in vitro. We found that, with three peptides that are good substrates of wild-type Kex2, the k(cal) of the Asp 314 enzyme was reduced approximately 4500-fold and its K(M) approximately 4-fold, relative to the wild-type enzyme. For the peptide substrate corresponding to the cleavage site of pro-alpha-factor, however, k(cat) of the Asp 314 enzyme was reduced only 125-fold, while the K(m) was increased 3-fold. Despite its reduced catalytic activity, however, processing of the mutant enzyme in vivo - by the intramolecular cleavage that removes its amino-terminal pro-domain - occurs at an unchanged rate. CONCLUSIONS: The effects of the Asn 314-Asp substitution reveal contributions to the reaction specificity of the Kex2 protease of substrate residues amino-terminal to the pair of basic residues at the cleavage site. Aspartate at the oxyanion hole appears to confer k(caf) discrimination between substrates by raising the energy barrier for productive substrate binding: this may have implications for pro-insulin processing by the PC2 protease, which has an aspartate at the equivalent position. The rate of intramolecular cleavage of pro-Kex2 may be limited by a step other than catalysis, presumably protein folding.     

Expression of the Saccharomyces cerevisiae Kex2p endoprotease in inset cells. Evidence for a carboxy-terminal autoprocessing event.

The pheromone-processing Kex2p endoprotease of Saccharomyces cerevisiae has been difficult to characterize due to its low level of expression in yeast cells. To overcome this problem, we have overexpressed Kex2p using the baculovirus/insect cell expression system. Spodoptera frugiperda Sf9 insect cells infected with a recombinant baculovirus, containing the complete KEX2 gene which encodes the Kex2p protease (814 amino acids), accumulate an 120-kDa functional form of the enzyme. The inhibition profile of the insect-cell-derived endoprotease is similar to that of the yeast enzyme. The recombinant infected insect cells also secrete into the medium about half of the total Kex2p activity produced. Deleting the carboxyl-terminal tail and the transmembrane domain of Kex2p (Kex2 delta p, 666 amino acids) does not measurably interfere with the enzyme characteristics and results in the secretion of up to 90% of the total enzyme activity. The truncated form, Kex2 delta p, of the endoprotease accumulates in the cell supernatant to 6.7 x 10(5) U/l. The molecular mass of the secreted forms for both the wild-type Kex2p and Kex2 delta p is the same (70 kDa) and is 50-kDa lower than the intracellular form. This result implicates a processing event which gives rise to shorter extracellular forms of both the wild-type Kex2p and Kex2 delta p and which trims their carboxy termini upsteam of amino acid 666. This processing event requires the integrity of the Ser385 of the Kex2p active site.     

Functional characterization of recombinant batroxobin, a snake venom thrombin-like enzyme, expressed from Pichia pastoris

A thrombin-like enzyme of Bothrops atrox moojeni venom, batroxobin, specifically cleaves fibrinogen alpha chain, resulting in the formation of non-crosslinked fibrin clots. The cDNA encoding batroxobin was cloned, expressed in Pichia pastoris and the molecular function of purified recombinant protein was also characterized. The recombinant batroxobin had an apparent molecular weight of 33 kDa by SDS-PAGE analysis and biochemical activities similar to those of native batroxobin. The purified recombinant protein strongly converted fibrinogen into fibrin clot in vitro, and shortened bleeding time and whole blood coagulation time in vivo. However, it did not make any considerable alterations on other blood coagulation factors. Several lines of experimental evidence in this study suggest that the recombinant batroxobin is a potent pro-coagulant agent.     

碱性蛋白酶工程菌发酵条件及重组酶的纯化和性质的研究

在5L发酵罐中对重组碱性蛋白酶工程菌株BP071高产碱性蛋白酶的条件进行了研究,通过提高通气量和改变搅拌转速,BP071可在发酵40 h内达到产酶高峰,酶活力最高可达24480 u/mL。利用快速蛋白液相层析（FPLC）技术,建立了快速高效纯化碱性蛋白酶的方案。发酵液通过硫酸铵沉淀、DEAE-A-50脱色及聚乙二醇浓缩得粗酶,再经过CM-Sephadex-C-50、Sephadex-G-75柱层析后得到了单一组份的重组碱性蛋白酶,酶纯度提高了76.2倍。SDS-PAGE显示重组碱性蛋白酶分子量为28 kD。酶学性质研究表明,酶的最适作用pH为11,最适作用温度为60℃,具有良好的pH稳定性和热稳定性。Ca²⁺、Mg²⁺对酶的稳定性有促进作用,Hg²⁺、Ag⁺、PMFS和DFP能强烈抑制酶的活力。SDS和Urea对酶的活力无影响。     

大肠杆菌外膜蛋白酶T及其突变体的表达、复性及生物活性分析

外膜蛋白酶T(Outer-membrane protease T,OmpT)是定位于大肠杆菌外膜,具有高度底物特异性的蛋白水解酶。本文旨在建立克隆表达膜蛋白OmpT和体外复性的方法,考察其蛋白酶活性。首先以大肠杆菌基因组DNA为模板,PCR扩增ompT基因,连接至pET28a(pET-ompT),引入点突变Asp85Ala,构建表达质粒pET-ompT85。然后将两种重组质粒转化入BL21(DE3),均以包涵体形式大量表达。纯化后的蛋白经稀释法复性,并加入粗制脂多糖(Lipopolysaccharide,LPS)恢复蛋白酶活性。通过SDS-PAGE、鱼精蛋白水解试验及生长曲线观察表明,重组蛋白OmpT在体外能水解抗菌肽鱼精蛋白和兔肌肉肌酸激酶,而OmpT突变体则无上述功能。上述结果表明本文获得了具有蛋白水解酶功能的重组蛋白OmpT,该蛋白在体外可保护大肠杆菌抵抗鱼精蛋白的杀菌作用。     

Mutation of a tyrosine localization signal in the cytosolic tail of yeast Kex2 protease disrupts Golgi retention and results in default transport to the vacuole.

Kex2 protease processes pro-alpha-factor in a late Golgi compartment in Saccharomyces cerevisiae. The first approximately 30 residues of the 115 amino acid CO2H-terminal cytosolic tail (C-tail) of the Kex2 protein (Kex2p) contain a Golgi retention signal that resembles coated-pit localization signals in mammalian cell surface receptors. Mutation of one (Tyr713) of two tyrosine residues in the C-tail or deletion of sequences adjacent to Tyr713 results in loss of normal Golgi localization. Surprisingly, loss of the Golgi retention signal resulted in transport of C-tail mutant Kex2p to the vacuole (yeast lysosome), as judged by kinetics of degradation and by indirect immunofluorescence. Analysis of the loss of Kex2 function in vivo after shutting off expression of wild-type or mutant forms proved that mutations that cause rapid vacuolar turnover do so by increasing the rate of exit of the enzyme from the pro-alpha-factor processing compartment. The most likely explanation for these results is that mutation of the Golgi retention signal in the C-tail results in transport of Kex2p to the vacuole by default. Wild-type Kex2p also was transported to the vacuole at an increased rate when overproduced, although apparently not due to saturation of a Golgi-retention mechanism. Instead, the wild-type and C-tail mutant forms of Kex2p may follow distinct paths to the vacuole.     

An extracellular exo-beta-(1,3)-glucanase from Pichia pastoris: purification, characterization, molecular cloning, and functional expression

An extracellular exo-beta-(1,3)-glucanase (designated EXG1) was purified to apparent homogeneity from Pichia pastoris X-33 cultures by ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration. The native enzyme is unglycosylated and monomeric with a molecular mass of approximately 47kDa. At its optimal pH of 6.0, the enzyme shows highest activity among physiological substrates toward laminarin (apparent Km, 3.5 mg/ml; Vmax, 192 micromole glucose produced/min/mg protein) but also hydrolyzes amygdalin and esculin, and the chromogenic substrates p-nitrophenyl-beta-D-glucopyranoside and p-nitrophenyl-beta-D-xylopyranoside. The P. pastoris EXG1 gene was cloned by a PCR-based strategy using genomic DNA as template. This intronless gene predicts an ORF that encodes a primary translation product of 414 amino acids. We believe that this preproprotein is processed sequentially by signal peptidase and a Kex2-like endoprotease to yield a mature protein of 392 amino acids (45,376 Da; pI, 4.46) that shares 36-64% amino acid identity with other yeast exo-beta-(1,3)-glucanases belonging to Glycoside Hydrolase Family 5. It also possesses the eight invariant residues and signature pattern [LIV]-[LIVMFYWGA](2)-[DNEQG]-[LIVMGST]-X-N-E-[PV]-[RHDNSTLIVFY] shown by all Family 5 members. Overexpression of the cloned EXG1 gene in Pichia cells, followed by Ni-CAM HC resin chromatography, yielded milligram quantities of homogeneous recombinant EXG1 in active form for further characterization studies.     

Production of recombinant Rhizopus oryzae lipase by the yeast Yarrowia lipolytica results in increased enzymatic thermostability

The gene encoding Rhizopus oryzae lipase (ROL) was expressed in the non-conventional yeast Yarrowia lipolytica under the control of the strong inducible XPR2 gene promoter. The effects of three different preprosequence variants were examined: a preprosequence of the Y. lipolytica alkaline extracellular protease (AEP) encoded by XPR2, the native preprosequence of ROL, and a hybrid variant of the presequence of AEP and the prosequence of ROL. Lipase production was highest (7.6 U/mL) with the hybrid prepropeptide. The recombinant protein was purified by ion-exchange chromatography. The ROL included 28 amino acids of the C-terminal region of the prosequence, indicating that proteolytic cleavage occurred below the KR site through the activity of the Kex2-like endoprotease. The optimum temperature for recombinant lipase activity was between 30 and 40 °C, and the optimum pH was 7.5. The enzyme was shown not to be glycosylated. Furthermore, recombinant ROL exhibited greater thermostability than previously reported, with the enzyme retaining 64% of its hydrolytic activity after 30 min of incubation at 55 °C.