血小板衍生生长因子BB亚型在毕赤酵母中的表达及纯化

目的:用毕赤酵母表达系统表达重组人血小板衍生生长因子BB亚型(PDGF-BB)。方法:采用PT-PCR从人早幼粒白血病细胞系HL-60细胞中获得目的基因,克隆到表达载体p MEX9K中,质粒线性化后转化酵母表达菌株GS115,筛选后的酵母表达菌株经BMGY/BMMY培养基体系诱导表达后,通过疏水作用、离子交换、凝胶过滤纯化获得目的蛋白,采用SDS-PAGE、Western印迹、N端氨基酸序列和MTT增殖活性测定等方法检测目的蛋白性质及生物活性。结果:重组人PDGF-BB为分泌表达,表达量大于100 mg/L;经三步纯化,获得纯度高于95%且具有较高活性(5.0×105IU/mg)的目标蛋白。结论:利用毕赤酵母表达系统表达重组人PDGF-BB,表达产量高,成本低,工艺简单,易于工业化放大,有良好的市场前景。     

人小肠三叶因子在毕赤酵母中克隆表达及对肠粘膜修复作用的研究

为了研究人肠三叶因子(hITF)对肠粘膜的保护作用,利用RT-PCR从肠粘膜中扩增出hITF基因片段,与诱导分泌型毕赤酵母载体pPIC9连接构建了重组质粒pPIC9hITF,重组质粒转化至宿主菌GS115,经过PCR鉴定和转化子发酵筛选,得到一个重组毕赤酵母高产菌株GS115/pPIC9hITF。在5L发酵罐中用基本盐培养基培养重组菌株,添加甲醇诱导表达hITF,离心收集的上清液通过离心交换层析纯化得到hITF。质谱鉴定结果表明纯化的hITF与天然提取产品在N端序列上完全相同。细胞实验和动物实验结果表明重组hITF能够促进细胞迁移,并可以保护肠粘膜免受有害因子的侵袭,保持了较好的生物学活性。     

狐狸生长激素基因的克隆及其在毕赤酵母中的表达

为制备重组狐狸生长激素(fGH),采用RT-PCR方法,从银狐垂体中扩增fGHcDNA基因,利用SnaBI和NotI位点将fGH基因插入到酵母分泌型表达载体pPIC9K中α-因子信号肽的下游,构建成fGH基因的酵母分泌型表达载体pPIC9K/fGH,载体经SalI酶切线性化后,通过电转移将线性化的pPIC9K/fGH转化到组氨酸缺陷型酵母宿主菌GS115中。然后利用不含氨基酸的以葡萄糖为碳源的培养基(MD)和以甲醇为碳源的培养基(MM)筛选出组氨酸His+型和甲醇利用正型(Mut+)酵母重组体,再经G418加压筛选出高拷贝fGH基因的重组酵母,经摇瓶发酵培养和甲醇诱导使fGH进行分泌表达。结果表明本实验扩增的fGH基因序列与GenBank发表的序列基本一致,发酵液经SDS-PAGE和Western blotting检测证明构建的重组酵母能够分泌表达fGH,表达的fGH占发酵液总蛋白的34%,表达量达119mg/L发酵液。     

毕赤酵母高效分泌长效人生长激素的研究

目的:为了延长人生长激素(HGH)在血浆中的半衰期,构建了高效分泌表达人血清白蛋白(HSA)与HGH融合蛋白(HSA-HGH)的工程毕赤酵母菌株。方法:从人胎肝cDNA文库中扩增HSA基因,从HGH工程菌载体中扩增HGH基因,将其克隆至真核表达载体pHIL-D2,载体线性化后采用电击法转化毕赤酵母GS115。通过原位双层膜法筛选高效分泌表达菌株。分别采用SDS-PAGE和Western印迹鉴定融合蛋白。对工程酵母培养条件进行研究,发酵液经离子交换层析、亲和层析和分子筛层析纯化。纯化的蛋白经N端序列测定,分子量测定和等电聚焦电泳进行鉴定。冻干的蛋白制剂经食蟹猴试验进行药代动力学和药效动力学测试。结果:确立了工程酵母的最佳培养工艺,融合蛋白表达量达100mg/L。纯化后蛋白纯度达95%以上,得率达42%。融合蛋白与预期结果一致,经食蟹猴试验,显示有良好的生物活性,与等摩尔剂量的重组人生长激素相比,半衰期延长6.8倍,清除率慢44倍。结论:融合蛋白呈现明显的长效动力学特征,为开发重组长效人生长激素HSA-HGH融合蛋白药物(rHSA-HGH)奠定了基础。     

复合干扰素突变体在毕赤酵母中的表达、纯化及活性分析

根据毕赤酵母密码子偏性合成了复合干扰素突变体基因 ,克隆至分泌型酵母表达载体pMEX9K ,将重组载体pMEX CIFNm用SacⅠ线性化后 ,转化毕赤酵母GS115 .转化子经诱导后 ,培养上清有抗病毒活性的蛋白产生 .经过离子交换 ,疏水层析 ,凝胶过滤三步层析纯化 ,得到了纯度大于95 %的重组复合干扰素突变体 ,经N端氨基酸序列分析表明 ,该蛋白N端序列与理论值一致 ,质谱测定分子量为 19 3kD ,与理论值一致 .用细胞病变抑制法测定其活性 ,并结合Lowry法蛋白定量计算其比活性为 6× 10 8IU mg ,与复合干扰素的比活相当 .     

水稻N-糖酰胺酶基因 (OsPNGase A) 的克隆及在毕赤酵母中的分泌表达

N-聚糖酶是一类广泛应用于糖蛋白的N-糖基化修饰研究中的去糖基化酶。本研究通过RT-PCR从水稻中克隆了一个高GC含量(69.48%)的N-聚糖酶基因(Os PNGase A,XM_015775832),通过无缝克隆技术构建酵母分泌型表达载体p PICZ(α)A-Os PNGase A,在毕赤酵母SMD1168H中进行诱导表达,发酵液经DEAE Sepharose阴离子交换层析和His Trap HP金属离子螯合层析纯化,产量可达到12.3 mg/L,比活力为258 U/mg。SDS-PAGE结果显示,纯化的Os PNGase A为单一条带且与预期分子量一致。Os PNGase A能作用于水稻中重组表达的人转铁蛋白(TRF)、玉米中重组表达的鸡蛋抗生物素蛋白(Avidin)以及辣根过氧化物酶(HRP),并且对Avidin的酶切效果优于商业化的PNGase F。Os PNGase A反应的最适p H和温度分别为p H 6.0和40℃,在中性和碱性以及含有100 mmol/L还原剂β-ME和DTT的条件下仍具有活性。水稻Os PNGase A的成功表达为植物糖蛋白的研究提供一个新的工具酶,酵母分泌表达体系的建立为PNGase A的大量制备奠定了基础。     

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。     

人血管抑制素在酿酒酵母中的分泌表达和活性鉴定

将酵母交配因子 (MF)α1信号肽编码序列和人血管抑制素 (hAGN)cDNA融合序列插入穿梭载体pYADE4 ,构建得到分泌型重组表达质粒pYADEMA18.转化酿酒酵母JG110 7后 ,用 2 %乙醇和2 %甘油联合诱导表达 .ELISA分析表明 ,在诱导 14h～ 30h期间 ,hAGN获得了表达并分泌至细胞外 .发酵上清液经 75 %饱和度硫酸铵沉淀、CM 5 2纤维素离子交换层析和Superdex 75凝胶过滤层析纯化 .SDS PAGE分析显示 ,表达产物重组人血管抑制素 (rhAGN)相对分子质量约 5 0kD ,电泳纯度达到 94 %.生物活性分析证明 ,rhAGN在 0 0 1mg L～ 3 0mg L浓度范围内能够抑制人真皮内皮细胞株HDMEC增殖 ,抑制作用随剂量的增加而增强 .     

人α降钙素基因相关肽基因在酵母细胞中的分泌表达与产物的分离纯化

化学合成的人α降钙素基因相关肽(CCRP)基因用PCR法改造后使其能正确融合在酵母分泌型表达载体pVT102U/α中的α交配因子前导肽序列之后,然后进行克隆并转化酵母宿主菌S－78进行表达．培养物的上清用酶标(ELlSA)鉴定为阳性,而对照S－78、pVT102u／α为阴性,表达量用ELISA定量大于2mg／L。表达产物经阳离子交按层析(CM—Sphadex C₂₅)和HPLC纯化得到了HPLC纯产品。纯化后的CGRP能引起小鼠血压的降低,说明表达的目的蛋白既有CGRP的免疫结合活性,又有CGRP的生理活性。测定其N－端10个氨基酸序列,证明人工合成的CGRP基因在酵母细胞中已正确表达。     

疏棉状嗜热丝孢菌脂肪酶在大肠杆菌中的表达与纯化

目的:实现疏棉状嗜热丝孢菌脂肪酶(Thermomyces lanuginosus Lipase,TLL)在大肠杆菌中的可溶性表达,并建立有效的纯化方法。方法:根据疏棉状嗜热丝孢菌脂肪酶序列和大肠杆菌密码子的偏爱性,合成了疏棉状嗜热丝孢菌脂肪酶DNA序列,克隆至大肠杆菌表达载体PET-32a(+)中,通过筛选得到阳性重组载体,并转化入大肠杆菌Rosetta(DE3),诱导表达并利用SDS-PAGE电泳检测重组脂肪酶表达情况,并通过Ni柱亲和层析对目的蛋白TLL进行纯化,透析脱盐后,TEV酶切重组融合蛋白,再利用亲和层析纯化得到酶切后的脂肪酶,检测其酶活。结果:得到了可溶性表达的TLL,检测酶切前后脂肪酶的比活性:酶切前达5.7×106U/mg,酶切后达8.9×105U/mg。结论:实现了疏棉状嗜热丝孢菌脂肪酶的可溶性表达,并得到有效纯化,同时证明TEV酶切目的蛋白对其活性影响微小,为之后对其进行环化打下基础。     

Secretion of pro- and mature Rhizopus arrhizus lipases by Pichia pastoris and properties of the proteins

The lipases of Rhizopus spp. share a high 1,3-regiospecificity toward triacylglycerols, which makes them important enzymes in lipid modification. In the present study, the extracellularly active production of recombinant Rhizopus arrhizus lipase was carried out with genes encoding the mature region (mRAL) and the mRAL having the prosequence (ProRAL) in Pichia pastoris. Two transformed P. pastoris clones containing the multicopy of mRAL and ProRAL genes were separately selected for the production of recombinant enzymes. In a fed-batch cultivation, where methanol feeding was controlled by an on-line methanol analyzer, the supernatant contained 91 mg/L recombinant pro-form lipase (rProRAL) and 80 mg/L recombinant mature lipase (rRAL) after 92 h of cultivation. rProRAL and rRAL were purified by ultrafiltration, SP-Sepharose Rast Flow chromatography, and Butyl-Sepharose Fast Flow chromatography. Molecular weights of rProRAL and rRAL are 32 kDa and 29 kDa, respectively. The amino-terminal analysis showed that the 32-kDa protein was mRAL attached with 28 amino acids of the carboxy-terminal part of the prosequence (rPro28RAL). The specific lipase activities of mRAL attached with 28 amino acids of the carboxy-terminal part of the prosequence (rPro28RAL) and rRAL were 1543 U/mg and 2437 U/mg. The rPro28RAL was more stable than rRAL at pH 4.0–7.0, whereas rRAL was more stable at pH 7.0–10.0. The rPro28RAL had the highest lipase activity toward tributyrin (C₄), whereas rRAL had the highest lipase activity toward tricaprylin (C₈).     

少根根霉Δ^6-脂肪酸脱氢酶基因在毕赤酵母中的表达

Δ^6-脂肪酸脱氢酶是一种膜整合蛋白,也是多不饱和脂肪酸合成途径中的限速酶。在前期工作中,通过RT-PCR和RACE技术,从少根根霉NK300037中克隆到一个潜在编码Δ^6-脂肪酸脱氢酶的序列,序列和功能分析结果表明该序列具有一个长度为1377bp、编码由458个氨基酸组成、大小为52kD的新的Δ^6-肪酸脱氢酶基因。把少根根霉Δ^6-脂肪酸脱氢酶基因（RAD6）亚克隆到表达载体pPIC3．5K,构建重组表达载体pPICRAD6,并转化到毕赤酵母菌株GS115进行表达。提取酵母细胞总脂肪酸和进行甲酯化,经气相色谱和气相色谱-质谱连用分析表明,目的基因的编码产物能将C16：1、C17：1、C18：1、亚油酸和α-亚麻酸在△6和7位间特异性脱氢而引入一个新的双键,生成更高不饱和的脂肪酸,该催化反应没有链长特异性,只有键位特异性。此外,按Kozak序列特点,改变目的基因转译起始密码子周边序列结构,并把改变后序列导入毕赤酵母GS115中进行功能表达分析,结果表明在毕赤酵母中这种改变同样能提高目的基因的表达水平。综合所有分析结果表明,巴斯德毕赤酵母更适合用来综合分析Δ^6-脂肪酸脱氢酶基因的功能。     

少根根霉△6-脂肪酸脱氢酶基因在毕赤酵母中的表达

△6-脂肪酸脱氢酶是一种膜整合蛋白,也是多不饱和脂肪酸合成途径中的限速酶.在前期工作中,通过RT-PCR和RACE技术,从少根根霉NK300037中克隆到一个潜在编码△6-脂肪酸脱氢酶的序列,序列和功能分析结果表明该序列具有一个长度为1377bp、编码由458个氨基酸组成、大小为52kD的新的△6-脂肪酸脱氢酶基因.把少根根霉△6-脂肪酸脱氢酶基因(RAD6)亚克隆到表达载体pPIC3.5K,构建重组表达载体pPICRAD6,并转化到毕赤酵母菌株GS115进行表达.提取酵母细胞总脂肪酸和进行甲酯化,经气相色谱和气相色谱-质谱连用分析表明,目的基因的编码产物能将C16:1、C17:1、C18:1、亚油酸和α-亚麻酸在△6和7位间特异性脱氢而引入一个新的双键,生成更高不饱和的脂肪酸,该催化反应没有链长特异性,只有键位特异性.此外,按Kozak序列特点,改变目的基因转译起始密码子周边序列结构,并把改变后序列导入毕赤酵母GS115中进行功能表达分析,结果表明在毕赤酵母中这种改变同样能提高目的基因的表达水平.综合所有分析结果表明,巴斯德毕赤酵母更适合用来综合分析△6-脂肪酸脱氢酶基因的功能.     

Optimization of the high-level production of Rhizopus oryzae lipase in Pichia pastoris

The lipases of the Rhizopus species family are important and versatile enzymes that are mainly used in fat and oil modification due to their strong 1,3-regiospecificity. Inexpensive synthetic medium was used for the production of Rhizopus oryzae lipase in the methylotrophic yeast Pichia pastoris. Methanol accumulation inside the bioreactor has previously been shown to negatively influence the production level. Three different methanol fed-batch strategies for maintaining the methanol concentration within optimal limits have been assayed in high-density cultures. One methanol feeding strategy, which is based on the monitoring of the methanol concentration by gas chromatography, resulted in a 2.5-fold higher productivity compared to an initial cultivation, where the feeding rate was adjusted according to the dissolved oxygen concentration (DO) in the supernatant. Finally, productivity could be further increased by introducing a transition phase that involved the simultaneous feeding of glycerol and methanol followed by a single methanol feed. This optimized strategy resulted in the highest productivity (12888 U l(-1) h(-1)), which is 13.6-fold higher than the DO-based strategy.     

Sorbitol co-feeding reduces metabolic burden caused by the overexpression of a Rhizopus oryzae lipase in Pichia pastoris

To improve the specific production rate of Rhizopus oryzae lipase (ROL) in Pichia pastoris, a protein that triggers the unfolded protein response in P. pastoris, the effect of sorbitol/methanol mixed substrates was tested in batch and fed-batch cultures. Remarkably, a different substrate consumption behaviour was observed depending on the host's phenotype (Mut(+) or Mut(s)) in batch cultures: when the methanol assimilation capacity is genetically reduced (Mut(s) phenotype), both substrates were consumed simultaneously, allowing not only a higher specific growth rate but also higher lipase levels (8.7-fold) compared to those obtained by cells growing on methanol as a sole carbon source in batch culture. This effect was not observed in Mut(+) phenotype, where the two substrates were consumed sequentially and the levels of heterologous product were only slightly higher (1.7-fold). A mixed substrate strategy was also applied to a Mut(s) fed-batch culture at a low methanol concentration set-point (0.5 gl(-1)). This resulted in a 2.2-fold increase in the heterologous protein level achieved, compared with the methanol-only feeding strategy. In addition, sorbitol co-feeding permitted the achievement of higher specific growth rates, and avoided the drastic decrease of the specific production rate observed after the start of the induction phase when methanol was used as sole carbon source This resulted in a significant increase in the overall bioprocess volumetric productivity (2.2-fold) and specific productivity (1.7-fold). Moreover, whereas increased ROL gene dosage in Mut(s) strains have been previously reported to be deleterious for P. pastoris cells growing on methanol, sorbitol co-feeding allowed for sustained cell growth and lipase production.     

BMMYA’平板-Fast blue RR顶层琼脂法高效筛选高酶活脂肪酶

建立了一种高效筛选高酶活或高产脂肪酶茵株的平板方法。该方法以华根霉（Rhizopus chinensis CCTCCM201021）脂肪酶基因proRCL在毕赤酵母中构建的基因突变文库为筛选对象,利用BMMYA’平板-Fast blue RR顶层琼脂法对其中高酶活或高产的脂肪酶突变株进行筛选,将待筛茵株接种至含有2％甲醇的BMMYA’平板上,30℃生长并诱导4～5d后,平板经脂肪酶致死温度65℃处理1h,冰浴、室温平衡后,向平板中倾入Fast blue RR顸层琼脂。2min内周围显示出明显的黑褐色的菌株为高酶活或高产突变株。该方法简便,快速,高效而且准确,筛选阳性率可达到90％。     

干扰素与转铁蛋白融合蛋白在毕赤酵母中的表达及鉴定

利用重叠 PCR 技术将干扰素 (interferon , IFN) 基因与转铁蛋白 N 端半分子 (transferrin N-terminal half-molecule , TFN) 基因在体外融合,融合基因和单独的 TFN 基因分别克隆至真核表达载体 pPIC9 中,转化毕赤酵母 GS115 ,得到的转化子经诱导表达后在发酵上清中均获得了表达 . 经 SP Sepharose Fast Flow 阳离子交换层析、 Phenyl Sepharose Fast Flow 疏水层析纯化,获得了纯度大于 93 ％的重组融合蛋白 IFN-TFN 和纯度大于 95 ％的重组 TFN 样品 . 生物活性实验证明融合蛋白 IFN-TFN 具有抗病毒活性 . 铁饱和实验证明融合蛋白 IFN-TFN 和单独的 TFN 具有相同的铁结合能力 . 因而 TFN 可望作为 IFN 的天然运输载体 .     

High yield and secretion of recombinant human apolipoprotein AI in Pichia pastoris

Apolipoprotein AI (ApoAI) is an important apolipoprotein in plasma and is known to have various physiological functions suitable for pharmaceutical applications. Human blood has been the only source of this protein for research and large-scale applications. To obtain large amounts of ApoAI a Pichia pastoris expression system was first used to obtain a high level of expression of secreted, recombinant protein. The human gene encoding ApoAI was inserted into the secretion vector pPIC9K and used to transform P. pastoris GS115. AP16, a high expression transformant with high G418 resistance, was obtained. After induction with methanol, the expression level of rhApoAI (recombinant human ApoAI) was 160 mg/L in a 14L fermentor. RhApoAI was purified by cold acetone precipitation followed by Q-Sepharose Fast Flow ion exchange column chromatography with 60% recovery. The N-terminal amino acid sequence and molecular weight (mass spec.) of rhApoAI are identical to native human ApoAI. Purified rhApoAI has specific binding activity with liver cells SMC7721 and binding can be inhibited by native human ApoAI.     

Expression of a Rhizopus oryzae lipase in Pichia pastoris under control of the nitrogen source-regulated formaldehyde dehydrogenase promoter

A Rhizopus oryzae lipase gene has been expressed in Pichia pastoris as a reporter using the formaldehyde dehydrogenase 1 promoter (PFLD1) of this organism, which has been reported to be strongly and independently induced by either methanol as sole carbon source or methylamine as sole nitrogen source. Levels of lipase expressed and secreted under the control of the PFLD1 at different induction conditions have been compared to those obtained with the commonly used alcohol oxidase 1 promoter (PAOX1) in small (shake flask) and 1l bioreactor batch cultures. PFLD1-controlled heterologous gene expression was strongly repressed by excess of either glycerol or glucose-but not sorbitol-during growth using methylamine both as sole nitrogen source and inducing substrate. Co-induction of PFLD1 with methanol and methylamine resulted in a synergistic effect on extracellular lipase expression levels. In all tested conditions, the substitution of ammonium for methylamine as carbon source provoked a clear decrease in the specific growth rate and yield of biomass per gram of carbon source. Overall, this study demonstrates that the PFLD1 promoter is at least as efficient as the PAOX1 for extracellular expression of heterologous proteins in P. pastoris bioreactor cultures and provides a first basis for the further design of methanol-free high cell density fed-batch cultivation strategies for controlled overproduction of foreign proteins in P. pastoris.     

甲状旁腺激素和转铁蛋白N端半分子融合蛋白在毕赤酵母中的表达

用重叠PCR技术将PTH（parathyroid hormone, 甲状旁腺激素）基因与TFN（transferrin N_terminal half_molecule, 转铁蛋白N端半分子）基因在体外融合,融合基因克隆至真核表达载体pPIC9中,转化毕赤酵母GS115。转化子经甲醇诱导后,融合蛋白得到了表达并分泌到发酵上清液中。经 SP Sepharose F F阳离子交换层析、Phenyl Sepharose Fast Flow疏水层析纯化获得了纯度大于95％的PTH_TFN样品。Western blot分析及腺苷酸环化酶实验证明融合蛋白中的PTH具有与抗PTH抗体结合能力及刺激腺苷酸环化酶的活性,铁饱和实验证明融合蛋白中的TFN和单独的TFN具有相同铁结合能力。因而TFN可望作为PTH的天然运输载体。