Cloning, expression and characterization of recombinant sweet-protein thaumatin II using the methylotrophic yeast Pichia pastoris

Thaumatin, an intensely sweet-tasting protein, was secreted by the methylotrophic yeast Pichia pastoris. The mature thaumatin II gene was directly cloned from Taq polymerase-amplified PCR products by using TA cloning methods and fused the pPIC9K expression vector that contains Saccharomyces cerevisiae prepro alpha-mating factor secretion signal. Several additional amino acid residues were introduced at both the N- and C-terminal ends by genetic modification to investigate the role of the terminal end region for elicitation of sweetness in the thaumatin molecule. The secondary and tertiary structures of purified recombinant thaumatin were almost identical to those of the plant thaumatin molecule. Recombinant thaumatin II elicited a sweet taste as native plant thaumatin II; its threshold value of sweetness to humans was around 50 nM, which is the same as that of plant thaumatin II. These results demonstrate that the functional expression of thaumatin II was attained by Pichia pastoris systems and that the N- and C-terminal regions of the thaumatin II molecule do not -play an important role in eliciting the sweet taste of thaumatin.     

单纯疱疹病毒Ⅰ型糖蛋白D在酵母中的表达

从提取的HSV-1基因组中扩增得到编码gD蛋白胞外区1～314aa的基因gDt,将其插入毕赤酵母表达质粒pPIC9K的醇氧化酶(AOX1)启动子下游,构建携带gDt的重组载体,经电转化GS115菌株和G418筛选,得到了高效分泌表达gD蛋白的毕赤酵母菌株,表达量达到250mg/L,该目的蛋白可被gD单抗(1-I-9)特异性识别。表达产物经离子交换、金属螯合、分子筛柱层析纯化后得到纯度较高的重组蛋白。重组gD蛋白免疫BALB/c小鼠可诱生一定水平的特异性抗体,表明该蛋白具有较好的免疫原性,能够诱导小鼠产生体液免疫应答。     

凡纳滨对虾溶菌酶基因在毕赤酵母中的分泌表达和活性检测

原核重组表达的凡纳滨对虾(Litopenaeus vannamei)溶菌酶蛋白主要以包涵体形式存在, 经变性和复性处理后活性仍较差。研究将凡纳滨对虾溶菌酶基因(Lvlyz基因)克隆至毕赤酵母分泌型表达载体pPIC9K中, 电击转化毕赤酵母GS115细胞, 经组氨酸营养缺陷培养基筛选和PCR检测获得转化子。对其进行连续甲醇诱导表达, 利用SDS-PAGE和C端携带的6×His标签,对发酵液上清进行Western blot检测, 结果表明19.3 kD左右的条带即是重组表达的溶菌酶蛋白。用溶壁微球菌平板抑菌法鉴定表达产物具有较强的抑菌能力。研究首次利用毕赤酵母真核表达系统实现对虾溶菌酶基因的可溶性表达, 并且表达产物的活性良好。       

中华眼镜蛇心脏毒素(CardiotoxinⅢ)的克隆及其在大肠杆菌和酵母中的表达

心脏毒素cardiotoxinⅢ（CTXⅢ）是中华眼镜蛇毒主要组分之一,根据Genbank中已有的CTXs序列设计特异性引物,从中华眼镜蛇毒中克隆出208bp的CTXⅢ片段,并将该基因片段分别克隆至大肠杆菌His—patch ThioredoxinB载体中,重组CTXⅢ经渗透休克分泌至胞外。将CTXⅢ基因插入酵母pPIC9K分泌型表达载体重组表达,N端带有3个氨基酸（GYT）残基的重组CTXⅢ（rCTXⅢ）分泌量达9．5mg／L,经一步凝胶过滤纯化,其纯度达90％以上,纯化率达65％。经细胞毒性实验检测,纯化的rCTXⅢ 12h的IC50为4．66μg／ml,证实重组蛋白具有良好的生物学活性。     

中华眼镜蛇心脏毒素（cardiotoxinⅢ, CTXⅢ）的克隆及其在大肠杆菌和酵母中的表达

心脏毒素cardiotoxinⅢ (CTXⅢ)是中华眼镜蛇毒主要组分之一,根据Genbank中已有的CTXs序列设计特异性引物,从中华眼镜蛇毒中克隆出208bp的CTXⅢ片段,并将该基因片段分别克隆至大肠杆菌His-patch ThioredoxinB载体中,重组CTXⅢ经渗透休克分泌至胞外。将CTXⅢ基因插入酵母pPIC9K分泌型表达载体重组表达,N端带有三个氨基酸（GYT）残基的重组CTXⅢ（rCTXⅢ）分泌量达9.5mg/L,经一步凝胶过滤纯化,其纯度达90％以上,纯化率达65％。经细胞毒性实验检测,纯化的rCTXⅢ 12h的IC50为4.66?g/ml,证实重组蛋白具有良好的生物学活性。     

Expression of Apostichopus japonicus lysozyme in the methylotrophic yeast Pichia pastoris

Apostichopus japonicus (sea cucumber) is one of the economically important farmed echinoderm species in Northern China. As a crucial enzyme in innate immunity, lysozyme plays a key role in the overall defense against pathogens in A. japonicus. In the present study, a lysozyme gene from A. japonicus was cloned by PCR and expressed in Pichia pastoris using the expression vector pPIC9K. The expressed lysozyme had a molecular mass of ～14 kD, as shown by SDS-PAGE and Western-blotting. The expression condition was optimized, and the highest expression level was achieved by induction with 1% methanol at pH 5.0 for 120 h. The recombinant lysozyme was purified by affinity chromatography using a Ni-NTA column. The specific activity of the purified lysozyme was 34,000 U/mg using Micrococcus lysodeikticus as substrates. It exhibited antimicrobial activity toward M.lysodeikticus, as detected by growth inhibition on agar plate and turbidity assay, suggesting a potential application of A. japonicus lysozyme as an antimicrobial agent in A. japonicus aquaculture.     

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.     

人源性抗bFGF单链抗体表达载体的构建与酵母表达

为了提高人源性抗bFGF抗体的表达量,从噬菌体抗体库筛选出的人源性抗bFGF抗体基因中亚克隆单链抗体(single chain fragment variable,ScFv)基因,并将其构建到酵母表达载体pPICZαA中。表达载体质粒经线性化后,电转化法转化至毕赤酵母GS115中,甲醇诱导表达。表达产物经镍离子亲和层析和阴离子交换层析纯化,并检测其生物学活性。酶切鉴定结果显示人源性的酵母表达载体构建成功。SDS-PAGE和Western blot结果显示,抗bFGF单链抗体获得了高效表达,表达量可达124mg/L,目的蛋白大小为36 kDa左右。通过两步纯化方案,目的蛋白的纯度可达95%以上。ELISA结果显示纯化的目的蛋白可与bFGF特异性结合。CCK8检测结果显示,纯化的抗bFGF单链抗体可剂量依赖性地抑制人肺腺癌细胞株A549的增殖。研究结果表明在毕赤酵母中可获得人源性抗bFGF单链抗体高效表达,表达产物具有很好的生物学活性。     

Posttransformational vector amplification in the yeast Pichia pastoris

Generating a high yield of recombinant protein is a major goal when expressing a foreign gene in any expression system. In the methylotrophic yeast Pichia pastoris , a common means of achieving this end is to select for transformants containing multiple integrated copies of an expression vector by plating them on high levels of a selectable marker drug followed by screening for rare colonies with multiple copies. We describe a more convenient method to select for such clones. Using Zeocin-resistance-based vectors, we demonstrate that strains transformed with only one or a few vector copies can, long after transformation, be subjected to further selection at high levels of drug. This resulted in the frequent selection of clones containing increased copy numbers of the vector. This posttransformational vector amplification (PTVA) process resulted in strains containing multiple head-to-tail copies of the entire vector integrated at a single locus in the genome. Of our PTVA selected clones, 40% showed a three- to fivefold increase in vector copy number. So-called 'jackpot' clones with >10 copies of the expression vector represented 5–6% of selected clones and had a proportional increase in recombinant protein.     

Expression,purification, and characterization of equine lactoferrin in Pichia pastoris

Lactoferrin is an 80kDa iron-binding glycoprotein. It is secreted by exocrine glands. Many functions such as iron sequestering, anti-bacterial activity, regulation of gene expression, and immunomodulation are attributed to it. In the present study, we report the production of recombinant equine lactoferrin (ELF) in the methylotropic yeast Pichia pastoris using pPIC9K vector. The recombinant protein was purified by one-step affinity chromatography using heparin-Sepharose column. The purified protein has a molecular weight of 80kDa and reacted with antibody raised against the native equine lactoferrin. Its N-terminal sequence was identical to that of the native ELF. The iron-binding behavior and circular dichroism studies of the purified protein indicate that it has folded properly. The recombinant protein appears to be hyperglycosylated by the host strain, GS115. This is the first heterologous expression of equine lactoferrin and also the first report of intact lactoferrin expression using P. pastoris system. An yield of 40mg/l obtained in shake-flask cultures with this system, which is higher than the reported values for other systems.     

Expression,purification, and characterization of an unstable lysozyme mutant in Pichia pastoris

To investigate the expression and purification of an unstable heterologous protein in Pichia pastoris, the cDNA of H5-lysozyme, a hen egg lysozyme mutant with a hydrophobic pentapeptide (Phe-Phe-Val-Ala-Pro) fused to the carboxyl terminus, was integrated into the genome of P. pastoris. It was found that medium composition, induction time, and fermenter type were important factors for the expression of H5-lysozyme. Substantially active H5-lysozyme was secreted by induction with methanol when the prepro-sequence of alpha-factor was used as secretion signal sequence. The amount secreted was 422-fold greater than that observed with Saccharomyces cerevisiae. Recombinant H5-lysozyme was recovered and purified by cation-exchange chromatography directly from fermentation broth. The mutant lysozyme showed bactericidal activity against Gram-positive as well as Gram-negative bacteria.     

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。     

重组人GALNT3-sol蛋白在毕赤酵母中的高效表达和活性鉴定

目的:为了获得有催化活性的人乙酰半乳糖胺转移酶3(GALNT3),构建了GALNT3可溶性区域(GALNT3-sol)的真核分泌表达载体,在巴斯德毕赤酵母中表达并纯化GALNT3-sol蛋白,体外检测其转糖基活性。方法:以构建好的pET15b/GALNT3-sol为模板进行PCR,扩增编码人GALNT3-sol的cDNA片段(1 755 bp),将其克隆至真核表达载体pPIC9K,载体线性化后采用电击法转化毕赤酵母GS115。通过MD平板和G418平板筛选出阳性高拷贝重组菌株。阳性菌株经过甲醇诱导表达人GALNT3-sol重组蛋白,表达上清进行Ni-NAT分离纯化。分别采用SDS-PAGE和Western blot鉴定纯化的重组蛋白,并使用HPLC和MALDI-TOF/MS分析其转糖基化反应的活性。结果:成功构建了能够分泌表达GALNT3-sol的毕赤酵母菌株。阳性表达菌株在BMMY培养基(pH 6.0)中20℃培养,经0.5%甲醇诱导表达96 h,摇瓶表达量可达5mg/L。SDS-PAGE和Western blot结果显示表达重组蛋白为糖基化形式。活性检测显示表达的重组蛋白具有转糖基活性。结论:成功获得可以高效分泌表达具有活性的人GALNT3-sol蛋白的毕赤酵母菌株,为进一步研究人GALNT3的性质及其应用提供了基础。     

慢病毒siRNA靶向干扰YAP基因胃癌细胞株的建立

目的：构建并鉴定YAP基因短发夹干扰RNA（shRNA）慢病毒载体,建立稳定干扰YAP基因表达的胃癌细胞株SGC7901。方法：荧光定量PCR检测YAP基因在多种胃癌细胞株中的表达情况。构建重组靶向YAP基因的shRNA慢病毒表达质粒PGC-shRNA-YAP,用脂质体转染的方法将载体导入胃癌细胞。经杀稻瘟菌素筛选后,建立稳定表达siRNA的细胞株。荧光定量PCR检测干扰效率。结果：在胃癌细胞株SGC7901中,YAP基因显示高表达。测序验证PGC-shRNA-YAP重组质粒构建成功。将重组质粒稳定转染入胃癌细胞株SGC7901后能明显抑制YAPmRNA表达水平。结论：成功构建了PGC-shRNA-YAP慢病毒重组质粒,建立了靶向稳定干扰YAP基因表达的siRNA胃癌细胞株SGC7901。     

慢病毒siRNA靶向干扰YAP基因胃癌细胞株的建立

目的:构建并鉴定YAP基因短发夹干扰RNA(shRNA)慢病毒载体,建立稳定干扰YAP基因表达的胃癌细胞株SGC7901。方法:荧光定量PCR检测YAP基因在多种胃癌细胞株中的表达情况。构建重组靶向YAP基因的shRNA慢病毒表达质粒PGC-shRNA-YAP,用脂质体转染的方法将载体导入胃癌细胞。经杀稻瘟菌素筛选后,建立稳定表达siRNA的细胞株。荧光定量PCR检测干扰效率。结果:在胃癌细胞株SGC7901中,YAP基因显示高表达。测序验证PGC-shRNA-YAP重组质粒构建成功。将重组质粒稳定转染入胃癌细胞株SGC7901后能明显抑制YAPmRNA表达水平。结论:成功构建了PGC-shRNA-YAP慢病毒重组质粒,建立了靶向稳定干扰YAP基因表达的siRNA胃癌细胞株SGC7901。     

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。     

重组人kallistatin蛋白在毕赤酵母中的高效表达及生物活性分析

为了研究kallistatin(Kal)的生物活性,本实验构建了可分泌表达Kal的毕赤酵母菌株。首先通过PCR方法从pAAV-Kal中扩增出KalcDNA,并克隆至酵母表达载体pPIC9,得到甲醇酵母分泌型表达载体pPIC9-Kal,然后将载体线性化并电击转化毕赤酵母GS115(his4),通过MD平板筛选出阳性表达菌株。阳性表达菌株在BMMY培养基(pH7.0)中29℃培养,经2%甲醇诱导表达96h,摇瓶表达量可达14mg/L。表达上清经PhenylSuperose、Heparin SepharoseFF分离纯化,目的蛋白纯度达到98%,分子量为58kDa。生物活性实验显示,所得到的Kal蛋白具有较好的抗氧化活性,过氧化物酶活性达到(163±4)U/(mg·min),可有效降低H2O2对LX-2细胞的氧化损伤。另外,重组产生的Kal还能抑制HUVEC细胞的增殖。本研究首次成功地利用毕赤酵母表达系统分泌表达了有生物活性的Kal,为继续开展其抗肿瘤活性奠定了基础。     

Expression of recombinant chinese bovine enterokinase catalytic subunit in P. pastoris and its purification and characterization

EK (enterokinase) is a serine proteinase which consistsof a heavy chain and a light chain linked by a disulfidebond. The light chain of EK contains a chymotrypsin-likeserine proteinase domain sufficient for the normal recog-nition and cleavage of EK subst…     

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。     

单链莫内甜蛋白基因的合成及其植物表达载体的构建

目的:合成单链莫内甜蛋白基因,构建其植物表达载体。方法与结果:根据已报道的单链莫内甜蛋白的氨基酸序列及甜味机理,重新设计合成了全长294bp的莫内甜蛋白基因,其中单链莫内甜蛋白氨基酸序列中的Asp69(原AspA16)突变为Asn。利用DNA重组技术,将莫内甜蛋白基因克隆到植物表达载体pBl221中,构建了莫内甜蛋白基因的植物表达载体pBI221-monellin。结论:构建了莫内甜蛋白基因的植物表达载体,为转化园艺植物以改善其口感奠定了基础。