人乳头瘤病毒16型L1基因在毕赤酵母中表达的影响因素分析

目的:优化人乳头瘤病毒16型主要衣壳蛋白L1(human papillomavirus type 16 major capsid protein L1,HPV16L1)在毕赤酵母中的表达,并考察可能的影响因素。方法:四个不同序列特征的HPV16L1基因M16、Y16、P16、W16(其中,M16和Y16按酵母密码子优化,P16为哺乳动物细胞密码子优化,而W16为野生型序列)分别克隆于毕赤酵母表达质粒p PinkTM-HC(高基因拷贝菌落筛选)和p PinkTM-LC(低基因拷贝菌落筛选),并转化不同蛋白酶缺陷的宿主菌。甲醇诱导24小时后,取菌体样品经Western blot分析L1蛋白的表达。结果:M16显示了最高的表达水平,其次是Y16与P16,而W16几乎无表达。基因序列密码子应用特征分析显示,4个基因的密码子适应指数从高到低依次为Y16、M16、W16和P16。通过自由能和GC含量分析4个序列的mRNA二级结构,Y16为-409.40 kcal/mol和43.85%;M16为-451.50 kcal/mol和47.83%;P 16为-606.50kcal/mol and 64.10%;W16为-384.70 kcal/mol and 38.01%。蛋白酶缺陷菌株L1表达高于野生型菌株,质粒p PinkTM-HC与p PinkTM-LC介导的表达无明显区别。结论:密码子优化操作显著改善了HPV16L1在毕赤酵母中的表达,但表达水平与密码子利用优劣并不完全对应,提示密码子优化仅是部分原因,而mRNA结构与稳定性变化值得探讨。蛋白酶缺陷菌株提高了HPV16L1蛋白的稳定性,显著影响了表达水平。研究证明基因剂量对HPV16L1的表达未产生明显影响。     

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 .     

植酸酶基因的多点突变及在毕赤酵母中的高效表达

根据毕赤酵母基因的密码子选择偏爱性,不改变其编码氨基酸序列,对来源于黑曲霉N25植酸酶phyA基因,进行了突变,构建了含有正确突变的酵母表达载体pPIC9k-phyAm-4,电击转化毕赤酵母,获得优化了密码子的重组酵母转化子。经PCR鉴定表明,植酸酶基因已整合到酵母基因组中; 表达产物的SDS-PAGE分析表明,酶蛋白分子大小为70.15KD。Southern blotting结果表明,phyA基因整合到酵母染色体DNA中;转化子酶活测定结果表明,经密码子优化的重组酵母PP-NPm-4-2酶活可达136900U/ml,比Arg没有优化的PP-NPm-8 (47600 Uoml-1)酶活高约2.8倍。     

甘油脱氢酶基因在大肠杆菌中的密码子优化表达

[目的]利用密码子优化技术,提高甘油脱氢酶基因gldA在大肠杆菌中的表达水平.[方法]针对gldA起始密码子下游区域,优先选择AT含量最高的同义密码子,从而在不改变氨基酸序列的前提下,提高该区域的AT含量.利用大引物PCR的方法对野生型gldA-WT进行定点突变,获得优化型基因gldA-4,与pET-32a(+)连接后...     

鸡α干扰素在毕赤酵母中的表达及糖基化对其表达的影响

目的:实现鸡α干扰素(ChIFNα)在毕赤酵母GS115中的表达,并考察糖基化对其表达的影响。方法:人工合成按照毕赤酵母偏好密码子优化的ChIFNα基因,克隆至分泌表达载体pPIC9,电转到毕赤酵母GS115中进行表达。利用定点突变对ChIFNα基因序列中4个糖基化位点进行缺失,SDS-PAGE分析N-糖基化对毕赤酵母表达ChIFNα的影响。结果:ChIFNα在GS115中获得了表达,在摇瓶发酵条件下,表达量为35.2 mg/L;构建了缺失1~4个糖基化位点的4个突变体,在GS115中实现了表达,分析表达结果显示,与未突变的ChIFNα对照相比,突变体的糖基化程度和表达量都有大幅度下降。结论:N-糖基化对于毕赤酵母表达ChIFNα具有重要影响,无糖基化的ChIFNα在毕赤酵母中表达量极低。     

基于密码子优化的蛋白酶K在毕赤酵母中的表达及分离纯化

【目的】提高蛋白酶K在毕赤酵母中的表达产量,建立分离纯化方法。【方法】首先对蛋白酶K密码子进行优化,将其导入毕赤酵母GS115中实现分泌表达。然后对甲醇浓度、发酵温度和p H等表达条件进行优化,再对硫酸铵沉淀、亲和层析等纯化工艺进行比对分析。【结果】蛋白酶K密码子优化后实现了在毕赤酵母中的高效表达。在甲醇量0.75%、温度25°C和p H 7.0条件下进行发酵罐培养,蛋白酶K表达量达到2.2 g/L。采用Ni-NTA亲和柱对发酵液进行纯化可以得到较好的纯化效果。【结论】密码子优化后的蛋白酶K在毕赤酵母中高效表达并可以利用Ni-NTA亲和柱进行有效分离纯化。     

高效表达具有生物学活性的植酸酶的毕赤酵母

对来源于Aspergillusniger 96 3的植酸酶基因 phyA2进行了改造 ,包括去掉基因中的内含子和信号肽编码序列 ,在不改变所编码氨基酸的情况下 ,定点突变优化了对此基因在酵母中高效表达起关键作用的Arg密码子 .经改造的植酸酶基因按正确的阅读框架融合到毕赤酵母 (Pichiapastoris)高效表达载体 pPIC9上的α 因子信号肽编码序列 3′端 ,重组表达载体电击转化毕赤酵母得到重组转化子 ,经Southernblotting分析证实了植酸酶基因已整合到酵母基因组中 ,并确定了基因整合的拷贝数 .Northernblotting分析证明植酸酶基因能进行正常的转录 .SDS PAGE分析和表达产物的酶活性研究证明 ,植酸酶能有效分泌和高效表达 (Arg密码子优化后其表达量可达每毫升培养液 1 5 0 0 0U ,比Arg密码子没有优化的表达量高约 37倍 ,比原菌株A .niger 96 3的表达量高约 30 0 0倍 ) ,表达产物具有正常的生物学活性 .这是迄今为止我国饲料工业中 ,构建的第 1株具有实用价值和应用前景的生产饲料添加剂的基因工程菌株     

角毛壳菌几丁质酶基因chi58多点突变及在毕赤酵母中的高效表达

应用重叠延伸PCR技术(gene splicing by overlap extension PCR,gene SOEing),简称SOE-PCR对角毛壳菌(Chaetomium cupreum)的几丁质酶基因chi58进行多点突变.依据毕赤酵母密码子偏爱性,将毕赤酵母中编码Arg使用频率几乎为0的密码子CGC突变为使用频率高的AGA,构建了含有正确突变的酵母表达载体pPIC9K-chi58A,电转化毕赤酵母GS115,获得的重组酵母株在诱导120 h酶活力最高,平均可达101.71 U/mL±3.33 U/mL;其活力比未优化重组酵母株(31.83 U/mL±4.85 U/mL)提高了约3倍,且经10代传代培养后遗传稳定性良好.表达产物的SDS-PAGE分析表明,酶蛋白分子大小为58 kD.     

合成耐高温α-淀粉酶PFA在巴斯德毕赤酵母中的分泌表达

PFA是来源于Pyrococcus furious的一种耐高温α-淀粉酶,为了使PFA能够在巴斯德毕赤酵母中高效表达,根据巴斯德毕赤酵母密码子的偏好性对PFA的基因序列进行密码子优化,人工合成耐高温淀粉酶PFA基因pfa,并连接到巴斯德毕赤酵母中表达载体pPIC9K上,得到重组质粒pPIC9K-pfa。重组质粒线性化后转化到巴斯德毕赤酵母菌株GS115中,重组菌株在摇瓶中用甲醇诱导表达,分泌表达酶活最高为220U／L。     

利用毕赤酵母系统表达重组人生长激素的研究

为了获得重组人生长激素在毕赤酵母中高表达的菌株,按毕赤酵母基因密码子偏爱性,人工合成hGH的全基因序列.该基因被克隆到穿梭质粒pPIC9K中,PEG1000介导转入毕赤酵母GS115细胞,通过G418筛选获得高拷贝转化子.在甲醇的诱导下.实现了hGH在毕赤酵母中的成功表达.通过发酵条件的优化.发酵上清中的表达量达1537 mg/L经过超滤和两步层析,重组蛋白的得率这35%,纯度为97%,相对分子质量测定表明重组蛋白的相对分子质量与理论值相近.N-端氨基酸测序证实hGH基因在毕赤酵母中获得正确的表达.     

毕赤酵母表达系统外源基因表达序列优化的软件设计

作为真核生物表达系统,毕赤酵母已成功表达了包括病毒、细菌和真核生物在内的多种外源蛋白。但并非所有的外源序列在毕赤酵母中都能得以高表达,外源序列含有毕赤酵母中低频密码子或是序列中A T含量不合理是限制高表达的重要原因。根据毕赤酵母密码子使用的有关特性,利用遗传算法,建立外源基因表达序列优化的数学模型,并依此设计一套毕赤酵母外源基因表达序列的优化软件。     

毕赤酵母的密码子用法分析

通过分析Pichia pastoris的28个蛋白编码基因的同义密码子使用情况并计算该酵母的密码子用法,首次确定出P.pastoris的19个高表达优越密码子。这些结果经与已知的Saccharomyces cerevisiae及Kluyveromyces lactis的密码子用法基本相似,但在氨基酸谷氨酸的密码子选择上截然相反,提示这可能属于P.pastoris所偏爱的密码子用法。     

来源于Escherichia coli的高比活植酸酶基因的高效表达

高效表达高比活植酸酶是进一步提高植酸酶发酵效价、降低植酸酶生产成本的一个有效途径。对源于Escherichiacoli的高比活植酸酶基因appA ,按照毕赤酵母 (Pichiapastoris)密码子的偏爱进行了密码子优化改造。该改造后的基因appA m按正确的阅读框架融合到毕赤酵母表达载体pPIC9上的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。对重组毕赤酵母的Southernblotting分析证实植酸酶基因已整合到酵母基因组中 ,并确定了整合基因的拷贝数。Northernblotting分析证实植酸酶基因得到了正常转录。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 2 5mg mL发酵液 ,酶活性 (发酵效价 )达到 7 5× 10 6 IU mL发酵液以上 ,大大高于目前报道的各种植酸酶基因工程菌株的发酵效价。     

Synthesis of Drosophila melanogaster acetylcholinesterase gene using yeast preferred codons and its expression in Pichia pastoris

To improve the expression level of recombinant Drosophila melanogaster AChE (R-DmAChE) in Pichia pastoris, the cDNA of DmAChE was first optimized and synthesized based on the preferred codon usage of P. pastoris. The synthesized AChE cDNA without glycosylphosphatidylinositol (GPI) signal peptide sequence was then ligated to the P. pastoris expression vector, generating the plasmid pPIC9K/DmAChE. The linearized plasmid was homologously integrated into the genome of P. pastoris GS115 via electrotransformation. Finally seven transformants with high expression level of R-DmAChE activity were obtained. The highest production of R-DmAChE in shake-flask culture after 5-day induction by methanol was 718.50units/mL, which was about three times higher than our previous expression level of native DmAChE gene in P. pastoris. Thus, these new strains with the ability to secret R-DmAChE in the medium could be used for production of R-DmAChE to decrease the cost of the enzyme expense for rapid detection of organophosphate and carbamate insecticide residues.     

基因表达水平与同义密码子使用关系的初步研究

提出一个预测基因表达水平和同义密码子使用的自洽信息聚类方法。将同义密码子分成最适密码子、非最适密码子和稀有密码子,认为三者的使用频率是调控基因表达水平的主要因素。基于这一观点,对Ｅｃｏｌｉ和Ｙｅａｓｔ两类生物的基因表达水平和密码子的使用,用自洽信息聚类方法进行了预测。发现高低表达基因明显分开,基因表达水平被分为四级;甚高表达基因（ＶＨ）、高表达基因（Ｈ）、较低表达基因（ＬＭ）和低表达基因（ＬＬ）;     

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     

Optimization of codon usage of the envelope protein E2 gene from various genotypes of hepatitis C virus to predict the expression level in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Hepatitis C virus infection (HCV) alarmingly increases worldwide; it causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, so there is urgent need of developing effective and sufficient quantity of vaccine. HCV envelope protein E2 is the main target for developing as a vaccine candidate. Presently recombinant proteins can successfully be used as a vaccine for many diseases. This concern, it is challenging to produce sufficient quantities of many recombinant proteins from their expression hosts. One of the main factors affecting the success of expression of foreign genes in heterologous hosts is the divergence of codon usage of the target gene from that used in the expression system. In this study, we optimized the various genotypes of HCV envelope protein E2 gene according to the codon usage of Pichia pastoris and predicted the expression level. Synonymous codon usage of E2 adapted to that used by P. pastoris was estimated using the relative synonymous codon usage value (RSCU), codon adaptation index (CAI) and effective number of codon (ENC). The CAI of optimized HCV E2 sequences was enhanced from 0.638 to 0.833 and %GC was decreased from 56.05 to 44.05; this was significantly (p < 0.01) different from the native sequences. Codon with RSCU value less than one was replaced with most preferred synonymous codons. The ENC values of optimized HCV E2 sequences varied from 47.00 to 47.50, with a mean value of 47.15 and an SD of 0.14. Our study suggested that, from the measured values of predicted expression level, the codon optimized HCV E2 protein could be produced in sufficient quantity in the expression host; knowledge of the codon usage patterns of E2 of various genotypes facilitate the production of a promising unique vaccine candidate for HCV.     

Attenuated secretion of the thermostable xylanase xynB from Pichia pastoris using synthesized sequences optimized from the preferred codon usage in yeast

Xylanase has been used extensively in the industrial and agricultural fields. However, the low-yield production of xylanase from native species cannot meet the increasing demand of the market. Therefore, improving the heterologous expression of xylanase through basic gene optimization may help to overcome the shortage. In this study, we synthesized a high-GC-content native sequence of the thermostable xylanase gene xynB from Streptomyces olivaceoviridis A1 and, also designed a slightly AT-biased sequence with codons completely optimized to be favorable to Pichia pastoris. The comparison of the sequences' expression efficiencies in P. pastoris X33 was determined through the detection of single-copy-number integrants, which were quantified using qPCR. Surprisingly, the high GC content did not appear to be detrimental to the heterologous expression of xynB in yeast, whereas the optimized sequence, with its extremely skewed codon usage, exhibited more abundant accumulation of synthesized recombinant proteins in the yeast cell, but an approximately 30% reduction of the secretion level, deduced from the enzymatic activity assay. In this study, we developed a more accurate method for comparing the expression levels of individual yeast transformants. Moreover, our results provide a practical example for further investigation of what constitutes a rational design strategy for a heterologously expressed and secreted protein.     

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。     

Codon optimization, expression, and characterization of an internalizing anti-ErbB2 single-chain antibody in Pichia pastoris

Anti-ErbB2 antibodies are used as convenient tools in exploration of ErbB2 functional mechanisms and in treatment of ErbB2-overexpressing tumors. When we employed the yeast Pichia pastoris to express an anti-ErbB2 single-chain antibody (scFv) derived from the tumor-inhibitory monoclonal antibody A21, the yield did not exceed 1-2 mg/L in shake flask cultures. As we considered that the poor codon usage bias may be one limiting factor leading to the inefficient translation and scFv production, we designed and synthesized the full-length scFv gene by choosing the P. pastoris preferred codons while keeping the G+C content at relatively low level. Codon optimization increased the scFv expression level 3- to 5-fold and up to 6-10 mg/L. Northern blotting further confirmed that the increase of scFv expression was mainly due to the enhancement of translation efficiency. Investigation of culture conditions revealed that the maximal cell growth and scFv expression were achieved at pH 6.5-7.0 with 2% casamino acids after 72 h methanol induction. Secreted scFv was easily purified (>95% homogeneous product) from culture supernatants in one step by using Ni2+ chelating affinity chromatography. The yield was approximately 10-15 mg/L. Functional studies showed that the A21 scFv could be internalized with high efficiency after binding to the ErbB2-overexpressing cells, suggesting this regent may prove especially useful for ErbB2-targeted immunotherapy.