不同猪胰岛素前体基因拷贝数Mut+型毕赤酵母的摇瓶发酵研究

毕赤酵母是优秀的外源蛋白的表达系统之一。本文对Mut~+型的不同PIP基因拷贝数的毕赤酵母进行了摇瓶试验。研究了生长特性以及对外源蛋白表达量的影响等。发现了低拷贝和高拷贝的蛋白表达量、生长情况有差异。G12重组菌的PIP表达量最高为181.6mg/L,是单拷贝重组菌表达量的12.6倍。对于基因拷贝数低于12的菌株,PIP表达水平与PIP基因拷贝数成线性关系(r=0.996)。     

一种增加毕赤酵母生产胰岛素前体的方法

为了提高胰岛素前体(PI)的产量,构建了p PIC9K-PI表达载体并电转化至毕赤酵母菌株GS115中,在浓度为4.0 mg/m L的G418抗性平板上筛选到了1株拷贝数为12的菌株CL012。将SNAREs(可溶性N-乙基马来酰亚胺敏感因子受体蛋白)组分中的SNC2和SNC2-SSO2分别转入菌株CL012中,并在摇瓶和5 L发酵罐水平上检测SNAREs对PI产量的影响。结果表明:摇瓶水平上,甲醇诱导96 h后,菌株CL012的PI产量为1.53 mg/L;表达SNC2和SNC2-SSO2的菌株的PI产量分别为1.89 mg/L和2.21 mg/L,分别比菌株CL012提高了23.53%和44.44%。在5 L发酵罐上进行高密度发酵,甲醇诱导96 h后菌株CL012的PI产量为53 mg/L,是摇瓶水平的34.64倍;表达SNC2和SNC2-SSO2的菌株的PI产量分别达到64 mg/L和78 mg/L,分别比菌株CL012提高了20.75%和47.17%。由此得出结论 SNAREs可以促进胰岛素前体的分泌,从而提高在毕赤酵母中的异源表达。     

一种增加重组毕赤酵母拷贝数提高胰岛素前体产量的策略

[目的] 为了进一步提高毕赤酵母表达生产人胰岛素前体的产量,[方法] 将构建的表达载体pPICZα-IP 电转至毕赤酵母X-33中,在100 μg/mL zeocin的YPDS抗性平板筛选获得过量表达人源胰岛素前体(IP)的重组菌株B4和S6.在此基础上,以过量表达胰岛素前体的B4和S6为出发菌株,以SacI线性化的表达载体pPICZα-IP进行重复电转化,并在1000 μg/mL zeocin的YPDS平板上筛选获得一株拷贝数为7的重组毕赤酵母2B4.[结果] 该菌株在5L规模发酵罐上,人源胰岛素前体产量是低拷贝数菌株B7的2.7倍,且菌体并未因拷贝数高而表现出生长不良的现象.实时定量PCR检测后,发现2B4菌株胰岛素前体基因的转录水平是B7的2倍.[结论] 综上结果表明,采用抗性筛选和重复电转相结合的高拷贝重组毕赤酵母构建策略,能有效促进目的基因的转录水平,最终显著提高目标蛋白的产量.     

人胰岛素在甲醇酵母Pichia pastoris中的分泌表达

将猪胰岛素前体基因和在其５’端引入９个氨基酸的间隔肽序列的ＰＩＰ基因插入到Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ的分泌表达质粒ｐＰＩＣ９中,得到分泌表达质粒ｐｐＩＣ９／ＰＩＰ和ｐＰＣ９／ｓｐ－ＰＩＰ７并用以转化ｐａｓｔｏｒｉｓ ＧＳ１１５。用点杂交筛选,获得高拷贝转化Ｐ３９（－ｓｐ）和Ｓ５１（＋ｓｐ）。     

一种多拷贝毕赤酵母表达载体的构建及人脑源性神经营养因子的表达

利用PCR技术扩增出pPIC9K载体的HIS4 Kan序列片段 ,与pPICZα重组 ,构建结合了两个载体特点的适合体外构建多拷贝基因表达盒的毕赤酵母表达载体pPICZα1,改造后的载体含HIS4 Kan序列 ,能整合到酵母染色体上 ,具有筛选方便、外源基因多拷贝组建迅速、蛋白产物分泌表达和易纯化等优点。将人脑源性神经营养因子(hBDNF)的cDNA(35 7bp)克隆入载体pPICZα1,利用同尾酶BglⅡ、BamHⅠ不可逆连接方式 ,分别构建含有 1、2、3、6个拷贝hBDNF表达盒的重组表达载体 ,电击法转化毕赤酵母GS115菌株 ,用G4 18和Zeocin筛选转化子 ,筛选到的阳性转化子用 0 5 %甲醇诱导 ,获得分泌型表达。表达产物类似于天然神经营养因子单体大小、分子量约 14kD。多拷贝hBDNF表达盒的表达水平亦高于单拷贝hBDNF表达盒 ,ELISA和Westernblot检测表明 :表达的蛋白能与鸡抗人脑源性神经营养因子抗体特异结合 ,证实该表达蛋白具有hBDNF的免疫原性     

Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris

We have found a direct relationship between protein production in Pichia pastoris and the number of introduced synthetic genes of miniproinsulin (MPI), fused to the Saccharomyces cerevisiae pre-pro alpha factor used as secretion signal, and inserted between the alcohol oxidase 1 (AOX1) promoter and terminator sequences. Two consecutive approaches were followed to increase the number of integrated cassettes: the head-to-tail expression cassette multimerization procedure and re-transformation with a dominant selection marker. This increased expression from 19 to 250 mg l^–1 when about 11 copies have been integrated. Further, the correct position of one of the disulphide bridges of the purified molecule was verified by digestion with Glu-C endoprotease, followed by mass spectrometry of the isolated fragments.     

中心复合实验设计法优化重组毕赤酵母表达猪胰岛素前体的培养基和诱导pH

毕赤酵母中猪胰岛素前体(Porcine Insulin Precursor,PIP)的表达受到培养基组成及培养环境的影响。本文首先通过部分因子法设计实验,筛选出影响PIP表达的显著因子。实验结果表明,在试验范围内,甲醇补料量和硫酸铵浓度为正效应因子,诱导pH为负效应因子。在此基础上,经过快速登高法逼近显著因子的最优值后,再在此值附近利用中心复合法设计实验,最终得到了PIP表达的最佳条件。经过多批次实验验证,在此条件下PIP的表达量为120.4mg/L,比优化前的值41.5mg/L提高了将近两倍。     

High-level secretory expression of immunologically active intact antibody from the yeast Pichia pastoris

We have produced a functional murine antibody to dioxin in the culture medium of the methylotrophic yeast Pichia pastoris. Complementary DNA copies encoding the light () and heavy () chains of the dioxin monoclonal antibody, DD1, were each placed under the control of P.pastoris alcohol oxidase (AOX1) promoter and Saccharomyces cerevisiae -mating factor secretion signal sequence. The resulting expression cassettes were assembled into a single plasmid (pPICZDD1) to permit co-expression of both light and heavy chains of the antibody molecule. P.pastoris SMD1168 (pep4, his4) transformed with pPICZDD1 was able to secrete intact antibody into the culture medium. As high as 36 mg l^–1 of the antibody was produced in shake-flask cultures after 96-h induction with methanol. Functional analysis using immunoassay confirmed murine nature of the recombinant antibody and its ability to bind dioxin.     

基因工程抗体在巴斯德毕赤酵母中表达的研究进展

简述了运用巴斯德毕赤酵母系统表达基因工程抗体从构建载体到表达的一般过程,及如何改善和提高抗体的表达。侧重介绍运用该系统获得抗体高表达、高产量的研究近况。     

Heterologous protein expression in the methylotrophic yeast Pichia pastoris

During the past 15 years, the methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of heterologous proteins. The increasing popularity of this particular expression system can be attributed to several factors, most importantly: (1) the simplicity of techniques needed for the molecular genetic manipulation of P. pastoris and their similarity to those of Saccharomyces cerevisiae, one of the most well-characterized experimental systems in modern biology; (2) the ability of P. pastoris to produce foreign proteins at high levels, either intracellularly or extracellularly; (3) the capability of performing many eukaryotic post-translational modifications, such as glycosylation, disulfide bond formation and proteolytic processing; and (4) the availability of the expression system as a commercially available kit. In this paper, we review the P. pastoris expression system: how it was developed, how it works, and what proteins have been produced. We also describe new promoters and auxotrophic marker/host strain combinations which extend the usefulness of the system.     

Expression of monomeric insulin precursor in Pichia pastoris and its conversion into monomeric B27 Lys destripeptide insulin by tryptic hydrolysis

Monomeric B27 Lys destripeptide insulin (B27 Lys DTrI) was designed and produced from its precursor expressed in Pichia pastoris through tryptic hydrolysis instead of the less efficient tryptic transpeptidation. The monomeric B27 Lys DTrI precursor (MIP) was purified from a cultured medium of P. pastoris by a combination of hydrophobic, size-exclusion, and ion-exchange chromatography. The purified MIP was converted, by tryptic hydrolysis, to B27 Lys DTrI, which was then purified by ion-exchange chromatography to homogeneity as assessed by native gel electrophoresis, HPLC, amino acid composition,and electrospray mass-spectrometric analysis. B27 Lys DTrI exhibited superior monomeric properties in size-exclusion chromatography. The yield of MIP was 200 mg per liter of culture, and the overall yield of purified B27 Lys DTrI from the crude MIP was 70%. The in vivo biological activity of B27 Lys DTrI as determined by the mouse convulsion assay was 21 U/mg, identical to that obtained by semisynthesis.     

基因拷贝数对重组毕赤酵母产谷氨酰胺转胺酶的影响

基于毕赤酵母核糖体DNA序列 (rDNA),构建多拷贝谷氨酰胺转胺酶基因表达载体pPICZα-rDNA- mtg,并转化到表达前导肽 (Pro peptide或pro) 的宿主菌pGAP9-pro/GS115,得到共表达菌株pro/rDNA-mtg (GS115)。实时荧光定量PCR (qPCR) 分析了4株阳性表达菌株中mtg基因拷贝数,进一步研究了不同基因拷贝数对重组毕赤酵母产酶的影响及高产菌株在3 L发酵罐高密度发酵。结果表明,被检测的4株阳性表达菌株中mtg拷贝数分别为2.21、3.36、5.72和7.62 (mtg-2c、mtg-3c、mtg-6c和mtg-8c),其发酵产酶能力和蛋白质表达水平为mtg-3c>mtg-2c>mtg-6c>mtg-8c;高密度发酵较低和较高拷贝数的两株菌mtg-3c和mtg-6c,发酵上清的最高酶活和单位菌体酶活分别为3.12 U/mL、52.1 U/g湿重和2.07 U/mL、36.5 U/g湿重,其中单位菌体酶活mtg-3c是mtg-6c的1.4倍;mtg-3c纯化酶的最高酶活达到7.21 U/mL,蛋白浓度为437.2 μg /mL。通过分析拷贝数对重组毕赤酵母产酶的影响,发现mtg-3c适合pro/rDNA-mtg中pro和mtg共表达,MTG高酶活与菌株较高分泌蛋白有关。     

甲醇／山梨醇共混诱导改善重组毕赤酵母表达猪a干扰素发酵过程和NADH再生效率

在10L发酵罐中利用重组毕赤酵母诱导表达猪a干扰素（pIFN-a）,考察甲醇／山梨醇共混诱导策略对pIFN-a表达水平提高和能量（NADH）再生效率的影响。结果表明：在诱导稳定期,甲醇／山梨醇共混诱导可弱化细胞的甲醇代谢,有利于缓解毒副中间产物（过氧化氢、甲醛等）的生成积累;以0．785g／（L·h）的速率缓慢共混流加山梨醇时,pIFN-a抗病毒活性最大,最高活性可达1．8×107IU／mL,与30℃常温甲醇单独诱导（最高活性1．0X10。IU／mL）和20℃低温甲醇单独诱导（最高活性1．4×lO6IU／mL）相比,活性均大幅提高,且胞外pIFN-a的降解减缓;发酵体系的抗高甲醇浓度冲击能力有效提高,发酵生产的稳定性增强;能量利用效率大幅提高,NADH的再生利用效率提高了29％-84％。     

猪干扰素-γ基因在毕赤酵母中的分泌表达

将去除信号肽的猪干扰素-γ（PoIFN-γ）基因置于酿酒酵母α因子分泌信号的DNA序列后, 构建成pPIC9K-α-PoIFN-γ分泌型重组表达载体, 电转化导入毕赤酵母GS115中,经G418筛选后获得2株多拷贝插入的重组子。SDS-PAGE和Western blot分析结果表明,所获得的重组子能够分泌表达出17kD和23kD左右的PoIFN-γ特异蛋白,其表达量为108mg/L,占培养液总蛋白的60%。实验首次在毕赤酵母表达系统中实现了PoIFN-γ基因的分泌表达。     

影响外源基因在巴氏毕赤酵母中表达的因素

要在一种宿主表达系统中成功表达外源蛋白并获得较高产量,必须要较为全面地了解影响其表达的许多因素。影响外源基因在巴氏毕赤酵母中表达的因素主要包括：外源基因的特性、表达框的染色体整合位点和方式、宿主菌的甲醇利用表型、基因剂量、分泌信号、产物稳定性和翻译后修饰等。本文就这些因素进行分析,并提出一定的对策和建议。     

重组毕赤酵母甲醇利用表型与基因拷贝数对外源基因表达的影响

毕赤酵母是目前最优秀的外源蛋白表达系统之一。本文着重对重组毕赤酵母甲醇利用表型（Mut+型、MutS型和Mut-型）、基因剂量对外源蛋白高效表达的影响机理进行综述。MutS型的比生长速率和蛋白产率比Mut+型低、发酵周期长、副产物（如乙醇、乙酸等）形成速率不同。外源基因拷贝数对外源蛋白的影响主要有三种情况：（1）高基因拷贝数对外源蛋白表达水平有明显的正效应作用;（2）基因拷贝数增加反而降低了表达水平,即负效应作用;（3）重组蛋白表达与基因剂正相关,之后则表现负相关关系,这可能与外源蛋白翻译后加工有关（如二硫键形成、折叠等）,而与分子伴侣共表达可促进外源蛋白的高表达。     

人血管内皮细胞生长抑制因子在巴斯德毕赤酵母中的分泌表达

血管内皮细胞生长抑制因子抑制肿瘤部位新生血管的形成,切断肿瘤细胞营养供应及废物排泄通道,抑制肿瘤细胞恶性增殖。将编码成熟的人血管内皮细胞生长抑制因子基因克隆到表达载体pPICZα中,电转化P.pastorisGS115菌株,抗生素ZeocinTM浓度梯度筛选高抗性转化子,PCR筛选阳性重组菌株。经表型鉴定后,用甲醇进行诱导表达,SDS-PAGE和Western印迹杂交结果证实了表达产物为重组人血管内皮细胞生长抑制因子-his6融合蛋白,表达量约为5mg/L。经细胞毒性试验测定,表达产物对人脐静脉内皮细胞HUVEC增殖具有较明显的抑制作用。     

利用毕赤酵母系统直接分泌表达具有活性的谷氨酰胺转胺酶

使用异源表达系统直接分泌表达具有活性的微生物谷氨酰胺转氨酶(Microbial transglutaminase,MTG)是目前最具前景的MTG生产方法之一,但由于产量较低无法实现工业化生产.毕赤酵母是近年来发展出的高效蛋白表达系统.通过采用pro序列与成熟MTG基因共表达的策略,成功地实现了用重组毕赤酵母分泌表达具有活性的茂原链霉菌Streptomyces mobaraense MTG.进一步通过对pro序列和MTG基因拷贝数以及重组酵母培养条件的优化,最终使得MTG在1L发酵罐中高密度发酵的酶活达到7.3 U/mL,为MTG的工业化生产奠定了基础.     

猪白细胞介素-6在巴斯德毕氏酵母(Pichia pastoris)中的分泌表达

白细胞介素6 (Interleukin_6 ,IL_6 )是一种具有多种生物学效应的细胞因子,在疾病诊断与疫苗佐剂领域有广阔的应用前景。在本试验中,猪白细胞介素_6 (pIL_6 )的cDNA序列被克隆入甲醇酵母(Pichiapastoris)分泌表达载体pPIC9K中,并转化入P .pastorisGS115菌株。其重组菌株GS115 pPIC9K_IL6经1%甲醇诱导后,能分泌表达分子量约为2 4 5KD的重组蛋白,Westernblot确证为pIL_6。该酵母表达产物无N端糖基化修饰。用依赖IL6生长的B9细胞株检测提纯后的pIL_6 ,其生物学活性可达8×10 4 IU mg。