Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].The vector used here (pPICZαA) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the α-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.Download video file.^{(116M, mp4)}     

影响外源蛋白在巴斯德毕赤酵母中表达和分泌的研究进展

巴斯德毕赤酵母(Pichia pastoris)表达系统是基因工程研究中广泛使用的外源蛋白表达系统.但外源基因在该系统中表达时,由于受自身特性及环境等诸多因素的影响,在表达过程中出现表达量不够稳定或较低,甚至不表达的情况.本文对影响巴斯德毕赤酵母表达的各种可能因素进行了分析,并就如何提高外源基因在巴斯德毕赤酵母中表达量的问题进行了简要的综述.     

毕赤酵母表达体系中重组蛋白的分离纯化

随着基因重组技术的快速发展,基因工程产品的利用越来越广泛,但其分离纯化的成本约占总成本的60%～70%.因此,探索一些简单有效的分离纯化方法尤为必要.简单介绍了目前较为流行的毕赤酵母表达体系,着重概述了重组蛋白分离纯化技术方法的应用情况.     

重组HSA-hG-CSF融合蛋白在毕赤酵母中的表达

为了延长G-CSF半衰期,我们利用甲醇酵母表达重组人血清白蛋白融合的集落细胞刺激因子（rHSA-G-CSF）。用PCR方法从人胎肝cDNA文库扩增出HSA cDNA序列,hG-CSFcDNA序列从大肠表达载体中酶切获取。将HSA和hG-CSF两片段连接后,克隆到酵母分泌型表达载体pGENYK中,酶切线性化后原生质体转化导入酵母细胞进行整合。工程菌经发酵灌培养表达,层析法分离纯化融合蛋白。纯化的融合蛋白经Western 印迹分析表明具有HSA和G-CSF的免役原性,体外生物学活性分析表明,同縻尔数的融合表达产物的活性为E.coli表达G-CSF单体的活性的50%以上。体内动物实验研究表明,经HSA融合的G-CSF的半衰期为G-CSF单体的15-20倍。甲醇酵母表达的融合HSA的G-CSF具有比G-CSF更长的半衰期,有良好的临床应用前景。     

Expression and One-Step Purification of Recombinant Proteins Using an Alternative Episomal Vector for the Expression of N-Tagged Heterologous Proteins in Pichia pastoris

Here we report the construction of an alternative episomal vector, pBGP3, which allows the expression of heterologous proteins with N-terminal hexahistidine and myc-epitope tags in Pichia pastoris. To test the usefulness of pBGP3, four cellulases from termites were expressed. Production was confirmed by activity assays and Western blot using anti-c-Myc antibody. Purification was performed by single-step Ni²⁺-affinity chromatography, which confirmed the efficiency of pBGP3.     

Arresten在毕赤酵母中的表达和鉴定

Arresten来自人Ⅳ型胶原α-1链非胶原末端,可抑制新血管生成。从人肝脏提取总RNA,RT-PCR扩增arres-ten的cDNA,T载体进一步扩增后与表达载体pPIC9连接,测序,确认后转入毕赤酵母,获得表达可溶性arresten的酵母细胞。表达产物经初步纯化后,用SDS-PAGE测定分子量为26kD,与理论计算值接近;表达产物对matrigel辅助的内皮细胞管化有明显抑制作用。上述结果表明用毕赤酵母表达了有活性的arresten。     

Production of Recombinant Proteins with Pichia pastoris in Integrated Processing

Devices and methods for Integrated Bioprocessing have been developed for production of recombinant proteins with the yeast Pichia pastoris. In doing so cross flow filtration techniques for cell separation and product concentration are connected directly to high instrumented cultivation processes. These are equipped with on‐line measuring techniques for substrates and products, e.g., glycerol, methanol and pyruvate as well as recombinant proteins, e.g., the chemokines 1–8del MCP‐1 and vMIP‐II. Complex automation structures allow for process development at virtual plants which can be used as the basis for establishing and implementing fully automated real processes. Experiments for determination of reaction kinetics, optimization of productivity in high‐cell density cultures and Integrated Bioprocessing are outlined, along with detailed illustration of the realization of the methods at industrial pilot plant scale.     

巴斯德毕赤酵母表达外源蛋白的优化策略

巴斯德毕赤酵母(Pichia pastoris)表达系统已成为外源蛋白最理想的表达系统之一,诸多的优点体现了其广泛的研究价值和应用价值。综述了P.pastoris表达外源蛋白时在载体选择与利用、外源基因改造、翻译后修饰及表达稳定性等方面的优化策略,以加速其应用。     

重组人白细胞介素11在毕氏酵母中的表达

将人白细胞介素11基因选用酵母偏爱密码子人工合成全基因,克隆到酵母分泌型表达载体pGENYk中,酶切线性化后原生质体转化导入酵母细胞进行整合,G418筛选得到多拷贝转化子,甲醇诱导表达,纯化制备产物,经过SDS－PAGE、Western印迹及体内外生物学活性等分析表明,产物活性与E.coli融合表达的Neumega一致。     

带有PreS的重组乙肝表面抗原在毕赤酵母中的表达

带有ＰｒｅＳ区的乙肝表面抗原（ＨＢｓＡｇ）有望成为新一代更高效的乙肝疫苗。利用毕赤属酵母（Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ）表达系统,表达了带有ＰｒｅＳ区免疫决定簇的理组乙肝表面抗原Ｓ１Ｓ、ＳＳ１和Ｓ２Ｓ。对表达产物的性质鉴定表明,产物可以形成２具有相应的Ｓ、ＰｒｅＳ１或ＰｒｅＳ２抗原性的颗粒,表达水平高于啤酒酵母（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）表达系统。     

人LYC5溶菌酶基因在毕赤酵母中的重组表达

LYC5是一种c型人溶菌酶蛋白。根据毕赤酵母密码子的偏爱性,对LYC5的mRNA编码序列进行优化设计,将优化后的基因序列克隆至毕赤酵母分泌型表达载体pPIC9K中,构建重组酵母表达质粒pPIC9K- LYC5 。重组质粒经线性化处理后转化毕赤酵母GS115,应用G418抗性筛选出高拷贝转化子,并对其进行摇瓶诱导表达,产物经SDS-PAGE电泳检测,发现在约15 kDa的位置出现了一条特异蛋白条带,此条带经LTQ Orbitra pelite MS鉴定,证明此蛋白即LYC5溶菌酶蛋白,表达量约为20 mg/L。对表达上清液进行活性分析,发现表达上清对溶壁微球菌具有较好的溶菌活性,活性约为40 000 U/mg,最适酶活反应温度为45℃,最适pH为5.0。采用基因工程方法,首次表达出了有生物学活性的人源LYC5溶菌酶蛋白,为深入探讨人溶菌酶家族成员的抗菌谱及其应用前景的研究奠定了基础。     

重组人载脂蛋白C-I在毕赤酵母中的分泌表达及鉴定

目的:在毕赤酵母中高效分泌表达与天然人载脂蛋白C-I具有相同结构和活性的重组人载脂蛋白C-I( rhApoC-I).方法:RT-PCR法自人肝组织调取编码人ApoC-I的cDNA,构建真核分泌型表达载体pPICZα/hApoC-I.重组质粒线性化后转化毕赤酵母感受态细胞,甲醇诱导表达,建立rhApoC-I的毕赤酵母表达体系.对rhApoC-I进行Western blot分析和体外活性研究.结果:经PCR法克隆的hApoC-I cDNA序列与GenBank登录序列一致.SDS-PAGE和Western blot分析均在分子量约6.6kDa出现特异性条带,2L发酵条件下表达量达到80mg/L.结论:首次在毕赤酵母菌中高效分泌表达rhApoC-I,并确定其具有抑制血小板衍生生长因子诱导的平滑肌细胞增殖的抑制作用,为进一步研究其结构与功能提供物质基础.     

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.     

利用毕赤酵母表达外源蛋白的研究

综述了毕赤酵母表达系统的优越性、表达受体菌和表达载体、酵母转化、分泌信号、翻译后加工和修饰等特点,以及广泛的医用、商业用途,在理论研究特别是蛋白质结构与功能：疗面的潜在应用价值。     

Recombinant protein expression in Pichia pastoris

The methylotrophic yeast Pichia pastoris is now one of the standard tools used in molecular biology for the generation of recombinant protein. P. pastoris has demonstrated its most powerful success as a large-scale (fermentation) recombinant protein production tool. What began more than 20 years ago as a program to convert abundant methanol to a protein source for animal feed has been developed into what is today two important biological tools: a model eukaryote used in cell biology research and a recombinant protein production system. To date well over 200 heterologous proteins have been expressed in P. pastoris. Significant advances in the development of new strains and vectors, improved techniques, and the commercial availability of these tools coupled with a better understanding of the biology of Pichia species have led to this microbe's value and power in commercial and research labs alike.     

人p53蛋白在巴斯德毕赤酵母中的表达

将人ｐ５３ 基因装入 Ｐｉｃｈｉａ 分泌型质粒ｐ Ｈ Ｉ Ｌ Ｓ１ 中,酶切线性化后电穿孔导入酵母细胞进行整合,经筛选得到一高表达ｐ５３ 蛋白的克隆。 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 显示表达量约占分泌总量的３０ ％ 。 Ｅ Ｌ Ｉ Ｓ Ａ 验证重组人ｐ５３ 存在免疫学活性。在诱导时就降低 Ｐｉｃｈｉａ 酵母系统水解酶活力等方面进行优化,经 Ｆ Ｐ Ｌ Ｃ 分离纯化得到约２００ ｍ ｇ／ Ｌ 表达量。     

抗HIV-1重组导向毒素SL120在毕赤酵母中的表达及其活性检测

以pPIC9K为载体,构建抗HIV 1gp12 0单链抗体scFv12 0与葡萄球菌肠毒素A(StaphylococcalenterotoxinA ,SEA)融合基因表达质粒,线性化、电转化法整合入巴斯德毕赤酵母菌,经表型鉴定、PCR分析和G418筛选得到Muts型多拷贝整合菌,甲醇诱导培养可分泌表达5 7kD的预期大小蛋白—重组导向毒素SL120 ,表达量达50.1mg/L。通过单链抗体亲和力测定,表明蛋白SEA和scFv120的构象有微弱的相互影响,但此重组导向毒素仍可高效介导CTLs杀伤HIV-1靶细胞。     

Expression,Purification and Characterization of Recombinant Human Angiogenin in Pichia pastoris

The potential of angiogenin (Ang) for clinical use has been highlighted in view of its important roles in inducing angiogenesis, facilitating cell proliferation, and inhibiting cell apoptosis. To produce soluble, correctly folded recombinant protein with a high yield, a DNA fragment encoding human Ang was inserted into eukaryotic expression vector pPIC9 and transformed into Pichia pastoris. The expression of recombinant human Ang (rhAng) accounted for about 70% of total secreted proteins. Purifying the Ang from the culture supernatant yielded 30 mg/L at 90% purity by chromatography with a SP Sepharose FF column. Biological assays indicated that rhAng can induce new blood-vessel formation, promote HeLa cell proliferation, increase Erk1/2 phosphorylation, and upregulate c-myc expression. Preparation of bioactive rhAng might lay the basis for further functional study, and might provide an effective strategy for large-scale production of soluble human Ang.     

重组人巨细胞病毒嵌合肽基因在毕赤酵母中的克隆和表达

为了在毕赤酵母中表达带有组氨酸纯化标记的重组人巨细胞病毒嵌合肽(rHCMVp),根据 其基因序列,设计引物从pPIC9K2rHCMVp 上扩增得到目的基因片段,并导入毕赤酵母诱导型表达 载体pPICZαA 中。通过电击将线性化的重组质粒转化到毕赤酵母X33 细胞中,筛选获得表达量 较高的重组菌株,研究了该菌株生长的培养条件,包括不同诱导时间、甲醇浓度、pH 值对人巨细 胞病毒嵌合肽表达的影响。2L 发酵罐进行了高密度发酵,经1 %的甲醇、pH610 的条件下诱导 48h,最终菌体密度OD600达到180,每升发酵液中含目的蛋白7817mg,产量比摇瓶提高了418 倍。 rHCMVp 可通过高密度发酵大量获得。     

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。