Secretory expression and purification of the recombinant duck interleukin-2 in Pichia pastoris

Interleukin-2 (IL-2) is a vital cytokine secreted by activated T lymphocytes, and plays an important role in the regulation of cellular functions and immunity of animals. In this study, the recombinant duck IL-2 (rduIL-2) was secretory expressed in Pichia pastoris (P. pastoris). The recombinant P. pastoris strain was cultured in shake flasks and then scaled up in a 5.0-l bioreactor. The result showed that the maximal fresh-cell-weight of 594.1 g/l and the maximal OD600 of 408 were achieved in the bioreactor. The rduIL-2 was purified by two steps of purification procedures, and approximately 311 mg of rduIL-2/L fermentation supernatant was obtained. SDS-PAGE showed that the purified rduIL-2 constituted a homogeneous band of ~16 kDa or ~14 kDa corresponding to the glycosylated or non-glycosylated duIL-2 protein in size, respectively. The bioactivity of rduIL-2 was determined by lymphocyte proliferation assay. The result indicated that the rduIL-2 greatly promoted the proliferation of ConA-stimulated lymphocytes in vitro. The P. pastoris expression system described here could provide promising, inexpensive, and large-scale production of the rduIL-2, which lays the foundation for development of novel immunoadjuvants to enhance both the immunity of ducks against various infectious pathogens and vaccine efficacy.     

透明颤菌血红蛋白基因vgb和腈水解酶基因bxn在毕赤酵母中的表达研究

为获得高效表达外源蛋白的Pichia pastoris菌株而设计了重组质粒pPIC9K—vgbbxn,其中透明颤菌血红蛋白基因vgb胞内表达以提高菌体的发酵密度,腈水解酶基因bxn分泌表达。转化GS115菌株后,通过PCR、SDS-PAGE检测证实两基因已经整合进酵母基因组且能高效表达,以及用准确的蛋白活性测定方法成功地检测到二所表达的产物均具有正常的活性。摇瓶发酵实验证明,血红蛋白在贫氧条件下可明显促进酵母菌体生长和bxn基因分泌表达。     

Expression, purification, and characterization of equistatin in Pichia pastoris

Equistatin (EI) is a cysteine protease inhibitor that was isolated from the sea anemone Actinia equina. It belongs to a recently discovered group of thyroglobulin type-I domain inhibitors called thyropins. Since native EI is found only in low amounts in the body of sea anemone and expression of recombinant EI in Escherichia coli yielded only 1 mg/liter of protein, we used the Pichia pastoris expression system to obtain higher yields. A cDNA encoding EI was inserted into pPIC9 vector and transformed into the P. pastoris, strain GS115. Clones expressing high levels of EI were selected from 48 transformants. Recombinant EI was produced in 2-liter shake flasks and recovered from the fermentation broth by affinity chromatography using CM-papain-Sepharose. SDS-PAGE and N-terminal sequence analysis revealed that EI was N-terminally intact and running at the expected molecular weight of 22 kDa. The equilibrium dissociation constants of EI with papain and bovine cathepsin D were determined and were found to be similar to the results for the native inhibitor. EI production was scaled up to a bench top fermentor with a 25 mg/liter yield of active EI.     

Production of large quantities of isotopically labeled protein in Pichia pastoris by fermentation

Heterologous expression in Pichia pastoris has many of the advantages of eukaryotic expression, proper folding and disulfide bond formation, glycosylation, and secretion. Contrary to other eukaryotic systems, protein production from P.pastoris occurs in simple minimal defined media making this system attractive for production of labeled proteins for NMR analysis. P.pastoris is therefore the expression system of choice for NMR of proteins that cannot be refolded from inclusion bodies or that require post-translational modifications for proper folding or function. The yield of expressed proteins from P.pastoris depends critically on growth conditions, and attainment of high cell densities by fermentation has been shown to improve protein yields by 10–100-fold. Unfortunately, the cost of the isotopically enriched fermentation media components, particularly 15NH4OH, is prohibitively high. We report fermentation methods that allow for both 15N- labeling from (15NH4)2SO4 and 13C-labeling from 13C-glucose or 13C-glycerol of proteins produced in Pichia pastoris. Expression of an 83 amino acid fragment of thrombomodulin with two N-linked glycosylation sites shows that fermentation is more cost effective than shake flask growth for isotopic enrichment.     

增加植酸酶基因appA-m的拷贝提高其在巴斯德毕赤酵母的表达量

为了进一步提高植酸酶的发酵效价,降低植酸酶生产成本,对毕赤酵母表达载体pGAPZα-A进行了改造。将表达载体pPIC9的AOX1启动子序列引入pGAPZα-A,使之成为甲醇可诱导型表达载体pAOXZα,插入植酸酶基因appA-m后得到重组载体pAOXZα-appA-m。以染色体上带有一个拷贝的appA-m基因、发酵效价可达到7.5×106IU/mL发酵液的重组酵母菌株74#为受体菌进行转化,在该重组菌株的染色体上的另一位点整合含有植酸酶基因的表达盒,经筛选到高表达植酸酶的重组子。通过PCR进行验证,植酸酶基因被整合到重组酵母的染色体上,且受体菌中原有的植酸酶基因结构未改变。重组菌在5L发酵罐经甲醇诱导120h植酸酶蛋白表达量达到4mg/mL发酵液,酶活性(发酵效价)达到1.2×107IU/mL发酵液以上,较含单拷贝植酸酶基因的受体菌株表达量有较大程度提高。PCR检测及表达量分析证明改良的菌株具有很好的遗传稳定性和表达稳定性。     

毕赤酵母工程菌高密度发酵研究与进展

毕赤酵母表达系统是近年来发展最为迅速的一种新型外源蛋白真核表达系统,被广泛应用于多种不同领域且成功表达了许多基因工程产品。高密度发酵技术已被广泛运用到毕赤酵母工程菌发酵工程当中。主要从毕赤酵母的表达常用菌株、载体及表型等方面介绍了其表达系统,从外源基因自身的特性、培养基的组成、温度、pH、溶氧量及补料流加策略方面阐述了对毕赤酵母高密度发酵的过程及蛋白质表达结果的影响,并对毕赤酵母工程菌高密度发酵进行了展望,为其今后的研究及应用提供借鉴。     

用GAP启动子在Pichia pastoris GS115中组成型表达鼠灰链霉菌腺苷酸脱氨酶

【目的】构建产AMP脱氨酶的重组毕赤酵母(Pichia pastoris GS115)菌株,并初步优化其发酵条件。【方法】以鼠灰链霉菌(Streptomyces murinus)基因组为模板PCR扩增获得腺苷酸脱氨酶基因AMPD,以pGAP9K为载体构建重组表达质粒pGAP9K-AMPD并通过电转化法转入Pichia pastoris GS115,筛选转化子对其酶活进行测定,并初步优化其发酵条件。【结果】构建了毕赤酵母重组菌,通过分光光度法测定,显示重组菌有明显的酶活;初步优化发酵条件为:该重组菌最适发酵培养基为:甘油2%,蛋白胨2%,酵母膏1%,KH2PO40.5%,MgSO4·7H2O0.05%,pH 6.0;发酵条件为:接种龄24 h,转接量3%,30°C﹑200 r/min培养96 h,取发酵上清液测定酶活,重组菌腺苷酸脱氨酶酶活达到2 230±60 U/mL。【结论】构建了一株产AMP脱氨酶活性较高的重组毕赤酵母菌株,并通过优化发酵条件使其酶活达到2 230±60 U/mL。为AMP脱氨酶工业化生产奠定了一定的基础。     

特异腐质霉内切葡聚糖酶Ⅱ基因在毕赤酵母中的表达及酶学性质

【目的】在毕赤酵母中表达特异腐质霉Humicola insolens的中性内切葡聚糖酶Ⅱ,并对其性质加以研究。【方法】利用RT-PCR的方法,以特异腐质霉(Humicola insolens)NC3总RNA为模板,克隆到中性内切葡聚糖酶Ⅱ基因(egⅡ)的cDNA。将其插入表达载体pPIC9K,重组质粒经线性化后电击转化毕赤酵母(Pichia pastoris)菌株GS115。【结果】SDS-PAGE和酶活的检测结果均表明:egⅡ基因在毕赤酵母中成功表达。重组酶的部分酶学性质研究表明,该酶的最适反应温度为70°C,且在65°C以下具有较好的热稳定性。最适反应pH为6.5,在pH 6.0?7.0之间有较好的稳定性。【结论】用重组毕赤酵母可高效表达外源中性内切葡聚糖酶,为其今后在工业应用奠定了基础。     

毕赤酵母工程菌高密度发酵的研究进展

近年来毕赤酵母已成为一种优越的异源蛋白表达系统。而提高目的蛋白的表达水平,高密度发酵已成为关键技术环节之一。我们从毕赤酵母工程菌的选择、培养基的优化设计,以及发酵工程过程控制等方面简要阐述毕赤酵母的高密度发酵,并提出了工程菌在高密度发酵过程中存在的问题。     

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。     

黑曲霉阿魏酸酯酶在毕赤酵母中的组成型表达

【目的】实现黑曲霉来源的阿魏酸酯酶在毕赤酵母(Pichia pastoris GS115)中的组成型表达。【方法】以黑曲霉(Aspergillus niger)基因组为模板,经重叠延伸PCR扩增得到阿魏酸酯酶基因(AnfaeA),将其与载体pGAP9K相连,构建重组表达载体p GAP9KAnfae A,经SalI线性化后电转入毕赤酵母GS115中,得到重组菌株。高效液相色谱法测定发酵液中阿魏酸酯酶活力,并对重组菌进行了发酵优化。【结果】克隆得到783 bp的阿魏酸酯酶编码基因并实现了其在毕赤酵母中的组成型表达。重组菌发酵84 h后,上清液中酶活达5.72±0.10 U/m L。重组酶(reAnfaeA)经分离纯化后比酶活为59.75 U/mg,大小约为40 k D。发酵优化结果为:葡萄糖40.0 g/L,蛋白胨10.0 g/L,酵母膏30.0 g/L,CaCO_3 0.2 g/L,种龄28 h,接种量3%(体积比),装液量50 m L/250 m L。在此条件下发酵培养,酶活达15.60±0.23 U/m L。【结论】阿魏酸酯酶在毕赤酵母中的组成型表达,对研究毕赤酵母组成型表达系统和阿魏酸酯酶的发酵生产具有一定的借鉴意义。     

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.     

Analysis of single-chain antibody production in Pichia pastoris using on-line methanol control in fed-batch and mixed-feed fermentations.

In the last few years the Pichia pastoris expression system has been gaining more and more interest for the expression of recombinant proteins. Many groups have employed fermentation technology in their investigations because the system is fairly easy to scale up and suitable for the production in the milligram to gram range. A large number of heterologous proteins from different sources has been expressed, but the fermentation process technology has been investigated to a lesser extent. A large number of fermentations are carried out in standard bioreactors that may be insufficiently equipped to meet the demands of high-cell-density fermentations of methylotrophic yeasts. In particular, the lack of on-line methanol analysis leads to fermentation protocols that may impair the optimal expression of the desired products. We have used a commercially available methanol sensor to investigate in detail the effects of supplementary glycerol feeding while maintaining a constant methanol concentration during the induction of a Mut(+) strain of Pichia pastoris. Specific glycerol feed rates in the range of 38-4.2 mg. g(-1). h(-1) (mg glycerol per gram fresh weight per hour) were investigated. Expression of the recombinant scFv antibody fragment was only observed at specific feed rates below 6 mg. g(-1). h(-1). At low specific feed rates, growth was even lower than with methanol as the sole carbon source and the harvest expression level of the scFv was only half of that found in the control fermentation. These results show that glycerol inhibits expression driven by the AOX1 promoter even at extremely limited availability and demonstrate the benefits of on-line methanol control in Pichia fermentation research.     

Generation of diploid Pichia pastoris strains by mating and their application for recombinant protein production

ABSTRACT: BACKGROUND: Yeast mating provides an efficient means for strain and library construction. However, biotechnological applications of mating in the methylotrophic yeast Pichia pastoris have been hampered because of concerns about strain stability of P. pastoris diploids. The aim of the study reported here is to investigate heterologous protein expression in diploid P. pastoris strains and to evaluate diploid strain stability using high cell density fermentation processes. RESULTS: By using a monoclonal antibody as a target protein, we demonstrate that recombinant protein production in both wild-type and glycoengineered P. pastoris diploids is stable and efficient during a nutrient rich shake flask cultivation. When diploid strains were cultivated under bioreactor conditions, sporulation was observed. Nevertheless, both wild-type and glycoengineered P. pastoris diploids showed robust productivity and secreted recombinant antibody of high quality. Specifically, the yeast culture maintained a diploid state for 240 h post-induction phase while protein titer and N-linked glycosylation profiles were comparable to that of a haploid strain expressing the same antibody. As an application of mating, we also constructed an antibody display library and used mating to generate novel full-length antibody sequences. CONCLUSIONS: To the best of our knowledge, this study reports for the first time a comprehensive characterization of recombinant protein expression and fermentation using diploid P. pastoris strains. Data presented here support the use of mating for various applications including strain consolidation, variable-region glycosylation antibody display library, and process optimization.     

Production of recombinant protein in Pichia pastoris by fermentation

This protocol is applicable to recombinant protein expression by small-scale fermentation using the Pichia pastoris expression system. P. pastoris has the capacity to produce large quantities of protein with eukaryotic processing. Expression is controlled by a methanol-inducible promoter, which allows a biomass-generation phase before protein production is initiated. The target protein is secreted directly into a protein-free mineral salt medium, and is relatively easy to purify. The protocol is readily interfaced with expanded bed adsorption for immediate capture and purification of recombinant protein. The setting up of the bioreactor plus the fermentation itself takes 1 wk. Making the master and user seed lots takes approximately 2 wk for each individual clone.     

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。     

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。     

强化表达SAM合成酶促进SAM在毕赤酵母中累积

S 腺苷甲硫氨酸 (S adenosyl L methionine ,SAM)是生物体硫代谢的重要中间代谢物质 ,在体内起着转甲基、转硫基、转氨丙基的作用 ,具有重要的药用和保健价值。将酿酒酵母来源的SAM合成酶 2基因置于GAP启动子调控下 ,构建胞内组成型表达质粒 ,并电转化至毕赤酵母菌株GS115。经Zeocin抗性和培养筛选到一株高产SAM的重组菌。对重组菌表达工艺的研究表明 ,碳源、氮源、pH和溶解氧对SAM的累积有较大影响。在优化条件下 ,重组细胞培养 3天 ,SAM累积量可达 2 .49g/L。     

High density fermentation and activity of a recombinant lumbrokinase (PI239) from Pichia pastoris

A system for the expression of recombinant lumbrokinase (rPI239) was developed in the yeast Pichia pastoris. A total supernatant protein content of 0.174 g/L of high density fermentation broth was obtained. The rPI239 exhibited in vitro fibrinolytic activity. The in vivo activity of rPI239 was measured by prothrombin time, kaolin part thrombin time, thrombin time, and fibrinolytic activity. This work presents the high-density fermentation of rPI239 from P. pastoris and shows that the recombinant protein has similar fibrinolytic activity both in vivo and in vitro.     

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。