外源基因在巴氏毕赤酵母中的表达

近年来,巴氏毕赤酵母(Pichia pastoris)已经发展成为一种优良的外源基因表达系统得到越来越广泛的应用。     

影响毕赤酵母高效表达外源蛋白的因素

分析了毕赤酵母高效表达外源蛋白的机理以及影响毕赤酵母表达外源蛋白的作用因素。     

毕赤酵母表达外源基因研究进展

毕赤酵母表达系统作为一种新型酵母表达系统,它既有原核生物的特点,又有真核生物的特性,可以对目的蛋白质进行糖基化、二硫键形成等翻译后修饰。在过去的20年中已经有200多种不同蛋白在毕赤酵母中实现了成功表达,其中许多已被广泛应用于临床诊断治疗或科研工作中。随着一系列新型酵母表达载体及菌株的构建及发酵技术的不断提高,毕赤酵母越来越吸引着科学家的关注,毕赤酵母己被发展成为一种细胞生物学研究及基因工程生产的模式真核生物。本文总结了毕赤酵母的载体与菌株构建及发酵技术和糖基化等方面近年的主要研究进展。     

表达外源蛋白的有利工具-毕赤酵母表达系统

巴斯得毕赤酵母(Pichiapastoris)作为80年代开发的一种表达系统,在外源蛋白的表达方面具有独特的优势,被广泛用于医药领域和科学研究.本文介绍了酵母的生物学特性、常用茵株和表达载体的特点及其研究进展,并从外源基因本身序列的内在特性和酵母发酵外源性因素两个方面,对影响外源蛋白表达的因素进行详细阐述.     

巴斯德毕赤酵母外源基因表达系统

巴斯德毕赤酵母是目前最为成功的外源基因表达系统之一。有关研究主要涉及以下几个方面。宿主菌株、表达载体、转化方法、外源基因的整合与影响外源蛋白表达的因素等。     

毕赤酵母外源基因表达系统研究进展

巴斯德毕赤酵母Pichia pastoris外源基因表达系统已经成功表达了很多胞间型和胞内型蛋白质。与酿酒酵母Saccharomyces ceresiviae相比,该系统所具有的很多优势使其应用越来越广泛。有关研究主要涉及以下几个方面：宿主菌株,表达载体,转化方法,外源基因整合,外源蛋白糖基化和高密度发酵培养等。     

巴氏毕赤酵母表达系统的特点及其研究进展

巴氏毕赤酵母表达系统具有真核生物表达的特点。本文综述了巴氏毕赤酵母菌及其表达载体的特点以及外源基因在该系统中表达存在的一些问题。     

提高外源基因在巴斯德毕赤酵母中表达量的研究进展

巴斯德毕赤酵母 (Pichiapastoris)表达系统是基因工程研究中广泛使用的真核表达系统 ,与现有的其它表达系统相比 ,巴斯德毕赤酵母在表达产物的糖基化修饰、折叠、加工、外分泌及表达量等方面有明显的优势。外源基因在该系统中表达时 ,由于受基因内部的结构、分泌信号、甲醇诱导的浓度及诱导时间、培养温度、启动子、表达环境的 pH值等诸多因素的影响 ,一些外源蛋白的表达也存在着表达不够稳定、表达量较低 ,甚至不表达的情况。对影响巴斯德毕赤酵母表达的各种可能因素进行了分析 ,结合具体实践经验 ,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综述。     

利用巴斯德毕赤酵母表达外源蛋白的研究进展

随着基因工程技术的迅速发展,已有数百种外源蛋白利用巴斯德毕赤酵母表达系统获得了成功表达。本综述了该表达系统的优点、系统的构成,外源基因转化该表达系统的方式及表达特点,阐述了该系统在生产外源蛋白上的广泛应用．并重点分析了影响外源蛋白在该表达系统中表达的因素及优化策略等。     

Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris

Improved expression of recombinant laccase by Pichia pastoris carrying the lcc1 cDNA isolated from Trametes versicolor was achieved by optimization of the cultivation conditions in a fermentor equipped with a methanol sensor system. The results indicated that the activity obtained in fermentor cultivations was at least 7 times higher than in shake-flask cultures. Three different strategies for fermentor cultivations were compared: A (30 degrees C, 1.0% methanol), B (20 degrees C, 1.0% methanol), and C (20 degrees C, 0.5% methanol). The laccase activity, particularly the specific activity, could be improved by decreasing the cultivation temperature. The mechanisms behind the temperature effect on the laccase activity may be ascribed to poor stability, release of more proteases from dead cells, and folding problems at higher temperature. The results showed that the methanol concentration had a marked effect on the production of active heterologous laccase. A fivefold higher volumetric laccase activity was obtained when the methanol concentration was kept at 0.5% instead of 1.0%. The detrimental effect of methanol on the production of recombinant laccase may be attributed to lower laccase stability, a higher proteolytic activity, and folding problems due to higher growth rate at 1.0% methanol.     

Optimisation of the expression of a Trametes versicolor laccase gene in Pichia pastoris

A cDNA encoding a laccase enzyme was isolated from a Trametes versicolor cDNA library. The gene was subcloned into the Pichia pastoris expression vector pPIC3.5 and transformed into the P. pastoris strains KM71 and GS115. Laccase-secreting transformants were selected by their ability to oxidise the substrate ABTS. No difference in laccase activity was observed between culture supernatants from GS115 (proteolytic) and KM71 (nonproteolytic) strains. The presence of at least 200 μM copper was necessary for optimal laccase activity in the culture supernatants. During growth of P. pastoris on minimal medium the pH of the medium was reduced to <3.0. If alanine was added to the medium the pH reduction was not as pronounced and at alanine concentrations >0.6% w/v the pH was kept constant for >7 days. Cultures in which the pH was maintained by alanine metabolism produced higher levels of laccase activity than those grown in the absence of alanine. This study describes the development of a medium that allows convenient pH control of P. pastoris without the need for continuous neutralisation. Journal of Industrial Microbiology & Biotechnology (2002) 29, 55–59 doi:10.1038/sj.jim.7000268 Received 08 August 2001/ Accepted in revised form 18 April 2002     

Mutation of cysteine residues increases heterologous expression of peach expansin in the methylotrophic yeast Pichia pastoris

The study of Carbohydrate-Active enZymes (CAZymes) associated with plant cell wall metabolism is important for elucidating the developmental mechanisms of plants and also for the utilization of plants as a biomass resource. The use of recombinant proteins is common in this context, but heterologous expression of plant proteins is particularly difficult, in part because the presence of many cysteine residues promotes denaturation, aggregation and/or protein misfolding. In this study, we evaluated two phenotypes of methylotrophic yeast Pichia pastoris as expression hosts for expansin from peach (Prunus persica (L.) Batsch, PpEXP1), which is one of the most challenging targets for heterologous expression. cDNAs encoding wild-type expansin (PpEXP1_WT) and a mutant in which all cysteine residues were replaced with serine (PpEXP1_CS) were each inserted into expression vectors, and the protein expression levels were compared. The total amount of secreted protein in PpEXP1_WT culture was approximately twice that of PpEXP1_CS. However, the amounts of recombinant expansin were 0.58 and 4.3 mg l⁻¹, corresponding to 0.18% and 2.37% of total expressed protein, respectively. This 13-fold increase in production of the mutant in P. pastoris indicates that the replacement of cysteine residues stabilizes recombinant PpEXP1.     

巴斯德毕赤酵母表达系统的研究进展和前景展望

巴斯德毕赤酵母经过近二十来年的发展,已经成为表达外源基因的优秀表达系统之一,成功地表达了许多重组异源蛋白。从表达菌株,表达载体等方面详细综述了毕赤酵母表达系统的优点,如：营养要求低、可高密度发酵、遗传稳定性高等;分析了可能影响巴斯德毕赤酵母表达系统的相关因素,这些因素包括外源基因的特性、基因拷贝数、产物稳定性及发酵策略等,结合这些因素和具体实践经验,就如何提高外源基因在巴斯德毕赤酵母中表达量进行了阐述;讨论了该表达系统存在的不足之处并且展望了其发展前景。     

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。     

桦褐孔菌膜二肽酶在巴斯德毕赤酵母中的分泌表达

本研究利用巴斯德毕赤酵母Pichia pastoris蛋白表达体系表达了药用担子菌桦褐孔菌的一个二肽酶基因。该二肽酶基因编码区全长1814bp,包含6个内含子,编码465个氨基酸。生物信息学分析发现,二肽酶基因编码的蛋白中不含信号肽序列,但在第55–77位氨基酸之间存在一个跨膜结构。将含跨膜结构和去跨膜结构蛋白的cDNA序列分别克隆到酵母分泌型表达载体pPICZαA上,电转化至巴斯德毕赤酵母X-33中,用1%(V/V)甲醇诱导重组菌株表达目标蛋白,采用SDS-PAGE和Western-blot检测表达蛋白。结果显示,巴斯德毕赤酵母可表达含跨膜结构的完整基因,但目标蛋白不能分泌到胞外,存在于破碎细胞的沉淀中,且没有催化活性;而去跨膜结构的蛋白则可分泌表达到胞外,并具有催化活性。Ni-NTA纯化去跨膜结构的桦褐孔菌二肽酶浓度可达0.12mg/mL,并发现其在pH 7.3、反应温度50℃、反应时间2h的条件下,以Gly-Gly为底物时,其比活为433U/mg。同时检测到其对Ile-Leu、Trp-Trp和Phe-Phe具有较高的水解活性。     

Molecular cloning and heterologous expression of an alkaline xylanase from Bacillus pumilus HBP8 in Pichia pastoris

The xynHB gene, encoding alkaline xylanase was cloned from Bacillus pumilus by a shot-gun method. The gene was cloned into vector pHBM905A, and expressed in Pichia pastoris GS115. Xylanase-secreting transformants were selected on plates containing RBB-xylan. Enzymatic activity in the culture supernatants was up to 644?U?mL^?1 and the optimal secretion time was 4 days at 25°C. SDS-PAGE showed two bands, of 32.2?kDa and 29.6?kDa, both larger than the predicted mass of 22.4?kDa based on its amino acid sequence. Zymogram analysis demonstrated that the enzyme in both bands could hydrolyze xylan. Deglycosylation by endoglycosidase H revealed that both were derived from the same protein but contain different extents of glycosylation (30 and 25%). The optimal pH and temperature of the enzyme was pH6–9 and 50°C, respectively.     

Molecular cloning and heterologous expression of an alkaline xylanase from Bacillus pumilus HBP8 in Pichia pastoris

The xynHB gene, encoding alkaline xylanase was cloned from Bacillus pumilus by a shot-gun method. The gene was cloned into vector pHBM905A, and expressed in Pichia pastoris GS115. Xylanase-secreting transformants were selected on plates containing RBB-xylan. Enzymatic activity in the culture supernatants was up to 644 U mL^-1 and the optimal secretion time was 4 days at 25°C. SDS-PAGE showed two bands, of 32.2 kDa and 29.6 kDa, both larger than the predicted mass of 22.4 kDa based on its amino acid sequence. Zymogram analysis demonstrated that the enzyme in both bands could hydrolyze xylan. Deglycosylation by endoglycosidase H revealed that both were derived from the same protein but contain different extents of glycosylation (30 and 25%). The optimal pH and temperature of the enzyme was pH6-9 and 50°C, respectively.     

阿维链霉菌来源的磷脂酰丝氨酸合成酶基因的克隆及其在毕氏酵母中的异源表达

本文旨在构建阿维链霉菌(Streptomyces avermitilis)来源的磷脂酰丝氨酸合成酶基因(pss)的重组质粒,研究其在毕氏酵母中的异源分泌型表达。利用PCR技术克隆阿维链霉菌来源的pss基因,再通过电转化方法将重组质粒pOG-01转入毕氏酵母KM71中,构建重组工程菌KP1。实验结果表明,阿维链霉菌来源的磷酯酰丝氨酸合成酶基因在毕氏酵母KM71中成功表达,2 mL菌体上清催化50 mmol/L卵磷脂,转酯反应的转化率为58%,酶活为4.83 U/mL。     

宇佐美曲霉木聚糖酶基因xynⅡ在不同毕赤酵母中的分泌表达

将宇佐美曲霉E001的内切-1,4-木聚糖酶基因克隆到毕赤酵母表达载体pPIC9K中,得到重组质粒pPXY-NII,将其经SalⅠ线性化后分别转化2株毕赤酵母GS115和KM71,xynⅡ基因通过同源重组被整合到毕赤酵母染色体上,并处于酵母α因子的下游,经筛选获得阳性重组菌PXGL98(Mut+)和PXKL29(Muts)。该木聚糖酶基因在2株毕赤酵母中均实现了分泌表达。同时对工程菌的发酵条件进行了优化,在甲醇诱导下,PXGL98与PXKL29培养物上清液中的酶活力分别可达1156.92 U/mL和1646.03 U/mL。