外源蛋白在毕赤酵母中的高效表达策略

毕赤酵母（Pichia pastoris）表达系统是目前应用最为广泛的蛋白表达系统之一,已成功表达了数百种外源蛋白。但不同外源蛋白的表达量差异较大,能够实现工业化生产的蛋白表达体系并不多,如何提高目的蛋白在该系统中的表达量有着重要的理论和现实意义。该文从发酵工艺方面对影响外源蛋白表达的各种因素做了具体阐述,主要包括培养基的选择与优化、发酵过程参数的控制、分泌型蛋白酶的降解抑制以及诱导型毕赤酵母的甲醇流加机制,旨在提高外源蛋白表达量。     

Direct selection of Pichia pastoris expression strains using new G418 resistance vectors

The methylotrophic yeast, Pichia pastoris, is widely used as a host organism for the expression of heterologous proteins. Currently, the Zeocin and blasticidin resistance genes are the only dominant selectable markers that can be used for primary selection of transformants. In this report we describe new expression vectors that can be used to select directly for P. pastoris transformants using G418 resistance conferred by a modified Tn903kan(r) gene. Compared to other dominant markers, this system is more economical and offers a higher transformation efficiency, due to the small sizes of the cloning vectors, pKAN B and pKANalpha B (GenBank Accession Nos EU285585 and EU285586, respectively). Additionally, multicopy transformants can be generated using these new vectors.     

Efficient expression and secretion of recombinant alpha amylase in Pichia pastoris using two different signal sequences

We have cloned and expressed a bacterial thermostable alpha amylase gene in Pichia pastoris using the methanol-controlled alcohol oxidase (AOX1) promoter. Two integrative vectors were constructed with two different secretion signal sequences in order to obtain efficient secretion of the protein. One vector contains the structural gene encoding the mature alpha amylase fused to the SUC2 gene signal sequence from Saccharomyces cerevisiae. In the other vector, the alpha amylase is expressed with its own signal sequence. In both cases, the alpha amylase were secreted into the culture medium with high efficiency, around 2·5 and 0·9 g/l respectively.     

源于 GAP 启动子的毕赤酵母组成型表达系统的研究进展

巴斯德毕赤酵母表达系统是目前应用非常广泛的外源基因真核表达系统。与源于醇氧化酶启动子(pAOX1)的诱导型表达系统相比, 源于三磷酸甘油醛脱氢酶启动子(pGAP)的毕赤酵母组成型表达系统在表达外源蛋白时不需使用甲醇, 表达时间短, 并且在高密度发酵时采用连续发酵模式, 因此成为近年研究的热点。本文分别从pGAP表达载体的构建、碳源、基因拷贝数、信号序列、高密度发酵等方面对该表达系统进行了综述, 以期为其在蛋白生产中的深入研究和应用提供参考。     

红平菇漆酶基因异源表达及对染料脱色的研究

以白腐菌红平菇Pleurotus djamor RNA为模板,通过RT-PCR得到漆酶Pd-lac1基因的完整CDS序列。构建毕赤酵母表达载体pPICZB-Pd-Lac1,并通过电转化法导入Pichia pastoris中,通过PCR结果证明,Pd-lac1的CDS序列部分阳性转化子已经整合到甲醇毕赤酵母染色体上,经摇瓶液体培养后筛选出蛋白表达较高的酵母工程菌株。漆酶活力达54 U/L。进而研究了重组漆酶对染料脱色的影响。结果表明：重组漆酶对蒽醌类SN4R脱色可达52.8%;杂环类中性红、偶氮类甲基橙和三苯基甲烷类孔雀绿的脱色率低于SN4R,脱色率分别为14.8%、14.5%、0.24%,显示出重组漆酶对SN4R脱色效率具有较大的应用潜力,从而对废水处理具有更广的应用前景。     

Cloning of the Penicillium minioluteum gene encoding dextranase and its expression in Pichia pastoris

The DEX gene encoding an extracellular dextranase was isolated from the genomic DNA library of Penicillium minioluteum by hybridization using the dextranase cDNA as a probe. Comparison of the gene and cDNA sequences revealed that the DEX gene does not contain introns. Amino acid sequences comparison of P. minioluteum dextranase with other reported dextranases reveals a significant homology (29% identity) with a dextranase from Arthrobacter sp. CB-8. The DEX gene fragment encoding a mature protein of 574 amino acids was expressed in the methylotrophic yeast Pichia pastoris by using the SUC2 gene signal sequence from Saccharomyces cerevisiae under control of the alcohol oxidase-1 (AOX1) promoter. Over 3·2g/l of enzymatically active dextranase was secreted into the medium after induction by methanol. The yeast product was indistinguishable from the native enzyme in specific activity and the N-terminus of both proteins were identical.     

Identification and characterization of novel promoters for recombinant protein production in yeast Pichia pastoris

Pichia pastoris expression system has been widely used in recombinant protein production. So far the majority of heterologous proteins are expressed by methanol inducible promoter P_AOX1 and constitutive promoter P_GAP. The use of other promoters is rather limited. Here we selected 16 potentially efficient and regulatory promoter candidates based on the RNA‐seq and RNA folding free energy ΔG data. GFP and recombinant amylase were inserted after these promoters to reveal their strength and efficiency under different carbon sources and culture scales. Two novel promoters were successfully identified and could possibly be applied in recombinant protein expression: the methanol‐inducible promoter P₀₅₄₇ and the constitutive promoter P₀₄₇₂.     

6-phosphofructokinase from Pichia pastoris: purification,kinetic and molecular characterization of the enzyme

6-Phosphofructokinase from Pichia pastoris was purified for the first time to homogeneity applying seven steps, including pseudo-affinity dye-ligand chromatography on Procion Blue H-5R-Sepharose. The specific activity of the purified enzyme was about 80 U/mg. It behaves as a typically allosteric 6-phosphofructokinase exhibiting activation by AMP and fructose 2,6-bis(phosphate), inhibition by ATP and cooperativity to fructose 6-phosphate. However, in comparison with the enzymes from Saccharomyces cerevisiae and Kluyveromyces lactis, the activation ratio of 6-phosphofructokinase from Pichia pastoris by AMP is several times higher, the ATP inhibition is stronger and the apparent affinity to fructose 6-phosphate is significantly lower. Aqueous two-phase affinity partitioning with Cibacron Blue F3G-A did not reflect remarkable structural differences of the nucleotide binding sites of the Pfks from Pichia pastoris and Saccharomyces cerevisiae. The structural organisation of the active enzyme seems to be different in comparison with hetero-octameric 6-phosphofructokinases from other yeast species. The enzyme was found to be a hetero-oligomer with an molecular mass of 975 kDa (sedimentation equilibrium measurements) consisting of two distinct types of subunits in an equimolar ratio with molecular masses of 113 kDa and 98 kDa (SDS-PAGE), respectively, and a third non-covalently complexed protein component (34 kDa, SDS-PAGE). The latter seems to be necessary for the catalytic activity of the enzyme. Sequencing of the N-terminus (VTKDSIXRDLEXENXGXXFF) and of peptide fragments by applying MALDI-TOF PSD, m/z 1517.3 (DAMNVVNH) and m/z 2177.2 [AQNCNVC(L/I)SVHEAHTM] gave no relevant information about the identity of this protein.     

蛹虫草中谷氨酰胺转氨酶基因的克隆及在毕赤酵母中的表达

谷氨酰胺转氨酶(TGase或TG)是一种可催化蛋白质间形成异肽键,使蛋白质改性的天然酶制剂。本文以蛹虫草基因组为模板,通过PCR扩增得到TGase相似蛋白的基因片段tgM,将其与大肠杆菌-毕赤酵母穿梭表达载体pPIC9K连接,构建重组表达载体pPIC9K-TG,在毕赤酵母中进行表达,实现目的蛋白的胞外分泌表达,结果发酵液中重组TGase的酶活力为100 U/L。本研究为TGase的异源表达及潜在的工业应用提供参考。     

重组毕赤酵母生产外源蛋白的过程控制与生理分析的研究进展

毕赤酵母是近年来应用广泛、极受青睐的一种外源蛋白表达系统。除了构建高效、稳定的毕赤酵母生产菌株外,研究不同发酵控制条件下,细胞的生理和代谢状态变化,为毕赤酵母细胞提供最优的生长和环境条件并综合考虑发酵成本是成功实现外源蛋白规模化、商业化生产的保障。本文从培养基组分、外界环境条件、细胞比生长速率、生长期甘油流加策略和诱导期甲醇流加策略等方面阐述了发酵过程对外源蛋白表达的影响,并进一步从细胞生理和代谢层面深入讨论了不同发酵过程控制条件的影响机制。      

产谷胱甘肽毕赤酵母工程菌的构建及能量调控

谷胱甘肽(glutathione,GSH)是生物体内重要非编码且含有巯基的三肽类物质,具有调节和保护等功能,在医药、食品等领域有着广泛的应用.目前,工业上主要通过高密度发酵生产GSH,ATP的供应往往成为GSH生产的限制因素.该文以毕赤酵母GS115为出发菌株,整合串联表达来源于酿酒酵母的Scgsh1和Scgsh2基因...     

菊糖果糖转移酶基因在毕赤酵母中的分泌表达

将PCR获得的不包含信号肽序列的菊糖果糖转移酶基因（ift）与毕赤酵母分泌表达载体pPIC9K进行连接,构建重组载体pPIC9K-IFTase。重组载体经线性化后电转入毕赤酵母GS115感受态细胞,利用G418抗性梯度筛选及PCR鉴定,获得一株高拷贝菊糖果糖转移酶重组毕赤酵母菌株。该重组菌株经甲醇诱导,能够分泌具有活性的菊糖果糖转移酶,且60h时酶活为10.3U/mL。结果表明,菊糖果糖转移酶可以实现在毕赤酵母的分泌表达。      

产木聚糖酶毕赤酵母工程菌株构建及表达条件优化研究

将黑曲霉XZ-3S木聚糖酶Xyn43A成熟肽基因插入表达载体p PIC9K,重组质粒SalⅠ线性化后分别电击转化2种毕赤酵母GS115和KM71,转化液经MD平板、G418浓度梯度平板和摇瓶复筛,获得两株重组菌GS115/Xyn43A（Mut⁺）和KM71/Xyn43A（Mut^s）。其中GS115/Xyn43A菌株最优表达条件为:甲醇浓度2.0%,接种时间24 h,诱导时间108 h,诱导温度30℃、诱导培养基初始p H6.3;KM71/Xyn43A菌株最优表达条件为:甲醇浓度1.75%,接种时间26 h,诱导时间132 h,诱导温度30℃、诱导培养基初始p H6.5。在最优表达条件下两重组菌GS115/Xyn43A和KM71/Xyn43A比酶活力分别可达139.36、143.29 U/mg。      

甜蛋白Monellin在毕赤酵母中的分泌表达

本研究采用毕赤酵母偏爱密码子,人工合成了高甜度Monellin基因,并构建了重组分泌型酵母表达载体pPIC9M,通过电击转化获得了可高效分泌表达有甜味活性高甜度Monellin的重组毕赤酵母GS115/pPIC9M.通过PCR检测证实Monellin基因已经整合进酵母基因组,SDS-PAGE和Western blot免疫杂交知所表达的蛋白是目的蛋白,最后通过双盲测定证实表达的蛋白存在正常活性.     

巴斯德毕赤酵母(Pichia pastoris)高效异源表达脂肪酶研究进展

脂肪酶作为一种常见的工业用酶,广泛应用于食品、化妆品等与生活密切相关的工业领域,但较高的生产成本和使用成本,在一定程度上限制了其进一步应用。通过巴斯德毕赤酵母(Pichia pastoris)异源表达系统重组表达脂肪酶,成为解决限制脂肪酶发展的方法之一。本文介绍了脂肪酶在P.pastoris表达系统中的异源表达及其优化策略,并对毕赤酵母表面展示异源脂肪酶的技术及应用进行了归纳。      

Hygromycin‐resistance vectors for gene expression in Pichia pastoris

Pichia pastoris is a common host organism for heterologous protein expression and metabolic engineering. Zeocin‐, G418‐, nourseothricin‐ and blasticidin‐resistance genes are the only dominant selectable markers currently available for selecting P. pastoris transformants. We describe here new P. pastoris expression vectors that confer a hygromycin resistance base on the Klebsiella pneumoniae hph gene. To demonstrate the application of the vectors for intracellular and secreted protein expression, green fluorescent protein (GFP) and human serum albumin (HSA) were cloned into the vectors and transformed into P. pastoris cells. The resulting strains expressed GFP and HSA constitutively or inducibly. The hygromycin resistance marker was also suitable for post‐transformational vector amplication (PTVA) for obtaining strains with high plasmid copy numbers. A strain with multiple copies of the HSA expression cassette after PTVA had increased HSA expression compared with a strain with a single copy of the plasmid. To demonstrate compatibility of the new vectors with other vectors bearing antibiotic‐resistance genes, P. pastoris was transformed with the Saccharomyces cerevisiae genes GSH1, GSH2 or SAM2 on plasmids containing genes for resistance to Zeocin, G418 or hygromycin. The resulting strain produced glutathione and S‐adenosyl‐l ‐methionine at levels approximately twice those of the parent strain. The new hygromycin‐resistance vectors allow greater flexibility and potential applications in recombinant protein production and other research using P. pastoris. Copyright © 2014 John Wiley & Sons, Ltd.     

Expression and purification of goat lactoferrin from Pichia pastoris expression system

ABSTRACT:  The recombinant goat lactoferrin (rGLF) was expressed in the methylotropic yeast Pichia pastoris using pGAPZαC vector, GAP as promoter, and Zeocin as the selective marker. After transformation of the GLF-pGAPZαC into Pichia pastoris X-33 expression host, the GLF-pGAPZαC vector was integrated into the GAP promoter locus of Pichia pastoris X-33 chromosome. The rGLF was expressed and secreted into the broth using α-factor preprosequence. SDS-PAGE and PAS staining analysis indicated that the rGLF could be purified to electrophoretic homogeneity by heparin-Sepharose 6 Fast Flow affinity chromatography and glycosylated by the expression host. The yield of purified rGLF was approximately 2.0 mg/L of culture broth. The N-terminal sequence was identical to the native goat lactoferrin (nGLF). The iron-binding behavior, papain-inhibiting property, and thermal stability of the purified rGLF were comparable to nGLF. This is the 1st report of intact goat lactoferrin expression using the P. pastoris system.     

类芽孢杆菌碱性果胶裂解酶Pel在毕赤酵母中的高效表达

本研究全基因合成了来源于类芽孢杆菌的碱性果胶酶基因pel,克隆至p HKA载体上,成功实现其在毕赤酵母GS115中的分泌表达;并对信号肽和启动子进行优化,使得摇瓶发酵诱导168 h果胶裂解酶酶活达到432.36 U/m L。初步测定了果胶裂解酶Pel的基本酶学性质,其最适反应温度和p H分别为60℃、10.0;在50℃下处理300 min裂解酶活力保留90%以上,而在60℃下处理210 min,酶活力剩余约50%;在p H 9~12的50 m M各缓冲液中处理酶液16 h,酶活力仍然保留90%以上,该酶耐碱性强,但在偏酸性缓冲液中处理该酶,酶活力有所下降;同时研究了二价金属离子对果胶裂解酶活性力的影响,Ca~(2+)对Pel发挥裂解作用是必需的,Fe~(2+)、Mg~(2+)、Ni~(2+)对Pel均有激活作用,而金属离子螯合物EDTA的存在则使酶活力完全丧失;该酶在碱性条件下较好的稳定性决定了其潜在的工业应用前景。     

棘孢青霉右旋糖酐酶基因克隆及其在毕赤酵母中的表达

以右旋糖酐酶产生菌棘孢青霉F1001基因组为模板反转录合成右旋糖酐酶的cDNA（dex）,基因全长1 866 bp,根据毕赤酵母密码子偏好性优化dex序列获得优化右旋糖酐酶基因（opt-dex）,分别构建dex-pPICZαA和opt-dex-pPICZαA重组质粒,电击转入毕赤酵母X33中构建重组子。通过蓝色右旋糖酐T-2000平板以及摇瓶发酵筛选获得产右旋糖酐酶的重组酵母菌株。重组酶的酶学性质分析显示,重组酶分子质量65?kDa、最适pH?5.0、最适温度35?℃,专一作用于α-1,6糖苷键。在摇瓶水平上对重组毕赤酵母表达条件进行优化,优化培养条件为培养温度25?℃、初始pH?5.0、每24?h甲醇添加量1%（体积分数）、每24?h山梨醇添加量5?g/L、吐温-80添加量4?g/L、摇瓶装液量50?mL/500?mL锥形瓶,优化后的重组右旋糖酐酶分泌表达酶活力提高到240.74?U/mL。重组酵母X33是一株适合外源表达棘孢青霉右旋糖酐酶基因的工程菌,该重组酶可替代棘孢青霉右旋糖酐酶直接应用于工业生产催化制备右旋糖酐。