酵母表达基因工程产物特性分析

本文阐述了酵母表达系统在表达外源基因特别是大分子真核生物基因方面的优越性;分析了酵母表达系统的局限性:聚合体的存在、信号肽加工不完全、内部降解等而造成的产物不均一现象。同时提出了相应的解决办法。     

酵母表达基因工程产物特性分析

本文阐述了在酵母表达系统在表达外源基因特别是大分子真核生物基因方面的优越性;分析了酵母表达系统的局限性;聚合体的存在、信号肽加工不完全、内部降解等而造成的产物不均一现象。同时提出了相应的解决办法。     

毕赤酵母表达系统优化策略概述

毕赤酵母(Pichia pastor)表达系统是近年发展起来的一种高效表达外源蛋白的系统,利用该系统表达外源基因具有良好的应用前景。尽管毕赤酵母表达系统具有比较完备的基因表达调控机制和对真核基因表达产物的加工修饰能力,但由于基因本身及表达系统等诸多因素,仍然存在外源蛋白表达产量很低甚至不表达的情况。针对毕赤酵母表达系统这一因素,对表达载体的优化,毕赤酵母菌株优化及发酵条件优化进行了综述,以期为外源基因在毕赤酵母中的高效表达提供理论基础。     

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.     

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。     

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。     

巴斯德毕赤酵母表达系统在外源基因表达中的研究进展

巴斯德毕赤酵母是目前应用最广泛的外源蛋白表达系统。分别从的菌株、载体、外源基因整合、表达产物糖基化和外源基因高效表达等方面综述了毕赤酵母表达系统的研究进展。     

巴斯德毕赤酵母表达外源蛋白的研究进展

毕赤酵母表达系统是近年来发展起来的一种高效表达外源基因的表达系统。综述了毕赤酵母表达系统的起源、生物学特性、融合蛋白的表达以及影响蛋白表达量的因素。     

基因工程菌表达D-氨基酸氧化酶研究进展

用分子克隆手段获得D-氨基酸氧化酶基因后,对其在不同表达系统如大肠杆菌系统、酿酒酵母和克鲁维乳酸酵母、博伊丁假丝酵母、巴斯德毕赤氏酵母系统及动物细胞内的表达作了介绍。     

外源基因在酵母中的表达

随着基因工程研究的发展,至今已建立了不少系统可以用来表达外源基因。酵母系统就是其中最常用的系统之一。酵母作为外源基因的表达系统的好处在于:①酵母是一种传统的工业微生物,广泛被用于酿酒和食品工业中。它不会产生毒素,因此用它来表达医药工业和食品工业上有用的蛋白质,极易被人们所接受。②酵母是一种真核生物。尽管这是一种低等的真核生物,但它仍具有一般真核生物的特点。在基因工程中需要表达的外源基因多数来自人类或其他真核生物。要使表达产物具有生物活性常常需要对表达产物进行加工。酵母基本上具备这种能力。因此用酵母表达的产物生物活性要比原核生物表达的好。③酵母的遗传学     

Replacement of the folC gene, encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli, with genes mutagenized in vitro.

The folylpolyglutamate synthetase-dihydrofolate synthetase gene (folC) in Escherichia coli was deleted from the bacterial chromosome and replaced by a selectable Kmr marker. The deletion strain required a complementing gene expressing folylpolyglutamate synthetase encoded on a plasmid for viability, indicating that folC is an essential gene in E. coli. The complementing folC gene was cloned into the vector pPM103 (pSC101, temperature sensitive for replication), which segregated spontaneously at 42 degrees C in the absence of selection. This complementing plasmid was replaced in the folC deletion strain by compatible pUC plasmids containing folC genes with mutations generated in vitro, producing strains which express only mutant folylpolyglutamate synthetase. Mutant folC genes expressing insufficient enzyme activity could not complement the chromosomal deletion, resulting in retention of the pPM103 plasmid. Some mutant genes expressing low levels of enzyme activity replaced the complementing plasmid, but the strains produced were auxotrophic for products of folate-dependent pathways. The folylpolyglutamate synthetase gene from Lactobacillus casei, which may lack dihydrofolate synthetase activity, replaced the complementing plasmid, but the strain was auxotrophic for all folate end products.     

The therapeutic potential of gene transfer for the treatment of peripheral neuropathies

Peripheral neuropathy is a common medical problem with numerous aetiologies. Unfortunately, for the majority of cases there is no available medical solution for the underlying cause, and the only option is to try to treat the resulting symptoms. Treatment options exist when neuropathy results in positive symptoms such as pain, but there is a significant lack of treatments for negative symptoms such as numbness and weakness. Systemic application of growth factor peptides has shown promise in protecting nerves from neuropathic insults in preclinical animal studies, but translation into human trials has been problematic and disappointing. Significant advancements have been made in the past few years in utilising gene therapy approaches to treat peripheral neuropathy by expressing neuroprotective gene products either systemically or in specific nervous tissues. For example, plasmids expressing vascular endothelial growth factor injected into muscle, or herpes-simplex-virus-based vectors expressing neurotrophin gene products delivered to dorsal root ganglion neurons, have been used to protect peripheral nerve function in animal models of diabetes-associated peripheral neuropathy. Many published studies support the feasibility of this approach, although several questions still need to be addressed as gene therapy to treat peripheral neuropathy moves out of the laboratory and into the clinic.     

Rescue and production of vaccine and therapeutic adenovirus vectors expressing inhibitory transgenes

Expression of certain transgenes from an adenovirus vector can be deleterious to its own replication. This can result in the inhibition of virus rescue, reduced viral yields, or, in the worst case, make it impossible to construct a vector expressing the inhibiting transgene product. A gene regulation system based on the tet operon was used to allow the rescue and efficient growth of adenovectors that express transgenes to high levels. A key advantage to this system is that repression of transgene expression is mediated by the packaging cell line, thus, expression of regulatory products from the adenovector are not required. This provides a simple, broadly applicable system wherein transgene repression is constitutive during vector rescue and growth and there is no effect on adenovector-mediated expression of gene products in transduced cells. Several high-level expression vectors based on first- and second-generation adenovectors were rescued and produced to high titer that otherwise could not be grown. Yields of adenovectors expressing inhibitory transgene products were increased, and the overgrowth of cultures by adenovectors with nonfunctional expression cassettes was prevented. The gene regulation system is a significant advancement for the development of adenovirus vectors for vaccine and other gene transfer applications.     

Nuclear and chloroplast transformation inChlamydomonas reinhardtii: strategies for genetic manipulation and gene expression

Transformation of the nuclear, chloroplast, and mitochondrial genomes can now be accomplished inChlamydomonas reinhardtii. Many biosynthetic pathways are carried out in the chloroplast, and efforts to manipulate these pathways will require that gene products be directed to this compartment. Chloroplast proteins are encoded in either the chloroplast or nuclear genome. In the latter case they are synthesized in the cytoplasm and imported post-translationally into the chloroplast. Thus, strategies for expressing foreign genes or overexpressing endogenous genes whose products reside in the chloroplast could involve either genome. This paper reviews the present status of transformation methodology for the nuclear and chloroplast genomes inChlamydomonas. Considerations for expressing gene products in the chloroplast are discussed. Experimental evidence for homologous recombination during transformation of the nuclear genome is presented.     

利用Red重组系统对大肠杆菌ClpP基因的敲除

利用含有质粒pKD4 6的菌株BW2 5 113,在阿拉伯糖诱导后 ,表达λ噬菌体的 3个重组蛋白 ,宿主菌就具有了同源重组的能力 .设计的引物 5′端有 5 0bp的拟敲除基因的同源臂 ,3′端为扩增引物 ,以pKD3为模板 ,扩增两侧含FRT位点的氯霉素抗性基因 ,将此线性片段电转入具重组功能的感受态细胞 ,利用氯霉素平板就可以筛选到阳性转化体 .再利用表达Flp重组酶的质粒pCP2 0 ,可将FRT位点之间的氯霉素抗性基因删除 .利用该重组系统 ,构建了ClpP蛋白酶缺失的大肠杆菌工程菌株 ,可望在减少外源蛋白的降解方面发挥一定的作用 .     

Stochastic Fluctuations and Distributed Control of Gene Expression Impact Cellular Memory

Despite the stochastic noise that characterizes all cellular processes the cells are able to maintain and transmit to their daughter cells the stable level of gene expression. In order to better understand this phenomenon, we investigated the temporal dynamics of gene expression variation using a double reporter gene model. We compared cell clones with transgenes coding for highly stable mRNA and fluorescent proteins with clones expressing destabilized mRNA-s and proteins. Both types of clones displayed strong heterogeneity of reporter gene expression levels. However, cells expressing stable gene products produced daughter cells with similar level of reporter proteins, while in cell clones with short mRNA and protein half-lives the epigenetic memory of the gene expression level was completely suppressed. Computer simulations also confirmed the role of mRNA and protein stability in the conservation of constant gene expression levels over several cell generations. These data indicate that the conservation of a stable phenotype in a cellular lineage may largely depend on the slow turnover of mRNA-s and proteins.     

Cloning and expression of Treponema pallidum antigens in Escherichia coli

A library of Treponema pallidum genomic DNA fragments produced by partial Sau3A digestion was established in Escherichia coli K12 using the plasmid vector pAT153. The library was screened using immune syphilitic rabbit serum and six recombinant phenotypes expressing eight treponemal polypeptides were detected. With two exceptions, all the recombinant gene products were the same size as polypeptides detected on Western immunoblots of T. pallidum. The genes encoding three novel gene products, with molecular masses in SDS-PAGE of 42, 17 and 15.5 kDa, which had not been cloned previously from T. pallidum were also identified. Monoclonal antibodies which reacted with four of the eight recombinant polypeptides were generated.