酵母表达基因工程产物特性分析

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,分析了由于酵母表达系统的局限性如聚合体的存在、信号肽加工不完全、内部降解等而造成的产物不均一现象,提出了相应的解决方法。     

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。     

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。     

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.     

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。     

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     

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。     

基因簇大片段克隆技术研究进展及挑战

天然产物结构复杂、活性多样,是新药开发的重要来源,对天然产物生物合成途径的研究,有利于探索酶催化的合成机制,促进复杂天然产物的应用。天然产物的生物合成由其对应的基因簇调控,其中大量天然产物生物合成基因簇(biosynthetic gene clusters,BGCs)在野生型菌株中无法表达或表达量低。对这些基因簇的研究,需要进行克隆表达,而如何克隆大片段基因簇并使其表达,从而发现新型天然产物是一个具有挑战性的问题。其中构建基因组文库、转化关联重组(transformation-associated recombination,TAR)、Red/ET重组等是克隆大片段基因簇的重要技术。本文从克隆技术的策略和应用两个方面,总结了这3种克隆技术目前的研究进展,讨论了目前大片段基因簇克隆技术面临的挑战,为研究大片段基因簇提供方法学借鉴。     

Evidence that the two Escherichia coli groE morphogenetic gene products interact in vivo.

The Escherichia coli groEL and groES gene products are essential for both phage morphogenesis and bacterial growth. Although the gene products have been identified, their exact roles in these processes are not known. We have isolated mutations in the groEL gene that suppress defects in the groES gene. These intergenic suppressors were shown to map in the groEL gene by a variety of genetic and biochemical analyses. These results suggest that the two morphogenetic gene products interact in vivo and help to explain why mutations in either gene exhibit the same phenotype with respect to lambda head assembly and bacterial growth.     

Potential influence of the first PCR cycles in real-time comparative gene quantifications

There is an underlying assumption in real-time PCR that the amplification efficiency is equal from the first cycles until a signal can be detected. In this study, we evaluated this assumption by analyzing genes with known gene copy number using real-time PCR comparative gene quantifications. Listeria monocytogenes has six 23S rRNA gene copies and one copy of the hlyA gene. We determined 23S rRNA gene copy numbers between 0.9 and 1.6 relative to hlyA when applying the comparative gene quantification approach. This paper focuses on the first cycles of PCR to explain the difference between known and determined gene copy numbers. Both theoretical and experimental evaluations were done. There are three different products (types 1-3) dominating in the first cycles. Type 1 is the original target, type 2 are undefined long products, while type 3 are products that accumulate during PCR. We evaluated the effects of type 1 and 2 products during the first cycles by cutting the target DNA with a restriction enzyme that cuts outside the boundaries of the PCR products. The digestion resulted in a presumed increased amplification efficiency for type 1 and 2 products. Differences in the amplification efficiencies between type 1, 2, and 3 products may explain part of the error in the gene copy number determinations using real-time PCR comparative gene quantifications. Future applications of real-time PCR quantifications should account for the effect of the first few PCR cycles on the conclusions drawn.     

A role for the umuDC gene products of Escherichia coli in increasing resistance to DNA damage in stationary phase by inhibiting the transition to exponential growth

The umuDC gene products, whose expression is induced by DNA-damaging treatments, have been extensively characterized for their role in SOS mutagenesis. We have recently presented evidence that supports a role for the umuDC gene products in the regulation of growth after DNA damage in exponentially growing cells, analogous to a prokaryotic DNA damage checkpoint. Our further characterization of the growth inhibition at 30 degrees C associated with constitutive expression of the umuDC gene products from a multicopy plasmid has shown that the umuDC gene products specifically inhibit the transition from stationary phase to exponential growth at the restrictive temperature of 30 degrees C and that this is correlated with a rapid inhibition of DNA synthesis. These observations led to the finding that physiologically relevant levels of the umuDC gene products, expressed from a single, SOS-regulated chromosomal copy of the operon, modulate the transition to rapid growth in E. coli cells that have experienced DNA damage while in stationary phase. This activity of the umuDC gene products is correlated with an increase in survival after UV irradiation. In a distinction from SOS mutagenesis, uncleaved UmuD together with UmuC is responsible for this activity. The umuDC-dependent increase in resistance in UV-irradiated stationary-phase cells appears to involve, at least in part, counteracting a Fis-dependent activity and thereby regulating the transition to rapid growth in cells that have experienced DNA damage. Thus, the umuDC gene products appear to increase DNA damage tolerance at least partially by regulating growth after DNA damage in both exponentially growing and stationary-phase cells.     

Env gene products of AKR dual-tropic viruses: examination of peptide maps and cell surface expression.

The env gene products of nine AKR dual-tropic murine leukemia viruses were compared by peptide mapping and were assayed for expression on the cell surface of infected fibroblasts. Seven virus isolates expressed the env gene polyprotein on the cell surface. The env gene products of six of the seven viruses had identical peptide maps. The analysis of structure and expression of env gene products carried out in this study characterizes a subset of dual-tropic murine leukemia viruses shown by others to be thymotropic.     

The function of macrophages in antigen recognition by guinea pig T lymphocytes. III. Genetic analysis of the antigens mediating macrophage-T lymphocyte interaction.

Activation of immune T lymphocytes by antigen-pulsed macrophages is mediated by the Ia antigens of the guinea pig MHC or the products of closely linked genes. Studies using combinations of macrophages and T cells derived from outbred animals with different Ia antigens and/or Ir gene products have demonstrated that sharing of Ir gene products between macrophage and T cells is not sufficient for effective macrophage-T cell interaction. The role of the Ia antigens in the absence of the linked Ir gene products could not be directly examined because we were unable to identify an animal which bore the full complement of Ia antigens in the absence of the Ir gene that is normally associated with them. The results of these studies support the concept of the functional expression of the Ir gene product in the macrophage.     

Nucleotide sequence and mutagenesis of the nifA gene from Azotobacter vinelandii

The nucleotide sequence of the nifA gene from Azotobacter vinelandii was determined. This gene encodes an Mr = 58,100 polypeptide that shares significant sequence identity when compared to nifA-encoded products from other organisms. Interspecies comparisons of nifA-encoded products reveal that they all have a consensus ATP binding site and a consensus DNA binding site in highly conserved regions of the respective polypeptides. The nifA gene immediately precedes the nifB-nifQ gene region but is unlinked to the major nif gene cluster from A. vinelandii. A potential regulatory gene precedes and is apparently cotranscribed with nifA. Mutant strains that have a deletion or a deletion plus an insertion within nifA are incapable of diazotrophic growth and they fail to accumulate nitrogenase structural gene products.