衣藻叶绿体表达重组蛋白及表达优化策略

近年来,转基因技术已日趋成熟,医学、工业上的应用也越来越广泛。以重组蛋白为基础的药物治疗是目前医药生产领域发展最快的一项技术。它们的高特异性和低副作用使得治疗效率十分突出。但是重组蛋白表达的复杂性也给生产带来了一定限制。为了促进重组蛋白的应用,人们对适宜其表达的系统和能促进其表达的策略进行了探索。研究发现,衣藻叶绿体作为重组蛋白的生物反应器,能实现重组蛋白快速、高效、低成本生产。同时,衣藻能在人工培养基和人为控制的条件下生长,降低了受污染的风险,与传统的生产系统比较具有不可比拟的优越性。因此,衣藻叶绿体作为医药重组蛋白生物反应器在未来的生物技术领域将发挥巨大作用。     

莱茵衣藻细胞核转化体系研究进展

莱茵衣藻(Chlamydomonas reinhardtii)是一种三套基因组都可以进行遗传转化的模式生物,具有培养条件简单、生长速度快、光合效率高等优点,细胞核转化体系相对更为成熟,将其开发为生物反应器具有广阔的应用前景。该文对衣藻核基因组特点及转化机理、转基因衣藻的筛选方法、外源基因的表达以及影响因素等方面进行了综述。     

外源基因在莱茵衣藻叶绿体中的表达

把莱茵衣藻(Chlamydomonas reinhardtii)叶绿体作为生物反应器来表达外源基因具有广阔的应用前景。人们利用莱茵衣藻叶绿体表达体系已成功表达多种重组蛋白,其中包括人类药用蛋白。综述了莱茵衣藻叶绿体转化的方法、影响外源基因表达的主要因素以及外源基因在莱茵衣藻叶绿体表达研究进展。     

叶绿体转基因植物--一种新型生物反应器

叶绿体转基因植物作为生物反应器,具有外源蛋白表达量高和环境安全性好等优点,近年来呈现出诱人的发展前景。本文综述了叶绿体基因工程的优越性,并重点介绍了叶绿体转基因植物作为生物反应器在生产疫苗、药用蛋白及生物可降解塑料等物质方面的最新研究进展。     

叶绿体转基因植物——一种新型生物反应器

叶绿体转基因植物作为生物反应器, 具有外源蛋白表达量高和环境安全性好等优点, 近年来呈现出诱人的发展前景。本文综述了叶绿体基因工程的优越性, 并重点介绍了叶绿体转基因植物作为生物反应器在生产疫苗、药用蛋白及生物可降解塑料等物质方面的最新研究进展。     

衣藻叶绿体表达系统的研究

真核藻类作为一种新型的蛋白表达系统,因其培养方法简单,成本低廉并且能大规模繁殖,最近几年成为人们关注的焦点.作为一种模式生物,单细胞的真核生物莱茵衣藻已经成为人们研究的重点.外源蛋白不仅能在衣藻核中进行表达而且也能在叶绿体中表达,但衣藻叶绿体的表达系统较之核表达有巨大的优越性.在到目前为止,已经有许多的药用蛋白在莱茵衣藻的叶绿体中成功表达的报道,证明了莱茵衣藻叶绿体作为生物反应器的能力.将对衣藻叶绿体的表达做详细的描述.     

利用转基因植物表达药用蛋白

随着药物生物技术和植物基因工程迅速发展 ,转基因植物被用作生物反应器生产具有医疗价值的多肽和蛋白质已成为生物医学研究的热点。研究表明转基因植物表达的蛋白质能够保持原有的结构和功能 ,这预示它将为药用蛋白的生产提供一条安全和廉价的新途径。主要概述了近年来国内外转基因植物生产诸如疫苗、抗体和其他药用蛋白或多肽等的研究进展 ,并着重探讨了存在的问题和解决策略。     

植物生物反应器研究进展

植物生物反应器是近年来生物技术领域新的研究方向,利用农作物进行疫苗、药用蛋白的生产,具有广阔的市场前景和商业价值。研究证明,用各种农作物为载体的植物生物反应器产品可通过种子、果实或块茎表达,便于贮藏、运输和利用。它拓宽了传统农业概念,成为现代生物农业重要的研究方向之一,推动了生物经济快速健康的前进,促进农业可持续发展。综述植物生物反应器的研究与应用现状,并对转基因作物作为植物生物反应器的发展前景作分析和展望。     

植物生物反应器表达药用蛋白研究新进展

植物生物反应器被称为"分子农田",它具有无限生产重组蛋白的巨大潜力。利用转基因植物表达的重组蛋白具备原有的理化性质和生物活性,从而为人类提供了一种大量生产药用蛋白的安全可靠、经济、方便的新生产体系。目前已广泛运用于工业、农业尤其是生命科学以及医学制造领域。用植物生物反应器产重组疫苗、重组抗体和其他药用蛋白已成为国内外基因工程研究热点之一。然而,转基因植物产物的表达量、下游加工等问题却也成为利用植物生物反应器应用的限制因素。本文就其优势、近三年内国内外转基因植物生产药用蛋白的研究进展、存在问题及对策作一综述。     

叶绿体基因工程：一种植物生物技术的新方法

以叶绿体转化为主的叶绿体基因工程,与传统的基因工程技术细胞核转化相比,在外源基因表达水平和转基因植物安全性等方面有明显的优势, 尤其是在控制转基因沉默和遗传稳定性方面,可以互补核转化带来的局限性。因此,叶绿体基因工程是一种很具有发展前景的植物转基因技术,并在未来工农业生物技术领域发挥重要作用。本文着重在叶绿体转化技术主要特点,应用领域及其未来的发展前景等方面进行了简单评述。     

The microalga Chlamydomonas reinhardtii as a platform for the production of human protein therapeutics

Microalgae are a diverse group of eukaryotic photosynthetic microorganisms. While microalgae play a crucial role in global carbon fixation and oxygen evolution, these organisms have recently gained much attention for their potential role in biotechnological and industrial applications, such as the production of biofuels. We investigated the potential of the microalga Chlamydomonas reinhardtii to be a platform for the production of human therapeutic proteins. C. reinhardtii is a unicellular freshwater green alga that has served as a popular model alga for physiological, molecular, biochemical and genetic studies. As such, the molecular toolkit for this microorganism is highly developed, including well-established methods for genetic transformation and recombinant gene expression. We transformed the chloroplast genome of C. reinhardtii with seven unrelated genes encoding for current or potential human therapeutic proteins and found that four of these genes supported protein accumulation to levels that are sufficient for commercial production. Furthermore, the algal-produced proteins were bioactive. Thus, the microalga C. reinhardtii has the potential to be a robust platform for human therapeutic protein production.     

Localization and function of SulP,a nuclear-encoded chloroplast sulfate permease in<Emphasis Type="Italic"> Chlamydomonas reinhardtii</Emphasis>

Recent work [H.-C. Chen et al. (2003) Planta 218:98-106] reported on the genomic, proteomic, phylogenetic and evolutionary aspects of a putative nuclear gene ( SulP) encoding a chloroplast sulfate permease in the model green alga Chlamydomonas reinhardtii. In this article, evidence is provided for the envelope localization of the SulP protein and its function in the uptake and assimilation of sulfate by the chloroplast. Localization of the SulP protein in the chloroplast envelope was concluded upon isolation of C. reinhardtii chloroplasts, followed by fractionation into envelope and thylakoid membranes and Western blotting of these fractions with specific polyclonal antibodies raised against the recombinant SulP protein. The function of the SulP protein was probed in antisense transformants of C. reinhardtii having lower expression levels of the SulP gene. Results showed that cellular sulfate uptake capacity was lowered as a consequence of attenuated SulP gene expression in the cell, directly affecting rates of de novo protein biosynthesis in the chloroplast. The antisense transformants exhibited phenotypes of sulfate-deprived cells, displaying slow rates of light-saturated oxygen evolution, low levels of Rubisco in the chloroplast and low steady-state levels of the photosystem-II D1 reaction-center protein. The role of the chloroplast sulfate transport in the uptake and assimilation of sulfate in C. reinhardtii is discussed along with its impact on the repair of photosystem-II from a frequently occurring photo-oxidative damage and potential use for the elucidation of the H(2)-evolution-related metabolism in this green alga.     

An exogenous chloroplast genome for complex sequence manipulation in algae

We demonstrate a system for cloning and modifying the chloroplast genome from the green alga, Chlamydomonas reinhardtii. Through extensive use of sequence stabilization strategies, the ex vivo genome is assembled in yeast from a collection of overlapping fragments. The assembled genome is then moved into bacteria for large-scale preparations and transformed into C. reinhardtii cells. This system also allows for the generation of simultaneous, systematic and complex genetic modifications at multiple loci in vivo. We use this system to substitute genes encoding core subunits of the photosynthetic apparatus with orthologs from a related alga, Scenedesmus obliquus. Once transformed into algae, the substituted genome recombines with the endogenous genome, resulting in a hybrid plastome comprising modifications in disparate loci. The in vivo function of the genomes described herein demonstrates that simultaneous engineering of multiple sites within the chloroplast genome is now possible. This work represents the first steps toward a novel approach for creating genetic diversity in any or all regions of a chloroplast genome.     

The flanking regions of PsaD drive efficient gene expression in the nucleus of the green alga Chlamydomonas reinhardtii

The nuclear gene PsaD encodes an abundant chloroplast protein located on the stromal side of the Photosystem I complex. We have cloned and sequenced a genomic fragment containing the PsaD gene from the green alga Chlamydomonas reinhardtii. Sequence comparison with its cDNA revealed that the PsaD ORF contains no introns. Thus, the regulatory sequences required for high-level expression of PsaD must lie in the flanking promoter and untranslated regions. We used this genomic fragment to construct a vector that allows for high-level expression of endogenous and exogenous genes, as well as cDNAs that could not be expressed from existing vectors. It is also possible to use the PsaD transit sequence to target the expressed protein to the chloroplast compartment.     

Functional genomics of plant photosynthesis in the fast lane using Chlamydomonas reinhardtii

Oxygenic photosynthesis by algae and plants supports much of life on Earth. Several model organisms are used to study this vital process, but the unicellular green alga Chlamydomonas reinhardtii offers significant advantages for the genetic dissection of photosynthesis. Recent experiments with Chlamydomonas have substantially advanced our understanding of several aspects of photosynthesis, including chloroplast biogenesis, structure-function relationships in photosynthetic complexes, and environmental regulation. Chlamydomonas is therefore the organism of choice for elucidating detailed functions of the hundreds of genes involved in plant photosynthesis.     

Chlamydomonas reinhardtii and photosynthesis: genetics to genomics

Genetic and physiological features of the green alga Chlamydomonas reinhardtii have provided a useful model for elucidating the function, biogenesis and regulation of the photosynthetic apparatus. Combining these characteristics with newly developed molecular technologies for engineering Chlamydomonas and the promise of global analyses of nuclear and chloroplast gene expression will add a new perspective to views on photosynthetic function and regulation.