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1.
Protein secretion in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and advances in protein production 总被引:1,自引:0,他引:1
Yeast expression systems have been successfully used for over 20 years for the production of recombinant proteins. With the
growing interest in recombinant protein expression for various uses, yeast expression systems, such as the popular Pichia pastoris, are becoming increasingly important. Although P. pastoris has been successfully used in the production of many secreted and intracellular recombinant proteins, there is still room
for improvement of this expression system. In particular, secretion of recombinant proteins is still one of the main reasons
for using P. pastoris. Therefore, endoplasmic reticulum protein folding, correct glycosylation, vesicular transport to the plasma membrane, gene
dosage, secretion signal sequences, and secretome studies are important considerations for improved recombinant protein production. 相似文献
2.
Several Aspergillus species, in particular Aspergillus niger and Aspergillus oryzae, are widely used as protein production hosts in various biotechnological applications. In order to improve the expression
and secretion of recombinant proteins in these filamentous fungi, several novel genetic engineering strategies have been developed
in recent years. This review describes state-of-the-art genetic manipulation technologies used for strain improvement, as
well as recent advances in designing the most appropriate engineering strategy for a particular protein production process.
Furthermore, current developments in identifying bottlenecks in the protein production and secretion pathways are described
and novel approaches to overcome these limitations are introduced. An appropriate combination of expression vectors and optimized
host strains will provide cell factories customized for each production process and expand the great potential of Aspergilli
as biotechnology workhorses to more complex multi-step industrial applications. 相似文献
3.
米曲霉作为一种重要的工业微生物,在异源蛋白表达方面已有广泛应用,受限于被表达蛋白的修饰及分泌过程,目前实际生产使用的基因供体主要局限于其他真菌,尤其是丝状真菌。当外源基因来源于植物、昆虫和哺乳动物时,米曲霉所生产的异源蛋白产量及生物活性往往不尽如人意。本文综述了米曲霉作为宿主表达异源蛋白的研究进展,包括其现有的遗传操作手段及异源表达方面的应用及探索,重点介绍了应用过程中面临的挑战和解决策略,另外,对米曲霉表达异源蛋白的应用前景及发展方向进行了展望。 相似文献
4.
The production of recombinant proteins is important in academic research to identify protein functions. Moreover, recombinant enzymes are used in the food and chemical industries, and high-quality proteins are required for diagnostic, therapeutic, and pharmaceutical applications. Though many recombinant proteins are produced by microbial or mammalian cell-based expression systems, plants have been promoted as alternative, cost-effective, scalable, safe, and sustainable expression systems. The development and improvement of transient expression systems have significantly reduced the period of protein production and increased the yield of recombinant proteins in plants. In this review, we consider the importance of plant-based expression systems for recombinant protein production and as genetic engineering tools. 相似文献
5.
The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels and chemicals, and it is also provides a platform for the production of many heterologous proteins of medical or industrial interest. Therefore, many studies have focused on metabolic engineering S. cerevisiae to improve the recombinant protein production, and with the development of systems biology, it is interesting to see how this approach can be applied both to gain further insight into protein production and secretion and to further engineer the cell for improved production of valuable proteins. In this review, the protein post-translational modification such as folding, trafficking, and secretion, steps that are traditionally studied in isolation will here be described in the context of the whole system of protein secretion. Furthermore, examples of engineering secretion pathways, high-throughput screening and systems biology applications of studying protein production and secretion are also given to show how the protein production can be improved by different approaches. The objective of the review is to describe individual biological processes in the context of the larger, complex protein synthesis network. 相似文献
6.
Sims AH Gent ME Lanthaler K Dunn-Coleman NS Oliver SG Robson GD 《Applied and environmental microbiology》2005,71(5):2737-2747
Filamentous fungi have a high capacity for producing large amounts of secreted proteins, a property that has been exploited for commercial production of recombinant proteins. However, the secretory pathway, which is key to the production of extracellular proteins, is rather poorly characterized in filamentous fungi compared to yeast. We report the effects of recombinant protein secretion on gene expression levels in Aspergillus nidulans by directly comparing a bovine chymosin-producing strain with its parental wild-type strain in continuous culture by using expressed sequence tag microarrays. This approach demonstrated more subtle and specific changes in gene expression than those observed when mimicking the effects of protein overproduction by using a secretion blocker. The impact of overexpressing a secreted recombinant protein more closely resembles the unfolded-protein response in vivo. 相似文献
7.
Live attenuated Salmonella strains have been extensively explored as oral delivery systems for recombinant vaccine antigens and effector proteins with immunoadjuvant and immunomodulatory potential. The feasibility of this approach was demonstrated in human vaccination trials for various antigens. However, immunization efficiencies with live vaccines are generally significantly lower compared to those monitored in parenteral immunizations with the same vaccine antigen. This is, at least partly, due to the lack of secretory expression systems, enabling large-scale extracellular delivery of vaccine and effector proteins by these strains. Because of their low complexity and the terminal location of the secretion signal in the secreted protein, Type I (ATP-binding cassette) secretion systems appear to be particularly suited for development of such recombinant extracellular expression systems. So far, the Escherichia coli hemolysin system is the only Type I secretion system, which has been adapted to recombinant protein secretion in Salmonella. However, this system has a number of disadvantages, including low secretion capacity, complex genetic regulation, and structural restriction to the secreted protein, which eventually hinder high-level in vivo delivery of recombinant vaccines and effector proteins. Thus, the development of more efficient recombinant protein secretion systems, based on Type I exporters can help to improve efficacies of live recombinant Salmonella vaccines. Type I secretion systems, mediating secretion of bacterial surface layer proteins, such as RsaA in Caulobacter crescentus, are discussed as promising candidates for improved secretory delivery systems. 相似文献
8.
Emmanuel Margolin Ros Chapman Anna‐Lise Williamson Edward P. Rybicki Ann E. Meyers 《Plant biotechnology journal》2018,16(9):1531-1545
Plant molecular farming offers a cost‐effective and scalable approach to the expression of recombinant proteins which has been proposed as an alternative to conventional production platforms for developing countries. In recent years, numerous proofs of concept have established that plants can produce biologically active recombinant proteins and immunologically relevant vaccine antigens that are comparable to those made in conventional expression systems. Driving many of these advances is the remarkable plasticity of the plant proteome which enables extensive engineering of the host cell, as well as the development of improved expression vectors facilitating higher levels of protein production. To date, the only plant‐derived viral glycoprotein to be tested in humans is the influenza haemagglutinin which expresses at ~50 mg/kg. However, many other viral glycoproteins that have potential as vaccine immunogens only accumulate at low levels in planta. A critical consideration for the production of many of these proteins in heterologous expression systems is the complexity of post‐translational modifications, such as control of folding, glycosylation and disulphide bridging, which is required to reproduce the native glycoprotein structure. In this review, we will address potential shortcomings of plant expression systems and discuss strategies to optimally exploit the technology for the production of immunologically relevant and structurally authentic glycoproteins for use as vaccine immunogens. 相似文献
9.
Filamentous fungi have long been used for production of a range of valuable products; with the advent of molecular biology, it became apparent that these fungi possess considerable potential as expression hosts for the production of heterologous proteins and small molecules. Aspergillus is an important genus, including well known species of economically significant molds, and widely used for basic genetic research. The development of a genetic engineering "toolkit" for Aspergillus, such as those existing for the simpler yeasts and bacteria, was delayed due to the added complexity of the filamentous fungi, and also to the lesser resources devoted to their study. History of the development of Aspergillus as an expression host, current state of the art and future directions are reviewed, touching on related research in other fungi when discussing the areas of greatest potential for future biotechnological applications, focusing on the large and diverse families of fungal secondary metabolites. 相似文献
10.
Engineering of protein secretion in yeast: strategies and impact on protein production 总被引:1,自引:0,他引:1
Alimjan Idiris Hideki Tohda Hiromichi Kumagai Kaoru Takegawa 《Applied microbiology and biotechnology》2010,86(2):403-417
Yeasts combine the ease of genetic manipulation and fermentation of a microorganism with the capability to secrete and modify
foreign proteins according to a general eukaryotic scheme. Their rapid growth, microbiological safety, and high-density fermentation
in simplified medium have a high impact particularly in the large-scale industrial production of foreign proteins, where secretory
expression is important for simplifying the downstream protein purification process. However, secretory expression of heterologous
proteins in yeast is often subject to several bottlenecks that limit yield. Thus, many studies on yeast secretion systems
have focused on the engineering of the fermentation process, vector systems, and host strains. Recently, strain engineering
by genetic modification has been the most useful and effective method for overcoming the drawbacks in yeast secretion pathways.
Such an approach is now being promoted strongly by current post-genomic technology and system biology tools. However, engineering
of the yeast secretion system is complicated by the involvement of many cross-reacting factors. Tight interdependence of each
of these factors makes genetic modification difficult. This indicates the necessity of developing a novel systematic modification
strategy for genetic engineering of the yeast secretion system. This mini-review focuses on recent strategies and their advantages
for systematic engineering of yeast strains for effective protein secretion. 相似文献
11.
Annamarie C. Dalton William A. Barton 《Protein science : a publication of the Protein Society》2014,23(5):517-525
Secreted mammalian proteins require the development of robust protein over‐expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large‐scale growth of cells in a variety of formats. 相似文献
12.
Ruchika Sharma Meenu Katoch P. S. Srivastava G. N. Qazi 《World journal of microbiology & biotechnology》2009,25(12):2083-2094
Fungi combine the advantages of a microbial system such as a simple fermentability with the capability of secreting proteins
that are modified according to a general eukaryotic scheme. Filamentous fungi such as Aspergillus niger efficiently secrete genuine proteins but the secretion of recombinant proteins turned out be a difficult task. Aspergillus niger is an attractive organism because of its high secretion capacity and is frequently used as a model organism. Whereas high
production yields can be obtained when homologous proteins are expressed, much lower amounts are obtained with the production
of heterologous proteins. To fully exploit the potential of filamentous fungi, understanding of the molecular genetics, their
physiology, and the glycosylation metabolism has to be investigated and clarified in more detail. This review summarizes recent
developments in heterologous protein production by filamentous fungi and also generalizes the possibilities of improving the
protein production by various genetic and bioprocessing approaches, thereby easing recognition of filamentous fungi as a relevant
and reliable expression platform. 相似文献
13.
Molecular farming in plants: host systems and expression technology 总被引:30,自引:0,他引:30
Plants provide an inexpensive and convenient system for the large-scale production of valuable recombinant proteins. This principle has been demonstrated by the commercial success of several first-generation products, and many others are currently under development. Over the past ten years, several efficient plant-based expression systems have emerged, and >100 recombinant proteins have now been produced in a range of different species. Plants have many advantages over other production systems, particularly in terms of practicality, economy and safety. However, several constraints that hinder the widespread use of plants as bioreactors remain to be addressed. Important factors include quality and homogeneity of the final product, the challenge of processing plant-derived pharmaceutical macromolecules under good manufacturing practice conditions and concerns about biosafety. Molecular farming in plants will only realize its huge potential if these constraints are removed through rigorous and detailed science-based studies. 相似文献
14.
15.
Almost all of the 200 or so approved biopharmaceuticals have been produced in one of three host systems: the bacterium Escherichia coli, yeasts (Saccharomyces cerevisiae, Pichia pastoris) and mammalian cells. We describe the most widely used methods for the expression of recombinant proteins in the cytoplasm or periplasm of E. coli, as well as strategies for secreting the product to the growth medium. Recombinant expression in E. coli influences the cell physiology and triggers a stress response, which has to be considered in process development. Increased expression of a functional protein can be achieved by optimizing the gene, plasmid, host cell, and fermentation process. Relevant properties of two yeast expression systems, S. cerevisiae and P. pastoris, are summarized. Optimization of expression in S. cerevisiae has focused mainly on increasing the secretion, which is otherwise limiting. P. pastoris was recently approved as a host for biopharmaceutical production for the first time. It enables high-level protein production and secretion. Additionally, genetic engineering has resulted in its ability to produce recombinant proteins with humanized glycosylation patterns. Several mammalian cell lines of either rodent or human origin are also used in biopharmaceutical production. Optimization of their expression has focused on clonal selection, interference with epigenetic factors and genetic engineering. Systemic optimization approaches are applied to all cell expression systems. They feature parallel high-throughput techniques, such as DNA microarray, next-generation sequencing and proteomics, and enable simultaneous monitoring of multiple parameters. Systemic approaches, together with technological advances such as disposable bioreactors and microbioreactors, are expected to lead to increased quality and quantity of biopharmaceuticals, as well as to reduced product development times. 相似文献
16.
Asgari S Arun S Slade MB Marshall J Williams KL Wheldrake JF 《Journal of molecular microbiology and biotechnology》2001,3(3):491-497
Growth factors and their binding proteins are important proteins regulating mammalian cell proliferation and differentiation so there is considerable interest in producing them as recombinant proteins, especially in hosts that do not already produce a complex mixture of growth factors. Many growth factors require post-translational modifications making them unsuitable for production in Escherichia coli or other prokaryotes. Since several expression vector systems have been recently developed for foreign protein production in the cellular slime mould, Dictyostelium discoideum, we attempted to use two of these systems to express human insulin-like growth factor binding protein 6 (hIGFBP6) and bovine beta-cellulin (bBTC) as secreted proteins. Although both proteins were successfully produced in stably transformed amoebae, no secretion was detected in spite of several attempts to facilitate this occurring. 相似文献
17.
Chloroplast-derived vaccines against human diseases: achievements, challenges and scopes 总被引:1,自引:0,他引:1
Infectious diseases represent a continuously growing menace that has severe impact on health of the people worldwide, particularly in the developing countries. Therefore, novel prevention and treatment strategies are urgently needed to reduce the rate of these diseases in humans. For this reason, different options can be considered for the production of affordable vaccines. Plants have been proved as an alternative expression system for various compounds of biological importance. Particularly, plastid genetic engineering can be potentially used as a tool for cost-effective vaccine production. Antigenic proteins from different viruses and bacteria have been expressed in plastids. Initial immunological studies of chloroplast-derived vaccines have yielded promising results in animal models. However, because of certain limitations, these vaccines face many challenges on production and application level. Adaptations to the novel approaches are needed, which comprise codon usage and choice of proven expression cassettes for the optimal yield of expressed proteins, use of inducible systems, marker gene removal, selection of specific antigens with high immunogenicity and development of tissue culture systems for edible crops to prove the concept of low-cost edible vaccines. As various aspects of plant-based vaccines have been discussed in recent reviews, here we will focus on certain aspects of chloroplast transformation related to vaccine production against human diseases. 相似文献
18.
The expression of high levels of stable and functional proteins remains a bottleneck in many scientific endeavors, including the determination of structures in a high-throughput fashion or the screening for novel active compounds in modern drug discovery. Recently, numerous developments have been made to improve the production of soluble and active proteins in heterologous expression systems. These include modifications to the expression constructs, the introduction of new and/or improved pro- and eukaryotic expression systems, and the development of improved cell-free protein synthesis systems. The introduction of robotics has enabled a massive parallelization of expression experiments, thereby vastly increasing the throughput and, hopefully, the output of such experiments. In addition, the big challenges of recombinant overexpression of membrane and secreted proteins are tackled, and some new methods are reviewed. 相似文献
19.
MMP-9信号肽高效诱导PEX重组蛋白在COS7细胞中分泌表达 总被引:1,自引:1,他引:1
为了便于收集和纯化, 重组蛋白常需要引导至真核细胞外。蛋白能否分泌主要取决于其是否含有信号肽, 由于不同信号肽诱导蛋白分泌的效率不同,高效信号肽的筛选已成为生物工程领域提高重组蛋白产量的重要策略之一。为了筛选诱导MMP-2 C末端PEX在COS7细胞中高效分泌表达的信号肽,在PEX的N末端分别融合大鼠生长激素(rGH)、小鼠IgG κ链和人基质金属蛋白酶-9(matrix metalloproteinase 9, MMP-9)的信号肽并比较三种信号肽引导PEX分泌表达的效率。Western免疫印迹和ELISA蛋白定量检测表明MMP-9的信号肽引导PEX蛋白分泌的效率约为其它两种信号肽的两倍。利用Ni-NTA亲和柱对细胞培养基中的PEX进行纯化,蛋白产量约为1mg/L,纯化的PEX重组蛋白具有抑制鸡尿囊膜(chorioallantoic membrane,CAM)血管发生的作用。以上结果提示MMP-9的信号肽有效诱导具有生物活性的PEX重组蛋白在COS7细胞中分泌表达。 相似文献
20.
As a protein expression vector,the baculovirus demonstrates many advantages over other vectors.With the development of biotechnology,baculoviral vectors have been genetically modified to facilitate hig... 相似文献