共查询到20条相似文献,搜索用时 15 毫秒
1.
Elizabeth Specht Shigeki Miyake-Stoner Stephen Mayfield 《Biotechnology letters》2010,32(10):1373-1383
A complete set of genetic tools is still being developed for the micro-alga Chlamydomonas reinhardtii. Yet even with this incomplete set, this photosynthetic single-celled plant has demonstrated significant promise as a platform
for recombinant protein expression. In recent years, techniques have been developed that allow for robust expression of genes
from both the nuclear and plastid genome. With these advances, many research groups have examined the pliability of this and
other micro-algae as biological machines capable of producing recombinant peptides and proteins. This review describes recent
successes in recombinant protein production in Chlamydomonas, including production of complex mammalian therapeutic proteins and monoclonal antibodies at levels sufficient for production
at economic parity with existing production platforms. These advances have also shed light on the details of algal protein
production at the molecular level, and provide insight into the next steps for optimizing micro-algae as a useful platform
for the production of therapeutic and industrially relevant recombinant proteins. 相似文献
2.
Thomas Rademacher Markus Sack Daniel Blessing Rainer Fischer Tanja Holland Johannes Buyel 《Plant biotechnology journal》2019,17(8):1560-1566
Industrial plant biotechnology applications include the production of sustainable fuels, complex metabolites and recombinant proteins, but process development can be impaired by a lack of reliable and scalable screening methods. Here, we describe a rapid and versatile expression system which involves the infusion of Agrobacterium tumefaciens into three‐dimensional, porous plant cell aggregates deprived of cultivation medium, which we have termed plant cell packs (PCPs). This approach is compatible with different plant species such as Nicotiana tabacum BY2, Nicotiana benthamiana or Daucus carota and 10‐times more effective than transient expression in liquid plant cell culture. We found that the expression of several proteins was similar in PCPs and intact plants, for example, 47 and 55 mg/kg for antibody 2G12 expressed in BY2 PCPs and N. tabacum plants respectively. Additionally, the expression of specific enzymes can either increase the content of natural plant metabolites or be used to synthesize novel small molecules in the PCPs. The PCP method is currently scalable from a microtiter plate format suitable for high‐throughput screening to 150‐mL columns suitable for initial product preparation. It therefore combined the speed of transient expression in plants with the throughput of microbial screening systems. Plant cell packs therefore provide a convenient new platform for synthetic biology approaches, metabolic engineering and conventional recombinant protein expression techniques that require the multiplex analysis of several dozen up to hundreds of constructs for efficient product and process development. 相似文献
3.
Background
Plant bioreactor offers an efficient and economical system for large-scale production of recombinant proteins. However, high cost and difficulty in scaling-up of downstream purification of the target protein, particularly the common involvement of affinity chromatography and protease in the purification process, has hampered its industrial scale application, therefore a cost-effective and easily scale-up purification method is highly desirable for further development of plant bioreactor.Methodology/Principal Findings
To tackle this problem, we investigated the ELP-intein coupling system for purification of recombinant proteins expressed in transgenic plants using a plant lectin (PAL) with anti-tumor bioactivity as example target protein and rice seeds as production platform. Results showed that ELP-intein-PAL (EiP) fusion protein formed novel irregular ER-derived protein bodies in endosperm cells by retention of endogenous prolamins. The fusion protein was partially self-cleaved in vivo, but only self-cleaved PAL protein was detected in total seed protein sample and deposited in protein storage vacuoles (PSV). The in vivo uncleaved EiP protein was accumulated up to 2–4.2% of the total seed protein. The target PAL protein could be purified by the ELP-intein system efficiently without using complicated instruments and expensive chemicals, and the yield of pure PAL protein by the current method was up to 1.1 mg/g total seed protein.Conclusion/Significance
This study successfully demonstrated the purification of an example recombinant protein from rice seeds by the ELP-intein system. The whole purification procedure can be easily scaled up for industrial production, providing the first evidence on applying the ELP-intein coupling system to achieve cost-effective purification of recombinant proteins expressed in plant bioreactors and its possible application in industry. 相似文献4.
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Large-scale transient expression in mammalian cells for recombinant protein production 总被引:12,自引:0,他引:12
Large-scale transient expression from mammalian cells is a new technology. Breakthroughs have been achieved for non-viral delivery methods: transfections can now be done at the 1-10 L scale with mammalian cells grown in suspension. Production of 1-20 mg/L of recombinant protein have been obtained in stirred bioreactors. Modified alphaviruses have provided a fast and efficient expression technology based on viral vectors. 相似文献
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Stability of protein production from recombinant mammalian cells 总被引:6,自引:0,他引:6
10.
Susan DJ Chapple Anna M Crofts S Paul Shadbolt John McCafferty Michael R Dyson 《BMC biotechnology》2006,6(1):49-15
Background
A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner. 相似文献11.
Shabnam Shamriz 《Biotechnology & genetic engineering reviews》2016,32(1-2):92-106
Microalgae, also called microphytes, are a vast group of microscopic photosynthetic organisms living in aquatic ecosystems. Microalgae have attracted the attention of biotechnology industry as a platform for extracting natural products with high commercial value. During last decades, microalgae have been also used as cost-effective and easily scalable platform for the production of recombinant proteins with medical and industrial applications. Most progress in this field has been made with Chlamydomonas reinhardtii as a model organism mainly because of its simple life cycle, well-established genetics and ease of cultivation. However, due to the scarcity of existing infrastructure for commercial production and processing together with relatively low product yields, no recombinant products from C. reinhardtii have gained approval for commercial production and most of them are still in research and development. In this review, we focus on the chloroplast of C. reinhardtii as an algal recombinant expression platform and compare its advantages and disadvantages to other currently used expression systems. We then discuss the strategies for engineering the chloroplast of C. reinhardtii to produce recombinant cells and present a comprehensive overview of works that have used this platform for the expression of high-value products. 相似文献
12.
The RNA polymerase factory: a robotic in vitro assembly platform for high-throughput production of recombinant protein complexes 总被引:1,自引:0,他引:1
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The in-depth structure/function analysis of large protein complexes, such as RNA polymerases (RNAPs), requires an experimental platform capable of assembling variants of such enzymes in large numbers in a reproducible manner under defined in vitro conditions. Here we describe a streamlined and integrated protocol for assembling recombinant archaeal RNAPs in a high-throughput 96-well format. All aspects of the procedure including construction of redesigned expression plasmids, development of automated protein extraction/in vitro assembly methods and activity assays were specifically adapted for implementation on robotic platforms. The optimized strategy allows the parallel assembly and activity assay of 96 recombinant RNAPs (including wild-type and mutant variants) with little or no human intervention within 24 h. We demonstrate the high-throughput potential of this system by evaluating the side-chain requirements of a single amino acid position of the RNAP Bridge Helix using saturation mutagenesis. 相似文献
13.
Belzhelarskaia SN 《Molekuliarnaia biologiia》2011,45(1):142-159
The baculovirus vector systems has been extensively used for the expression of foreign gene products in insect and mammalian cells. New advances increase the possibilities and applications of the baculovirus expression system, which has the capability to express multiple genes simultaneously within a single infected insect cells and to use recombinant virus with mammalian cell-active expression cassettes to permit expression of recombinant proteins in mammalian cells in vitro and in vivo. Future investigations of the baculovirus expression system designed for specific target cells, can open wide variety of applications. This review summarizes the recent achievements in applications the baculovirus vector systems and optimization recombinant protein expression in both insect and mammalian cell lines. 相似文献
14.
Large-Scale transfection of mammalian cells for the fast production of recombinant protein 总被引:2,自引:0,他引:2
Recombinant proteins (r-proteins) are increasingly important in fundamental research and for clinical applications. As many of these r-proteins are of human or animal origin, cultivated mammalian cells are the host of choice to ensure their functional folding and proper posttranslational modifications. Large-scale transfection of human embryonic kidney 293 or Chinese hamster ovary cells is now an established technology that can be used in the production of hundreds of milligram to gram quantities of a r-protein in less than 1 mo from cloning of its cDNA. This chapter aims to provide an overview of large-scale transfection technology with a particular emphasis on calcium phosphate and polyethylenimine-mediated gene transfer. 相似文献
15.
Summary Two-phase cultivation of insect cells was carried out to produce recombinant -galactosidase and evaluate factors influencing high cell density and high expression. The primary factor limiting specific expression at high cell concentration was not the cell concentration but the medium condition. The addition of 4 g/L yeastolate during the growth phase increased cell density while the addition of 0.6 g/L glutamine in the production phase raised the specific expression rate. Productivity of recombinant protein was consequently improved seven-fold by this operation. 相似文献
16.
Kuprijanov A Schaepe S Aehle M Simutis R Lübbert A 《Bioprocess and biosystems engineering》2012,35(3):333-340
An new cascade control system is presented that reproducibly keeps the cultivation part of recombinant protein production
processes on its predetermined track. While the system directly controls carbon dioxide production mass and carbon dioxide
production rates along their setpoint profiles in fed-batch cultivation, it simultaneously keeps the specific biomass growth
rates and the biomass profiles on their desired paths. The control scheme was designed and tuned using a virtual plant environment
based on the industrial process control system SIMATIC PCS 7 (Siemens AG). It is shown by means of validation experiments
that the simulations in this straightforward approach directly reflect the experimentally observed controller behaviour. Within
the virtual plant environment, it was shown that the cascade control is considerably better than previously used control approaches.
The controller significantly improved the batch-to-batch reproducibility of the fermentations. Experimental tests confirmed
that it is particularly suited for cultivation processes suffering from long response times and delays. The performance of
the new controller is demonstrated during its application in Escherichia coli fed-batch cultivations as well as in animal cell cultures with CHO cells. The technique is a simple and reliable alternative
to more sophisticate model-supported controllers. 相似文献
17.
Li Y 《Biotechnology letters》2011,33(5):869-881
Fusion expression is a common practice for recombinant protein production. Some fusion tags confer solubility on the target
protein whereas others provide affinity handles that facilitate purification. However, the tag usually needs to be removed
from the final product, which involves using expensive proteases or hazardous chemicals and requires additional chromatography
steps. Self-cleaving tags are a special group of fusion tags that possess inducible proteolytic activity. Combined with appropriate
affinity tags, they enable fusion purification, cleavage and target separation to be achieved in a single step, which saves
time, labor and cost. This paper reviews currently available self-cleaving fusion tags for recombinant protein production.
For each system, an introduction of its key characteristics and a brief discussion of its advantages and disadvantages is
given. 相似文献
18.
Leander Blaas Monica Musteanu Robert Eferl Anton Bauer Emilio Casanova 《BMC biotechnology》2009,9(1):3-5
Background
The development of appropriate expression vectors for large scale protein production constitutes a critical step in recombinant protein production. The use of conventional expression vectors to obtain cell lines is a cumbersome procedure. Often, stable cell lines produce low protein yields and production is not stable over the time. These problems are due to silencing of randomly integrated expression vectors by the surrounding chromatin. To overcome these chromatin effects, we have employed a Bacterial Artificial Chromosome (BAC) as expression vector to obtain stable cell lines suitable for protein production. 相似文献19.
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
20.
We describe a platform that utilizes wheat germ cell-free technology to produce protein samples for NMR structure determinations. In the first stage, cloned DNA molecules coding for proteins of interest are transcribed and translated on a small scale (25 microL) to determine levels of protein expression and solubility. The amount of protein produced (typically 2-10 microg) is sufficient to be visualized by polyacrylamide gel electrophoresis. The fraction of soluble protein is estimated by comparing gel scans of total protein and soluble protein. Targets that pass this first screen by exhibiting high protein production and solubility move to the second stage. In the second stage, the DNA is transcribed on a larger scale, and labeled proteins are produced by incorporation of [(15)N]-labeled amino acids in a 4 mL translation reaction that typically produces 1-3 mg of protein. The [(15)N]-labeled proteins are screened by (1)H-(15)N correlated NMR spectroscopy to determine whether the protein is a good candidate for solution structure determination. Targets that pass this second screen are then translated in a medium containing amino acids doubly labeled with (15)N and (13)C. We describe the automation of these steps and their application to targets chosen from a variety of eukaryotic genomes: Arabidopsis thaliana, human, mouse, rat, and zebrafish. We present protein yields and costs and compare the wheat germ cell-free approach with alternative methods. Finally, we discuss remaining bottlenecks and approaches to their solution. 相似文献