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1.
Nearly 30% of currently approved recombinant therapeutic proteins are produced in Escherichia coli. Due to its well-characterized genetics, rapid growth and high-yield production, E. coli has been a preferred choice and a workhorse for expression of non-glycosylated proteins in the biotech industry. There is
a wealth of knowledge and comprehensive tools for E. coli systems, such as expression vectors, production strains, protein folding and fermentation technologies, that are well tailored
for industrial applications. Advancement of the systems continues to meet the current industry needs, which are best illustrated
by the recent drug approval of E. coli produced antibody fragments and Fc-fusion proteins by the FDA. Even more, recent progress in expression of complex proteins
such as full-length aglycosylated antibodies, novel strain engineering, bacterial N-glycosylation and cell-free systems further
suggests that complex proteins and humanized glycoproteins may be produced in E. coli in large quantities. This review summarizes the current technology used for commercial production of recombinant therapeutics
in E. coli and recent advances that can potentially expand the use of this system toward more sophisticated protein therapeutics. 相似文献
2.
Production of recombinant proteins in Mycobacterium smegmatis for structural and functional studies
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Ghader Bashiri Edward N. Baker 《Protein science : a publication of the Protein Society》2015,24(1):1-10
Protein production using recombinant DNA technology has a fundamental impact on our understanding of biology through providing proteins for structural and functional studies. Escherichia coli (E. coli) has been traditionally used as the default expression host to over‐express and purify proteins from many different organisms. E. coli does, however, have known shortcomings for obtaining soluble, properly folded proteins suitable for downstream studies. These shortcomings are even more pronounced for the mycobacterial pathogen Mycobacterium tuberculosis, the bacterium that causes tuberculosis, with typically only one third of proteins expressed in E. coli produced as soluble proteins. Mycobacterium smegmatis (M. smegmatis) is a closely related and non‐pathogenic species that has been successfully used as an expression host for production of proteins from various mycobacterial species. In this review, we describe the early attempts to produce mycobacterial proteins in alternative expression hosts and then focus on available expression systems in M. smegmatis. The advantages of using M. smegmatis as an expression host, its application in structural biology and some practical aspects of protein production are also discussed. M. smegmatis provides an effective expression platform for enhanced understanding of mycobacterial biology and pathogenesis and for developing novel and better therapeutics and diagnostics. 相似文献
3.
Nonribosomal peptide synthetases (NRPS) and type I polyketide synthases (PKS-I) are biosynthetic systems involved in the synthesis of a large number of important biologically active compounds produced by microorganisms, among others by actinomycetes. In order to assess the occurrence of these biosynthetic systems in this metabolically active bacterial group, we designed new PCR primers targeted to specifically amplify NRPS and PKS-I gene sequences from actinomycetes. The sequence analysis of amplified products cloned from two model systems and used to validate these molecular tools has shown the extreme richness of NRPS or PKS-I-like sequences in the actinomycete genome. When these PCR primers were tested on a large collection of 210 reference strains encompassing all major families and genera in actinomycetes, we observed that the wide distribution of these genes in the well-known productive Streptomyces species is also extended to other minor lineages where in some cases very few bioactive compounds have been identified to date. 相似文献
4.
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
5.
6.
Several unique protein families have been identified that play a role in the control of developmental cell division in streptomycetes. The SsgA-like proteins or SALPs, of which streptomycetes typically have at least five paralogues, control specific steps of sporulation-specific cell division in streptomycetes, affecting cell wall-related events such as septum localization and synthesis, thickening of the spore wall and autolytic spore separation. The expression level of SsgA, the best studied SALP, has a rather dramatic effect on septation and on hyphal morphology, which is not only of relevance for our understanding of (developmental) cell division but has also been successfully applied in industrial fermentation, to improve growth and production of filamentous actinomycetes. Recent observations suggest that SsgB most likely is the archetypal SALP, with only SsgB orthologues occurring in all morphologically complex actinomycetes. Here we review 10 years of research on the SsgA-like proteins in actinomycetes and discuss the most interesting regulatory, functional, phylogenetic and applied aspects of this relatively unknown protein family. 相似文献
7.
Joy A. Colquhoun Stephen C. Heald Lina Li J. Tamaoka Chiaki Kato Koki Horikoshi A. T. Bull 《Extremophiles : life under extreme conditions》1998,2(3):269-277
Deep-sea soft sediments from trench systems and depths in the northwestern Pacific Ocean ranging from less than 300 to 10
897 m in depth have been analyzed for three target genera of actinomycetes: Micromonospora, Rhodococcus, and Streptomyces. Only culturable strains, recovered at atmospheric pressure on selective isolation media, have been examined to date. Maximum
recoveries of culturable bacteria were greater that 107/ml wet g sediment, but actinomycetes comprised a small proportion of this population (usually less than 1%). The target actinomycetes
were isolated at all depths except from the Mariana Trench sediments. Actinomycete colonies were defined initially on the
basis of colony morphologies, and preliminary identification then was made by chemotaxonomic tests. Pyrolysis mass spectrometry
(PyMS) of deep-sea mycolic acid-containing actinomycetes gave excellent correspondence with numerical (phenetic) taxonomic
analyses and subsequently was adopted as a rapid procedure for assessing taxonomic diversity. PyMS analysis enabled several
clusters of deep-sea rhodococci to be distinguished that are quite distinct from all type strains. 16S rRNA gene sequence
analysis has revealed that several of these marine rhodococci have sequences that are very similar to certain terrestrial
species of Rhodococcus and to Dietzia. There is evidence for the intrusion of terrestrial runoff into these deep trench systems, and the inconsistency of the phenotypic
and molecular taxonomies may reflect recent speciation events in actinomycetes under the high-pressure conditions of the deep
sea. The results of DNA-DNA pairing experiments point to the novelty of Rhodococcus strains recovered from hadal depths in the Izu Bonin Trench. Biotransformation studies of deep-sea bacteria have focused
on nitrile compounds. Nitrile-metabolizing bacteria, closely related to rhodococci, have been isolated that grow well at low
temperature, high salt concentrations, and high pressures, suggesting that they are of marine origin or have adapted to the
deep-sea environment.
Received: January 22, 1998 / Accepted: February 16, 1998 相似文献
8.
Hua Ling 《Biologia》2007,62(2):119-123
For the production of recombinant proteins, product purification is potentially difficult and expensive. Plant oleosins are
capable of anchoring onto the surface of natural or artificial oil bodies. The oleosin fusion expression systems allow products
to be extracted with oil bodies. In vivo, oleosin fusions are produced and directly localized to natural oil bodies in transgenic plant seeds. Via the oleosin fusion
technology the thrombin inhibitor hirudin has been successfully produced and commercially used in Canada. In vitro, artificial oil bodies have been used as “carriers” for the recombinant proteins expressed in transformed microbes. In this
article, plant oleosins, strategies and limitations of the oleosin fusion expression systems are summarized, alongside with
progress and applications. The oleosin fusion expression systems reveal an available way to produce recombinant biopharmaceuticals
at large scale. 相似文献
9.
In the ever-expanding search for novel bioactive molecules and enzymes, marine actinomycetes have proven to be a productive
source. While open reef sediment and sponge-associated actinomycetes have been extensively examined, their marine cave counterparts
remain unevaluated. Anchialine cave systems in the Bahamas offered an ideal setting to evaluate the occurrence and variation
within sediment-associated actinomycete communities. While in close geographical proximity to open reef environments, these
systems provide a specialized environmental niche devoid of light and direct exposure to nutrient input. In the present study,
selective isolation techniques and molecular methods were used to test the hypothesis that variable distribution of actinomycetes
and secondary metabolite gene clusters occur between open reef and marine cave systems. The results indicated that differences
exist within the culturable sediment-associated actinomycete communities between marine caves and open reef systems, with
members of the genus Streptomyces dominating cultures from open reef sediments and a more diverse suite of actinomycetes isolated from marine cave sediment
samples. Within the cave isolates, members of the proposed genus Solwaraspora were the most represented. Based on PKS- and NRPS-gene-targeted PCR amplification and sequencing, geographic variation in
the occurrence of these biosynthetic pathways was also observed. These findings indicate that marine cave systems are a lucrative
source in the search for novel secondary metabolite producers with biotechnological applications and that environmental and
geographic factors likely affect the occurrence of these biosynthetic pathways. 相似文献
10.
Samaniego-Hernández M De León-Rodriguez A Aparicio-Fabre R Arias-Ortiz C Barba de la Rosa AP 《Cell biochemistry and biophysics》2006,44(3):336-341
Rotaviruses are one of the worldwide leading causes of gastroenteritis in children under 5 yr old. The rotavirus nonstructural
NSP5 is a phosphoprotein implicated in viroplasms formation, whereas NSP6 could have a possible regulatory role of NSP5. It
has been reported that N- and C-termini of NSP5 are important for amount of protein is required for structural analysis, efficient
expression systems are required. His-tag fusion at the C-terminus and glutathione-S-transferase (GST)-fusion at the N-terminus were used as expression systems, and conditions for recombinant proteins expression
were obtained. His-tag fusion was not efficient to produce NSP5 (2% of total protein), but NSP6 was expressed in higher amounts
(11% of total protein). In contrast, GST-NSP5 and GST-NSP6 proteins correspond to 34 and 31% of the total proteins, respectively.
GST-fusions seem to have a protective effect against nonstructural rotavirus protein toxicity in Escherichia coli; however, in both systems, NSP5 and NSP6 recombinant proteins were expressed as inclusion bodies. Conditions for solubilization
and purification of recombinant proteins were achieved. This is the first report of expression and purification of NSP5 and
NSP6 recombinant proteins in suitable amounts for further structural analysis. 相似文献
11.
Baltz RH 《Journal of industrial microbiology & biotechnology》2011,38(6):657-666
With the recent advances in DNA sequencing technologies, it is now feasible to sequence multiple actinomycete genomes rapidly
and inexpensively. An important observation that emerged from early Streptomyces genome sequencing projects was that each strain contains genes that encode 20 or more potential secondary metabolites, only
a fraction of which are expressed during fermentation. More recently, this observation has been extended to many other actinomycetes
with large genomes. The discovery of a wealth of orphan or cryptic secondary metabolite biosynthetic gene clusters has suggested
that sequencing large numbers of actinomycete genomes may provide the starting materials for a productive new approach to
discover novel secondary metabolites. The key issue for this approach to be successful is to find ways to turn on or turn
up the expression of cryptic or poorly expressed pathways to provide material for structure elucidation and biological testing.
In this review, I discuss several genetic approaches that are potentially applicable to many actinomycetes for this application. 相似文献
12.
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
13.
Georg Schmidt Gabriele Gadermaier Heidi Pertl Marc Siegert Kirsi-Marja Oksman-Caldentey Anneli Ritala Martin Himly Gerhard Obermeyer Fatima Ferreira 《Phytochemistry Reviews》2008,7(3):539-552
A large percentage of allergenic proteins are of plant origin. Hence, plant-based expression systems are considered ideal
for the recombinant production of certain allergens. First attempts to establish production of plant-derived allergens in
plants focused on transient expression in Nicotiana benthamiana infected with recombinant viral vectors. Accordingly, allergens from birch and mugwort pollen, as well as from apple have
been expressed in plants. Production of house dust mite allergens has been achieved by Agrobacterium-mediated transformation of tobacco plants. Beside the use of plants as production systems, other approaches have focused
on the development of edible vaccines expressing allergens or epitopes thereof, which bypasses the need of allergen purification.
The potential of this approach has been convincingly demonstrated for transgenic rice seeds expressing seven dominant human
T cell epitopes derived from Japanese cedar pollen allergens. Parallel to efforts in developing recombinant-based diagnostic
and therapeutic reagents, different gene-silencing approaches have been used to decrease the expression of allergenic proteins
in allergen sources. In this way hypoallergenic ryegrass, soybean, rice, apple, and tomato were developed. 相似文献
14.
15.
Chlamydomonas reinhardtii has many advantages compared with traditional systems for the molecular farming of recombinant proteins. These include low
production costs, rapid scalability at pilot level, absence of human pathogens and the ability to fold and assemble complex
proteins accurately. Currently, the successful expression of several proteins with pharmaceutical relevance has been reported
from the nuclear and the chloroplastic genome of this alga, demonstrating its usefulness for biotechnological applications.
However, several factors affect the level of recombinant protein expression in Chlamydomonas such as enhancer elements, codon dependency, sensitivity to proteases and transformation-associated genotypic modification.
The present review outlines a number of strategies to increase protein yields and summarizes recent achievements in algal
protein production including biopharmaceuticals such as vaccines, antibodies, hormones and enzymes with implications on health-related
approaches. The current status of bioreactor developments for algal culture and the challenges of scale-up and optimization
processes are also discussed. 相似文献
16.
Mohsen Karbalaei Seyed A. Rezaee Hadi Farsiani 《Journal of cellular physiology》2020,235(9):5867-5881
One of the most important branches of genetic engineering is the expression of recombinant proteins using biological expression systems. Nowadays, different expression systems are used for the production of recombinant proteins including bacteria, yeasts, molds, mammals, plants, and insects. Yeast expression systems such as Saccharomyces cerevisiae (S. cerevisiae) and Pichia pastoris (P. pastoris) are more popular. P. pastoris expression system is one of the most popular and standard tools for the production of recombinant protein in molecular biology. Overall, the benefits of protein production by P. pastoris system include appropriate folding (in the endoplasmic reticulum) and secretion (by Kex2 as signal peptidase) of recombinant proteins to the external environment of the cell. Moreover, in the P. pastoris expression system due to its limited production of endogenous secretory proteins, the purification of recombinant protein is easy. It is also considered a unique host for the expression of subunit vaccines which could significantly affect the growing market of medical biotechnology. Although P. pastoris expression systems are impressive and easy to use with well-defined process protocols, some degree of process optimization is required to achieve maximum production of the target proteins. Methanol and sorbitol concentration, Mut forms, temperature and incubation time have to be adjusted to obtain optimal conditions, which might vary among different strains and externally expressed protein. Eventually, optimal conditions for the production of a recombinant protein in P. pastoris expression system differ according to the target protein. 相似文献
17.
The preparation of stable isotope-labeled proteins is necessary for the application of a wide variety of NMR methods, to study
the structures and dynamics of proteins and protein complexes. The E. coli expression system is generally used for the production of isotope-labeled proteins, because of the advantages of ease of
handling, rapid growth, high-level protein production, and low cost for isotope-labeling. However, many eukaryotic proteins
are not functionally expressed in E. coli, due to problems related to disulfide bond formation, post-translational modifications, and folding. In such cases, other
expression systems are required for producing proteins for biomolecular NMR analyses. In this paper, we review the recent
advances in expression systems for isotopically labeled heterologous proteins, utilizing non-E. coli prokaryotic and eukaryotic cells. 相似文献
18.
Richard H. Baltz 《Journal of industrial microbiology & biotechnology》2010,37(8):759-772
Natural products discovery from actinomycetes has been on the decline in recent years, and has suffered from a lack of innovative
ways to discover new secondary metabolites within a background of the thousands of known compounds. Recent advances in whole
genome sequencing have revealed that actinomycetes with large genomes encode multiple secondary metabolite pathways, most
of which remain cryptic. One approach to address the expression of cryptic pathways is to first identify novel pathways by
bioinformatics, then clone and express them in well-characterized hosts with known secondary metabolomes. This process should
eliminate the tedious dereplication process that has hampered natural products discovery. Several laboratory and industrial
production strains have been used for heterologous production of secondary metabolite pathways. This review discusses the
results of these studies, and the pros and cons of using various Streptomyces and one Saccharopolyspora strain for heterologous expression. This information should provide an experimental basis to help researchers choose hosts
for current application and future development to express heterologous secondary metabolite pathways in yields sufficient
for rapid scale-up, biological testing, and commercial production. 相似文献
19.
Brian J. Green Masaaki Fujiki Valentina Mett Jon Kaczmarczyk Moneim Shamloul Konstantin Musiychuk Susan Underkoffler Vidadi Yusibov Vadim Mett Dr. 《Biotechnology journal》2009,4(2):230-237
The expression of proteins in plants both transiently and via permanently transformed lines has been demonstrated by a number of groups. Transient plant expression systems, due to high expression levels and speed of production, show greater promise for the manufacturing of biopharmaceuticals when compared to permanent transformants. Expression vectors based on a tobacco mosaic virus (TMV) are the most commonly utilized and the primary plant used, Nicotiana benthamiana, has demonstrated the ability to express a wide range of proteins at levels amenable to purification. N. benthamiana has two limitations for its use; one is its relatively slow growth, and the other is its low biomass. To address these limitations we screened a number of legumes for transient protein expression. Using the alfalfa mosaic virus (AMV) and the cucumber mosaic virus (CMV) vectors, delivered via Agrobacterium, we were able to identify three Pisum sativum varieties that demonstrated protein expression transiently. Expression levels of 420 ( 26.24 mg GFP/kgFW in the green pea variety speckled pea were achieved. We were also able to express three therapeutic proteins indicating promise for this system in the production of biopharmaceuticals. 相似文献
20.
The genus Amycolatopsis is a member of the phylogenetic group nocardioform actinomycetes. Most of the members of the genus Amycolatopsis are known to produce antibiotics. Additionally, members of this genus have been reported to metabolize aromatic compounds
as the sole sources of carbon and energy. Development of genetic manipulation in Amycolatopsis has progressed slowly due to paucity of genetic tools and methods. The occurrence of indigenous plasmids in different species
of Amycolatopsis is not very common. Till date, only three indigenous plasmids viz., pMEA100, pMEA300 and pA387 have been reported in Amycolatopsis species. Various vectors based on the indigenous plasmids, pMEA100, pMEA300 and pA387, have been constructed. These vectors
have proved useful for molecular genetics studies of actinomycetes. Molecular genetic work with Amycolatopsis strains is not easy, since transformation methods have to be developed, or at least optimized, for each particular strain.
Nonetheless, methods for efficient transformation (polyethyleneglycol (PEG) induced protoplast transformation, transformation
by electroporation and direct transformation) have been developed and used successfully for the introduction of DNA into several
Amycolatopsis species. The construction of plasmid cloning vectors and the development of gene transfer systems has opened up possibilities
for studying the molecular genetics of these bacteria. 相似文献