共查询到20条相似文献,搜索用时 463 毫秒
1.
Eva Jordan Michael Hust Andreas Roth Rebekka Biedendieck Thomas Schirrmann Dieter Jahn Stefan Dübel 《Microbial cell factories》2007,6(1):2
Background
Recombinant antibodies are essential reagents for research, diagnostics and therapy. The well established production host Escherichia coli relies on the secretion into the periplasmic space for antibody synthesis. Due to the outer membrane of Gram-negative bacteria, only a fraction of this material reaches the medium. Recently, the Gram-positive bacterium Bacillus megaterium was shown to efficiently secrete recombinant proteins into the growth medium. Here we evaluated B. megaterium for the recombinant production of antibody fragments. 相似文献2.
J. Wu B.A. Boghigian M. Myint H. Zhang S. Zhang B.A. Pfeifer 《Letters in applied microbiology》2010,51(2):196-204
Aims: Escherichia coli has emerged as a viable heterologous host for the production of complex, polyketide natural compounds. In this study, polyketide biosynthesis was compared between different E. coli strains for the purpose of better understanding and improving heterologous production. Methods and Results: Both B and K‐12 E. coli strains were genetically modified to support heterologous polyketide biosynthesis [specifically, 6‐deoxyerythronolide B (6dEB)]. Polyketide production was analysed using a helper plasmid designed to overcome rare codon usage within E. coli. Each strain was analysed for recombinant protein production, precursor consumption, by‐product production, and 6dEB biosynthesis. Of the strains tested for biosynthesis, 6dEB production was greatest for E. coli B strains. When comparing biosynthetic improvements as a function of mRNA stability vs codon bias, increased 6dEB titres were observed when additional rare codon tRNA molecules were provided. Conclusions: Escherichia coli B strains and the use of tRNA supplementation led to improved 6dEB polyketide titres. Significance and Impact of the Study: Given the medicinal potential and growing field of polyketide heterologous biosynthesis, the current study provides insight into host‐specific genetic backgrounds and gene expression parameters aiding polyketide production through E. coli. 相似文献
3.
Escherichia coli is the most commonly used host for recombinant protein production and metabolic engineering. Extracellular production of
enzymes and proteins is advantageous as it could greatly reduce the complexity of a bioprocess and improve product quality.
Extracellular production of proteins is necessary for metabolic engineering applications in which substrates are polymers
such as lignocelluloses or xenobiotics since adequate uptake of these substrates is often an issue. The dogma that E. coli secretes no protein has been challenged by the recognition of both its natural ability to secrete protein in common laboratory
strains and increased ability to secrete proteins in engineered cells. The very existence of this review dedicated to extracellular
production is a testimony for outstanding achievements made collectively by the community in this regard. Four strategies
have emerged to engineer E. coli cells to secrete recombinant proteins. In some cases, impressive secretion levels, several grams per liter, were reached.
This secretion level is on par with other eukaryotic expression systems. Amid the optimism, it is important to recognize that
significant challenges remain, especially when considering the success cannot be predicted a priori and involves much trials
and errors. This review provides an overview of recent developments in engineering E. coli for extracellular production of recombinant proteins and an analysis of pros and cons of each strategy. 相似文献
4.
Lingqia Su Sheng Chen Li Yi Ronald W Woodard Jian Chen Jing Wu 《Microbial cell factories》2012,11(1):8
Background
Extracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, Thermobifida fusca cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in Escherichia coli BL21(DE3), and it was found that parts of the expressed protein was accumulated in the periplasmic space. Due to the fact that alpha-hemolysin secretion system can export target proteins directly from cytoplasm across both cell membrane of E. coli to the culture medium, thus in the present study we investigated the expression of cutinase using this alpha-hemolysin secretion system. 相似文献5.
Background
Escherichia coli has been most widely used for the production of valuable recombinant proteins. However, over-production of heterologous proteins in E. coli frequently leads to their misfolding and aggregation yielding inclusion bodies. Previous attempts to refold the inclusion bodies into bioactive forms usually result in poor recovery and account for the major cost in industrial production of desired proteins from recombinant E. coli. Here, we describe the successful use of the immobilized folding machineries for in vitro refolding with the examples of high yield refolding of a ribonuclease A (RNase A) and cyclohexanone monooxygenase (CHMO). 相似文献6.
Gabriele R. M. Kleiner‐Grote Joe M. Risse Karl Friehs 《Engineering in Life Science》2018,18(8):532-550
The microorganism Escherichia coli is commonly used for recombinant protein production. Despite several advantageous characteristics like fast growth and high protein yields, its inability to easily secrete recombinant proteins into the extracellular medium remains a drawback for industrial production processes. To overcome this limitation, a multitude of approaches to enhance the extracellular yield and the secretion efficiency of recombinant proteins have been developed in recent years. Here, a comprehensive overview of secretion mechanisms for recombinant proteins from E. coli is given and divided into three main sections. First, the structure of the E. coli cell envelope and the known natural secretion systems are described. Second, the use and optimization of different one‐ or two‐step secretion systems for recombinant protein production, as well as further permeabilization methods are discussed. Finally, the often‐overlooked role of cell lysis in secretion studies and its analysis are addressed. So far, effective approaches for increasing the extracellular protein concentration to more than 10 g/L and almost 100% secretion efficiency exist, however, the large range of optimization methods and their combinations suggests that the potential for secretory protein production from E. coli has not yet been fully realized. 相似文献
7.
Background
Production of heterologous proteins in the E. coli periplasm, or into the extracellular fluid has many advantages; therefore naturally occurring signal peptides are selected for proteins translocation. The aim of this study was the production in high yields of a recombinant pectin lyase that is efficiently secreted and the encapsulation of transformed E. coli cells for pectin degradation in a biotechnological process. 相似文献8.
Background
Escherichia coli as a frequently utilized host organism for recombinant protein production offers different cellular locations with distinct qualities. The periplasmic space is often favored for the production of complex proteins due to enhanced disulfide bond formation, increased target product stability and simplified downstream processing. To direct proteins to the periplasmic space rather small proteinaceus tags that can be used for affinity purification would be advantageous. 相似文献9.
A gene encoding the xylanase from Bacillus subtilis strain R5 containing the native signal sequence was cloned and expressed in Escherichia coli. The heterologous expression of the gene resulted in the production of the recombinant protein in the cytoplasm as well as
its secretion into the culture medium. The xylanase activity in the culture medium increased with time after induction up
to 90% of the total activity in 14 h. Molecular mass and N-terminal amino acid sequence determinations of the purified recombinant
xylanase revealed that the native signal peptide was cleaved off by E. coli signal peptidases between Ala28 and Ala29. 相似文献
10.
11.
Charles Van der Henst Caroline Charlier Michaël Deghelt Johan Wouters Jean-Yves Matroule Jean-Jacques Letesson Xavier De Bolle 《BMC microbiology》2010,10(1):248
Background
When heterologous recombinant proteins are produced in Escherichia coli, they often precipitate to form insoluble aggregates of unfolded polypeptides called inclusion bodies. These structures are associated with chaperones like IbpA. However, there are reported cases of "non-classical" inclusion bodies in which proteins are soluble, folded and active. 相似文献12.
Background
The expression of heterologous proteins in Escherichia coli is strongly affected by codon bias. This phenomenon occurs when the codon usage of the mRNA coding for the foreign protein differs from that of the bacterium. The ribosome pauses upon encountering a rare codon and may detach from the mRNA, thereby the yield of protein expression is reduced. Several bacterial strains have been engineered to overcome this effect. However, the increased rate of translation may lead to protein misfolding and insolubilization. In order to prove this assumption, the solubility of several recombinant proteins from plants was studied in a codon bias-adjusted E. coli strain. 相似文献13.
Jens Kastenhofer Lukas Rettenbacher Lukas Feuchtenhofer Juergen Mairhofer Oliver Spadiut 《Biotechnology journal》2021,16(3):2000274
With the growing interest in continuous cultivation of Escherichia coli, secretion of product to the medium is not only a benefit, but a necessity in future bioprocessing. In this study, it is shown that induced decoupling of growth and heterologous gene expression in the E. coli X-press strain (derived from BL21(DE3)) facilitates extracellular recombinant protein production. The effect of the process parameters temperature and specific glucose consumption rate (qS) on growth, productivity, lysis and leakiness, is investigated, to find the parameter space allowing extracellular protein production. Two model proteins are used, Protein A (SpA) and a heavy-chain single-domain antibody (VHH), and performance is compared to the industrial standard strain BL21(DE3). It is shown that inducible growth repression in the X-press strain greatly mitigates the effect of metabolic burden under different process conditions. Furthermore, temperature and qS are used to control productivity and leakiness. In the X-press strain, extracellular SpA and VHH titer reach up to 349 and 19.6 mg g−1, respectively, comprising up to 90% of the total soluble product, while keeping cell lysis at a minimum. The findings demonstrate that the X-press strain constitutes a valuable host for extracellular production of recombinant protein with E. coli. 相似文献
14.
Alejandro Hochkoeppler 《Biotechnology letters》2013,35(12):1971-1981
Over the years, several vectors and host strains have been constructed to improve the overexpression of recombinant proteins in Escherichia coli. More recently, attention has focused on the co-expression of genes in E. coli, either by means of a single vector or by cotransformation with multiple compatible plasmids. Co-expression was initially designed to generate protein complexes in vivo, and later served to extend the use of E. coli as a platform for the production of heterologous proteins. This review shows how the co-expression of genes in E. coli is challenging the production of protein complexes and proteins bearing post-translational modifications or unnatural amino acids. In addition, the importance of co-expression to achieve efficient secretion of recombinant proteins in E. coli is discussed, with recent insights into the use of co-expression to overproduce membrane proteins. 相似文献
15.
Daiane D Hartwig Thaís L Oliveira Fabiana K Seixas Karine M Forster Caroline Rizzi Cláudia P Hartleben Alan JA McBride Odir A Dellagostin 《Microbial cell factories》2010,9(1):98
Background
Leptospirosis, a zoonosis caused by Leptospira spp., is recognized as an emergent infectious disease. Due to the lack of adequate diagnostic tools, vaccines are an attractive intervention strategy. Recombinant proteins produced in Escherichia coli have demonstrated promising results, albeit with variable efficacy. Pichia pastoris is an alternative host with several advantages for the production of recombinant proteins. 相似文献16.
Background
Genetic fusion of the major birch pollen allergen (Bet v1) to bacterial surface-(S)-layer proteins resulted in recombinant proteins exhibiting reduced allergenicity as well as immunomodulatory capacity. Thus, S-layer/allergen fusion proteins were considered as suitable carriers for new immunotherapeutical vaccines for treatment of Type I hypersensitivity. Up to now, endotoxin contamination of the fusion protein which occurred after isolation from the gram-negative expression host E. coli had to be removed by an expensive and time consuming procedure. In the present study, in order to achieve expression of pyrogen-free, recombinant S-layer/allergen fusion protein and to study the secretion of a protein capable to self-assemble, the S-layer/allergen fusion protein rSbpA/Bet v1 was produced in the gram-positive organism Bacillus subtilis 1012. 相似文献17.
Background
Despite advances in expression technologies, the efficient production of heterologous secreted proteins in Escherichia coli remains a challenge. One frequent limitation relies on their inability to be exported to the E. coli periplasm. However, recent studies have suggested that translational kinetics and signal sequences act in concert to modulate the export process. 相似文献18.
Escherichia coli (E. coli) is the most widely used prokaryotic host system for the synthesis of recombinant proteins. The overproduction of recombinant
proteins is sometimes lethal to the host cells. In the present study, we expressed the ligand binding domain (LBD) of mouse
estrogen receptor alpha (mouse ERα) using an expression vector (pIVEX) in E. coli BL21(DE3) and examined the effect of production of this protein on bacterial growth. The expressed protein was immunologically
detected as a 30 kD histidine-tagged protein in the soluble part of the bacterial lysate. The overproduction of mouse ERα-LBD,
as reflected by total protein content and expression pattern, resulted in the decrease of bacterial growth. 相似文献
19.
Background
Use of lactose-rich concentrates from dairy processes for the induction of recombinant gene's expression has not received much attention although they are interesting low cost substrates for production of recombinant enzymes. Applicability of dairy waste for induction of recombinant genes in Escherichia coli was studied. Clones expressing Lactobacillus phage muramidase and Lactobacillus alcohol dehydrogenase were used for the experiments. 相似文献20.
Nina Ihling Andreas Uhde Romy Scholz Christian Schwarz Lutz Schmitt Jochen Büchs 《Biotechnology progress》2020,36(2):e2911
Secretion of heterologous proteins into the culture supernatant in laboratory strains of Escherichia coli is possible by utilizing a Type I secretion system (T1SS). One prominent example for a T1SS is based on the hemolysin A toxin. With this system, heterologous protein secretion has already been achieved. However, no cultivations in a defined mineral medium and in stirred tank bioreactors have been described in literature up to now, hampering the broad applicability of the system. In this study, a mineral medium was developed for cultivation under defined conditions. With this medium, the full potential and advantage of a secretion system in E. coli (low secretion of host proteins, no contamination with proteins from complex media compounds) can now be exploited. Additionally, quantification of the protein amount in the supernatant was demonstrated by application of the Bradford assay. In this work, host cell behavior was described in small scale by online monitoring of the oxygen transfer rate. Scalability was demonstrated by stirred tank fermentation yielding 540 mg/L HlyA1 in the supernatant. This work enhances the applicability of a protein secretion system in E. coli and paves the way for an industrial application. 相似文献