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1.
Background
The formation of native disulfide bonds is a complex and essential post-translational modification for many proteins. The large scale production of these proteins can be difficult and depends on targeting the protein to a compartment in which disulfide bond formation naturally occurs, usually the endoplasmic reticulum of eukaryotes or the periplasm of prokaryotes. It is currently thought to be impossible to produce large amounts of disulfide bond containing protein in the cytoplasm of wild-type bacteria such as E. coli due to the presence of multiple pathways for their reduction. 相似文献2.
Bacillus subtilis is a well-established cellular factory for proteins and fine chemicals. In particular, the direct secretion of proteinaceous
products into the growth medium greatly facilitates their downstream processing, which is an important advantage of B. subtilis over other biotechnological production hosts, such as Escherichia coli. The application spectrum of B. subtilis is, however, often confined to proteins from Bacillus or closely related species. One of the major reasons for this (current) limitation is the inefficient formation of disulfide
bonds, which are found in many, especially eukaryotic, proteins. Future exploitation of B. subtilis to fulfill the ever-growing demand for pharmaceutical and other high-value proteins will therefore depend on overcoming this
particular hurdle. Recently, promising advances in this area have been achieved, which focus attention on the need to modulate
the cellular levels and activity of thiol-disulfide oxidoreductases (TDORs). These TDORs are enzymes that control the cleavage
or formation of disulfide bonds. This review will discuss readily applicable approaches for TDOR modulation and aims to provide
leads for further improvement of the Bacillus cell factory for production of disulfide bond-containing proteins. 相似文献
3.
Van Dat Nguyen Feras Hatahet Kirsi EH Salo Eveliina Enlund Chi Zhang Lloyd W Ruddock 《Microbial cell factories》2011,10(1):1
Background
Disulfide bonds are one of the most common post-translational modifications found in proteins. The production of proteins that contain native disulfide bonds is challenging, especially on a large scale. Either the protein needs to be targeted to the endoplasmic reticulum in eukaryotes or to the prokaryotic periplasm. These compartments that are specialised for disulfide bond formation have an active catalyst for their formation, along with catalysts for isomerization to the native state. We have recently shown that it is possible to produce large amounts of prokaryotic disulfide bond containing proteins in the cytoplasm of wild-type bacteria such as E. coli by the introduction of catalysts for both of these processes. 相似文献4.
Building bridges: disulphide bond formation in the cell 总被引:26,自引:1,他引:25
James C. A. Bardwell 《Molecular microbiology》1994,14(2):199-205
Disulphides are often vital for the folding and stability of proteins. Dedicated enzymatic systems have been discovered that catalyse the formation of disulphides in the periplasm of prokaryotes. These discoveries provide compelling evidence for the actual catalysis of protein folding in vivo. Disulphide bond formation in Escherichia coli is catalysed by at least three ‘Dsb’ proteins; DsbA, -B and -C. The DsbA protein has an extremely reactive, oxidizing disulphide which it simply donates directly to other proteins. DsbB is required for the reoxidation of DsbA. DsbC is active in disulphide rearrangements and appears to work synergistically with DsbA. The relative rarity of disulphides in cytoplasmic proteins appears to be dependent upon a disulphide-destruction machine. One pivotal cog in this machine is thioredoxin reductase. 相似文献
5.
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6.
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
7.
Daniel Hoffmann Mehrdad Ebrahimi Doreen Gerlach Peter Czermak 《Critical reviews in biotechnology》2018,38(5):729-744
The production of recombinant proteins in the microbial host Escherichia coli often results in the formation of cytoplasmic protein inclusion bodies (IBs). Proteins forming IBs are often branded as difficult-to-express, neglecting that IBs can be an opportunity for their production. IBs are resistant to proteolytic degradation and contain up to 90% pure recombinant protein, which does not interfere with the host metabolism. This is especially advantageous for host-toxic proteins like antimicrobial peptides (AMPs). IBs can be easily isolated by cell disruption followed by filtration and/or centrifugation, but conventional techniques for the recovery of soluble proteins from IBs are laborious. New approaches therefore simplify protein recovery by optimizing the production process conditions, and often include mild resolubilization methods that either increase the yield after refolding or avoid the necessity of refolding all together. For the AMP production, the IB-based approach is ideal, because these peptides often have simple structures and are easy to refold. The intentional IB production of almost every protein can be achieved by fusing recombinant proteins to pull-down tags. This review discusses the techniques available for IB-based protein production before considering technical approaches for the isolation of IBs from E. coli lysates followed by efficient protein resolubilization which ideally omits further refolding. The techniques are evaluated in terms of their suitability for the process-scale production and downstream processing of recombinant proteins and are discussed for AMP production as an example. 相似文献
8.
Bor-Ruei Lin Lila M. Gierasch Chun Jiang Phang C. Tai 《The Journal of membrane biology》2006,214(1-2):103-113
Protein translocation in Escherichia coli requires protein-conducting channels in cytoplasmic membranes to allow precursor peptides to pass through with adenosine
triphosphate (ATP) hydrolysis. Here, we report a novel, sensitive method that detects the opening of the SecA-dependent protein-conducting
channels at the nanogram level. E. coli inverted membrane vesicles were injected into Xenopus oocytes, and ionic currents were recorded using the two-electrode voltage clamp. Currents were observed only in the presence
of E. coli SecA in conjunction with E. coli membranes. Observed currents showed outward rectification in the presence of KCl as permeable ions and were significantly
enhanced by coinjection with the precursor protein proOmpA or active LamB signal peptide. Channel activity was blockable with
sodium azide or adenylyl 5′-(β,γ-methylene)-diphosphonate, a nonhydrolyzable ATP analogue, both of which are known to inhibit
SecA protein activity. Endogenous oocyte precursor proteins also stimulated ion current activity and can be inhibited by puromycin.
In the presence of puromycin, exogenous proOmpA or LamB signal peptides continued to enhance ionic currents. Thus, the requirement
of signal peptides and ATP hydrolysis for the SecA-dependent currents resembles biochemical protein translocation assay with
E. coli membrane vesicles, indicating that the Xenopus oocyte system provides a sensitive assay to study the role of Sec and precursor proteins in the formation of protein-conducting
channels using electrophysiological methods. 相似文献
9.
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
10.
11.
12.
Tatsurou Shibui Kaoru Munakata Rieko Matsumoto Kunihiko Ohta Rika Matsushima Yuuki Morimoto Kenji Nagahari 《Applied microbiology and biotechnology》1993,38(6):770-775
A high-level secretion system for the production of mouse-human chimeric antibody 21B2 (MHC 21B2) Fab fragment specific for human carcino embryonic antigen (hCEA) in Escherichia coli has been constructed. The genes encoding a light chain and an Fd fragment (a variable region and the CH1 domain of a heavy chain) of a mouse-human chimeric antibody were directly fused to the signal peptide of the E. coli ompF gene sequence. E. coli cells containing expression vectors in which each of the two genes are located downstream of a separate tac promoter were able to secrete the light chain and Fd fragment as two of their major cellular proteins. The signal peptides were efficiently removed from the primary products by post-translational processing, although they formed insoluble aggregates, possibly in the periplasm. In high-cell-density culture experiments using a jar fermentor, the amount of light chain and Fd fragment produced was at levels of up to 2.88 g/l and 1.28 g/l culture, respectively. By optimizing the conditions that encourage correct folding, formation of disulphide bonds, and association of the light chain with the Fd fragment, we have established a procedure that can purify, refold, and combine aggregated products to electrophoretically homogeneous Fab fragment with a yield of approximately 47%. Fab fragment produced in this manner shows essentially the same antigen-binding activity and specificity to hCEA as the parental mouse antibody 21B2 (MoAb 21B2).
Correspondence to: T. Shibui 相似文献
13.
Cristina Landeta Laura McPartland Ngoc Q. Tran Brian M. Meehan Yifan Zhang Zaidi Tanweer Shoko Wakabayashi Jeremy Rock Taehyun Kim Deepak Balasubramanian Rebecca Audette Melody Toosky Jessica Pinkham Eric J. Rubin Stephen Lory Gerald Pier Dana Boyd Jon Beckwith 《Molecular microbiology》2019,111(4):918-937
In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond, including bacterial virulence factors. Thus, proteins involved in disulfide bond formation represent good targets for the development of inhibitors that can act as antibiotics or anti‐virulence agents, resulting in the simultaneous inactivation of several types of virulence factors. Here, we present evidence that the disulfide bond forming enzymes, DsbB and VKOR, are required for Pseudomonas aeruginosa pathogenicity and Mycobacterium tuberculosis survival respectively. We also report the results of a HTS of 216,767 compounds tested against P. aeruginosa DsbB1 and M. tuberculosis VKOR using Escherichia coli cells. Since both P. aeruginosa DsbB1 and M. tuberculosis VKOR complement an E. coli dsbB knockout, we screened simultaneously for inhibitors of each complemented E. coli strain expressing a disulfide‐bond sensitive β ‐galactosidase reported previously. The properties of several inhibitors obtained from these screens suggest they are a starting point for chemical modifications with potential for future antibacterial development. 相似文献
14.
The essential cell division protein FtsN contains a critical disulfide bond in a non‐essential domain
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Disulfide bonds are found in many proteins associated with the cell wall of Escherichia coli, and for some of these proteins the disulfide bond is critical to their stability and function. One protein found to contain a disulfide bond is the essential cell division protein FtsN, but the importance of this bond to the protein's structural integrity is unclear. While it evidently plays a role in the proper folding of the SPOR domain of FtsN, this domain is non‐essential, suggesting that the disulfide bond might also be dispensable. However, we find that FtsN mutants lacking cysteines give rise to filamentous growth. Furthermore, FtsN protein levels in strains expressing these mutants were significantly lower than in a strain expressing the wild‐type allele, as were FtsN levels in strains incapable of making disulfide bonds (dsb‐) exposed to anaerobic conditions. These results strongly suggest that FtsN lacking a disulfide bond is unstable, thereby making this disulfide critical for function. We have previously found that dsb‐ strains fail to grow anaerobically, and the results presented here suggest that this growth defect may be due in part to misfolded FtsN. Thus, proper cell division in E. coli is dependent upon disulfide bond formation. 相似文献
15.
Overexpression of Protein Disulfide Isomerase DsbC Stabilizes Multiple-Disulfide-Bonded Recombinant Protein Produced and Transported to the Periplasm in Escherichia coli
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Dsb proteins (DsbA, DsbB, DsbC, and DsbD) catalyze formation and isomerization of protein disulfide bonds in the periplasm of Escherichia coli. By using a set of Dsb coexpression plasmids constructed recently, we analyzed the effects of Dsb overexpression on production of horseradish peroxidase (HRP) isozyme C that contains complex disulfide bonds and tends to aggregate when produced in E. coli. When transported to the periplasm, HRP was unstable but was markedly stabilized upon simultaneous overexpression of the set of Dsb proteins (DsbABCD). Whereas total HRP production increased severalfold upon overexpression of at least disulfide-bonded isomerase DsbC, maximum transport of HRP to the periplasm seemed to require overexpression of all DsbABCD proteins, suggesting that excess Dsb proteins exert synergistic effects in assisting folding and transport of HRP. Periplasmic production of HRP also increased when calcium, thought to play an essential role in folding of nascent HRP polypeptide, was added to the medium with or without Dsb overexpression. These results suggest that Dsb proteins and calcium play distinct roles in periplasmic production of HRP, presumably through facilitating correct folding. The present Dsb expression plasmids should be useful in assessing and dissecting periplasmic production of proteins that contain multiple disulfide bonds in E. coli. 相似文献
16.
Tek-Hyung Lee Timothy S. Carpenter Patrik D'haeseleer David F. Savage Mimi C. Yung 《Biotechnology and bioengineering》2020,117(3):603-613
Antimicrobial peptides (AMPs) are regarded as attractive alternatives to conventional antibiotics, but their production in microbes remains challenging due to their inherent bactericidal nature. To address these limitations, we have developed a novel AMP fusion protein system based on an encapsulin nanocompartment protein and have demonstrated its utility in enhancing expression of HBCM2, an AMP with activity against Gram-negative bacteria. Here, HBCM2 was fused to the N-terminus of several Encapsulin monomer (Enc) variants engineered with multiple TEV protease recognition site insertions to facilitate proteolytic release of the fused HBCM2. Fusion of HBCM2 to the Enc variants, but not other common carrier proteins, enabled robust overexpression in Escherichia coli C43(DE3) cells. Interestingly, variants with a TEV site insertion following residue K71 in Enc exhibited the highest overexpression and HBCM2 release efficiencies compared to other variants but were deficient in cage formation. HBCM2 was purified from the highest expressing variant following TEV protease digestion and was found to be highly active in inhibiting E. coli growth (MIC = 5 μg/ml). Our study demonstrates the potential use of the Enc system to enhance expression of AMPs for biomanufacturing and therapeutic applications. 相似文献
17.
Dmitry N. Ivankov Samuel H. Payne Michael Y. Galperin Stefano Bonissone Pavel A. Pevzner Dmitrij Frishman 《Environmental microbiology》2013,15(4):983-990
Over the last 5 years proteogenomics (using mass spectroscopy to identify proteins predicted from genomic sequences) has emerged as a promising approach to the high‐throughput identification of protein N‐termini, which remains a problem in genome annotation. Comparison of the experimentally determined N‐termini with those predicted by sequence analysis tools allows identification of the signal peptides and therefore conclusions on the cytoplasmic or extracytoplasmic (periplasmic or extracellular) localization of the respective proteins. We present here the results of a proteogenomic study of the signal peptides in Escherichia coli K‐12 and compare its results with the available experimental data and predictions by such software tools as SignalP and Phobius. A single proteogenomics experiment recovered more than a third of all signal peptides that had been experimentally determined during the past three decades and confirmed at least 31 additional signal peptides, mostly in the known exported proteins, which had been previously predicted but not validated. The filtering of putative signal peptides for the peptide length and the presence of an eight‐residue hydrophobic patch and a typical signal peptidase cleavage site proved sufficient to eliminate the false‐positive hits. Surprisingly, the results of this proteogenomics study, as well as a re‐analysis of the E. coli genome with the latest version of SignalP program, show that the fraction of proteins containing signal peptides is only about 10%, or half of previous estimates. 相似文献
18.
The methylotrophic yeast Pichia pastoris is a popular heterologous expression host for the recombinant production of a variety of prokaryotic and eukaryotic proteins.
The rapid emergence of P. pastoris as a robust heterologous expression host was facilitated by the ease with which it can be manipulated and propagated, which
is comparable to that of Escherichia coli and Saccharomyces cerevisiae. P. pastoris offers further advantages such as the tightly-regulated alcohol oxidase promoter that is particularly suitable for heterologous
expression of foreign genes. While recombinant production of bacterial toxins and their derivatives is highly desirable, attempts
at their heterologous expression using the traditional E. coli expression system can be problematic due to the formation of inclusion bodies that often severely limit the final yields
of biologically active products. However, recent literature now suggests that P. pastoris may be an attractive alternative host for the heterologous production of bacterial toxins, such as those from the genera
Bacillus, Clostridium, and Corynebacterium, as well as their more complex derivatives. Here, we review the recombinant production of bacterial toxins and their derivatives
in P. pastoris with special emphasis on their potential clinical applications. Considering that de novo design and construction of synthetic toxin genes have often been necessary to achieve optimal heterologous expression in
P. pastoris, we also present general guidelines to this end based on our experience with the P. pastoris expression of the Bacillus thuringiensis Cyt2Aa1 toxin. 相似文献
19.
Germn L. Rosano Enrique S. Morales Eduardo A. Ceccarelli 《Protein science : a publication of the Protein Society》2019,28(8):1412-1422
The production of proteins in sufficient amounts is key for their study or use as biotherapeutic agents. Escherichia coli is the host of choice for recombinant protein production given its fast growth, easy manipulation, and cost‐effectiveness. As such, its protein production capabilities are continuously being improved. Also, the associated tools (such as plasmids and cultivation conditions) are subject of ongoing research to optimize product yield. In this work, we review the latest advances in recombinant protein production in E. coli. 相似文献
20.
Vassilis J. Marmaras Stavros N. Bournazos Panagiotis G. Katsoris Maria Lambropoulou 《Archives of insect biochemistry and physiology》1993,23(4):169-180
A defense mechanism in the cuticle of developing C. capitata was demonstrated using an in vitro system consisting of isolated cuticular tyrosinase from C. capitata, cuticular tyrosinase-free proteins, tyrosine, and E. coli. The simultaneous presence of the above components resulted in the formation of large immobilized E. coli aggregates. By contrast, omission of any of the above components failed to produce such aggregates. In other words, E. coli retained their mobility and viability. The results indicate that certain cuticular proteins are responsible for the nonself-recognition, since they are able to bind to the E. coli surface in vitro, and a reactive tyrosine derivative is generated by the action of cuticular tyrosinase for the immobilization and probably killing of E. coli. Based on these studies the most likely explanation for the nonself-recognition and immobilization and/or killing of bacteria is the production of E. coli-protein complexes and their crosslinking through quinone intermediate. © 1993 Wiley-Liss, Inc. 相似文献