共查询到20条相似文献,搜索用时 15 毫秒
1.
Neophytou I Harvey R Lawrence J Marsh P Panaretou B Barlow D 《Applied microbiology and biotechnology》2007,77(2):375-381
A fusion protein expression system is described that allows for production of eukaryotic integral membrane proteins in Escherichia coli (E. coli). The eukaryotic membrane protein targets are fused to the C terminus of the highly expressed E. coli inner membrane protein, GlpF (the glycerol-conducting channel protein). The generic utility of this system for heterologous
membrane-protein expression is demonstrated by the expression and insertion into the E. coli cell membrane of the human membrane proteins: occludin, claudin 4, duodenal ferric reductase and a J-type inwardly rectifying
potassium channel. The proteins are produced with C-terminal hexahistidine tags (to permit purification of the expressed fusion
proteins using immobilized metal affinity chromatography) and a peptidase cleavage site (to allow recovery of the unfused
eukaryotic protein). 相似文献
2.
Shalaka Samant Gunja Gupta Subbulakshmi Karthikeyan Saiful F. Haq Ayyappan Nair Ganesh Sambasivam Sunilkumar Sukumaran 《Journal of industrial microbiology & biotechnology》2014,41(9):1435-1442
Recombinant proteins can be targeted to the Escherichia coli periplasm by fusing them to signal peptides. The popular pET vectors facilitate fusion of target proteins to the PelB signal. A systematic comparison of the PelB signal with native E. coli signal peptides for recombinant protein expression and periplasmic localization is not reported. We chose the Bacillus stearothermophilus maltogenic amylase (MA), an industrial enzyme widely used in the baking and brewing industry, as a model protein and analyzed the competence of seven, codon-optimized, E. coli signal sequences to translocate MA to the E. coli periplasm compared to PelB. MA fusions to three of the signals facilitated enhanced periplasmic localization of MA compared to the PelB fusion. Interestingly, these three fusions showed greatly improved MA yields and between 18- and 50-fold improved amylase activities compared to the PelB fusion. Previously, non-optimal codon usage in native E. coli signal peptide sequences has been reported to be important for protein stability and activity. Our results suggest that E. coli signal peptides with optimal codon usage could also be beneficial for heterologous protein secretion to the periplasm. Moreover, such fusions could even enhance activity rather than diminish it. This effect, to our knowledge has not been previously documented. In addition, the seven vector platform reported here could also be used as a screen to identify the best signal peptide partner for other recombinant targets of interest. 相似文献
3.
《Journal of molecular biology》2023,435(17):168191
Albumin is the most abundant protein in the blood serum of mammals and has essential carrier and physiological roles. Albumins are also used in a wide variety of molecular and cellular experiments and in the cultivated meat industry. Despite their importance, however, albumins are challenging for heterologous expression in microbial hosts, likely due to 17 conserved intramolecular disulfide bonds. Therefore, albumins used in research and biotechnological applications either derive from animal serum, despite severe ethical and reproducibility concerns, or from recombinant expression in yeast or rice. We use the PROSS algorithm to stabilize human and bovine serum albumins, finding that all are highly expressed in E. coli. Design accuracy is verified by crystallographic analysis of a human albumin variant with 16 mutations. This albumin variant exhibits ligand binding properties similar to those of the wild type. Remarkably, a design with 73 mutations relative to human albumin exhibits over 40 °C improved stability and is stable beyond the boiling point of water. Our results suggest that proteins with many disulfide bridges have the potential to exhibit extreme stability when subjected to design. The designed albumins may be used to make economical, reproducible, and animal-free reagents for molecular and cell biology. They also open the way to high-throughput screening to study and enhance albumin carrier properties. 相似文献
4.
Through the proteome analysis of Escherichia coli BL21(DE3), we previously identified the stress-responsive protein, arsenate reductase (ArsC), that showed a high cytoplasmic solubility and a folding capacity even in the presence of stress-inducing reagents. In this study, we used ArsC as an N-terminal fusion partner to synthesize nine aggregation-prone proteins as water-soluble forms. As a result, solubility of the aggregation-prone proteins increased dramatically by the fusion of ArsC, due presumably to its tendency to facilitate the folding of target proteins. Also, we evaluated and confirmed the efficacy of ArsC-fusion expression in making the fusion-expressed target proteins have their own native function or structure. That is, the self-assembly function of human ferritin light chain, l-arginine-degrading function of arginine deiminase, and the correct secondary structure of human granulocyte colony stimulating factor were clearly observed through transmission electron microscope analysis, colorimetric enzyme activity assay, and circular dichroism, respectively. It is strongly suggested that ArsC can be in general an efficient fusion expression partner for the production of soluble and active heterologous proteins in E. coli. 相似文献
5.
《Gene》1997,193(2):229-237
Because of the complexities involved in the regulation of gene expression in Escherichia coli and mammalian cells, it is considered general practice to use different vectors for heterologous expression of recombinant proteins in these host systems. However, we have developed and report a shuttle vector system, pGFLEX, that provides high-level expression of recombinant glutathione S-transferase (GST) fusion proteins in E. coli and mammalian cells. pGFLEX contains the cytomegaloma virus (CMV) immediate-early promoter in tandem with the E. coli lacZpo system. The sequences involved in gene expression have been appropriately modified to enable high-level production of fusion proteins in either cell type. The pGFLEX expression system allows production of target proteins fused to either the N or C terminus of the GST π protein and provides rapid purification of target proteins as either GST fusions or native proteins after cleavage with thrombin. The utility of this vector in identifying and purifying a component of a multi-protein complex is demonstrated with cyclin A. The pGFLEX expression system provides a singular and widely applicable tool for laboratory or industrial production of biologically active recombinant proteins in E. coli and mammalian cells. 相似文献
6.
In the recent past years, a large number of proteins have been expressed in Escherichia coli with high productivity due to rapid development of genetic engineering technologies. There are many hosts used for the production of recombinant protein but the preferred choice is E. coli due to its easier culture, short life cycle, well-known genetics, and easy genetic manipulation. We often face a problem in the expression of foreign genes in E. coli. Soluble recombinant protein is a prerequisite for structural, functional and biochemical studies of a protein. Researchers often face problems producing soluble recombinant proteins for over-expression, mainly the expression and solubility of heterologous proteins. There is no universal strategy to solve these problems but there are a few methods that can improve the level of expression, non-expression, or less expression of the gene of interest in E. coli. This review addresses these issues properly. Five levels of strategies can be used to increase the expression and solubility of over-expressed protein; (1) changing the vector, (2) changing the host, (3) changing the culture parameters of the recombinant host strain, (4) co-expression of other genes and (5) changing the gene sequences, which may help increase expression and the proper folding of desired protein. Here we present the resources available for the expression of a gene in E. coli to get a substantial amount of good quality recombinant protein. The resources include different strains of E. coli, different E. coli expression vectors, different physical and chemical agents and the co expression of chaperone interacting proteins. Perhaps it would be the solutions to such problems that will finally lead to the maturity of the application of recombinant proteins. The proposed solutions to such problems will finally lead to the maturity of the application of recombinant proteins. 相似文献
7.
《Journal of molecular biology》2021,433(24):167321
Obligate symbionts typically exhibit high evolutionary rates. Consequently, their proteins may differ considerably from their modern and ancestral homologs in terms of both sequence and properties, thus providing excellent models to study protein evolution. Also, obligate symbionts are challenging to culture in the lab and proteins from uncultured organisms must be produced in heterologous hosts using recombinant DNA technology. Obligate symbionts thus replicate a fundamental scenario of metagenomics studies aimed at the functional characterization and biotechnological exploitation of proteins from the bacteria in soil. Here, we use the thioredoxin from Candidatus Photodesmus katoptron, an uncultured symbiont of flashlight fish, to explore evolutionary and engineering aspects of protein folding in heterologous hosts. The symbiont protein is a standard thioredoxin in terms of 3D-structure, stability and redox activity. However, its folding outside the original host is severely impaired, as shown by a very slow refolding in vitro and an inefficient expression in E. coli that leads mostly to insoluble protein. By contrast, resurrected Precambrian thioredoxins express efficiently in E. coli, plausibly reflecting an ancient adaptation to unassisted folding. We have used a statistical-mechanical model of the folding landscape to guide back-to-ancestor engineering of the symbiont protein. Remarkably, we find that the efficiency of heterologous expression correlates with the in vitro (i.e., unassisted) folding rate and that the ancestral expression efficiency can be achieved with only 1–2 back-to-ancestor replacements. These results demonstrate a minimal-perturbation, sequence-engineering approach to rescue inefficient heterologous expression which may potentially be useful in metagenomics efforts targeting recent adaptations. 相似文献
8.
We recently developed a display method for the directed evolution of integral membrane proteins in the inner membrane of Escherichia coli for higher expression and stability. For the neurotensin receptor 1, a G-protein-coupled receptor (GPCR), we had evolved a mutant with a 10-fold increase in functional expression that largely retains wild-type binding and signaling properties and shows higher stability in detergent-solubilized form. We have now evolved three additional human GPCRs. Unmodified wild-type receptor cDNA was subjected to successive cycles of mutagenesis and fluorescence-activated cell sorting, and functional expression could be increased for all three GPCR targets. We also present a new stability screening method in a 96-well assay format to quickly identify evolved receptors showing increased thermal stability in detergent-solubilized form and rapidly evaluate them quantitatively. Combining the two methods turned out to be very powerful; even for the most challenging GPCR target—the tachykinin receptor NK1, which is hardly expressed in E. coli and cannot be functionally solubilized—receptor mutants that are functionally expressed at 1 mg/l levels in E. coli and are stable in detergent solution could be quickly evolved. The improvements result from cumulative small changes in the receptor sequence. This combinatorial approach does not require preconceived notions for designing mutations. Our results suggest that this method is generally applicable to GPCRs. Existing roadblocks in structural and biophysical studies can now be removed by providing sufficient quantities of correctly folded and stable receptor protein. 相似文献
9.
10.
Ann E. Lin Julian A. Guttman 《The international journal of biochemistry & cell biology》2012,44(12):2223-2232
Ubiquitylation is a widespread post-translational global regulatory system that is essential for the proper functioning of various cellular events. Recent studies have shown that certain types of Escherichia coli can exploit specific aspects of the ubiquitylation system to influence downstream targets. Despite these findings, examination of the effects pathogenic E. coli have on the overall host ubiquitylation system remain unexplored. To study the impact that pathogenic E. coli have on the ubiquitylation levels of host proteins during infections, we analyzed the entire ubiquitylation system during enteropathogenic E. coli infections of cultured cells. We found that these microbes caused a dramatic decrease in ubiquitylated host proteins during these infections. This occurred with a concomitant reduction in the expression of essential E1 activating enzymes in the host, which are integral for the initiation of the ubiquitylation cascade. Control of host E1 enzyme levels was dependent on the E. coli adherence factor plasmid which acted on host aspartyl proteases within enteropathogenic E. coli. Hijacking of the ubiquitylation system did not require the plasmid-encoded regulator or bundle forming pilus expression, as enteropathogenic E. coli mutated in those factors did not revert the ubiquitylation of host proteins or the abundance of E1 enzyme proteins to uninfected levels. Our work shows that E. coli have developed strategies to usurp post-translational systems by targeting crucial enzymes. The ability of enteropathogenic E. coli to inactivate host protein ubiquitylation could enable more efficient effector protein functionality, providing increased bacterial control of host cells during enteropathogenic E. coli pathogenesis. 相似文献
11.
12.
Barbara Maertens Anne Spriestersbach Uritza von Groll Udo Roth Jan Kubicek Michael Gerrits Marcus Graf Michael Liss Daniela Daubert Ralf Wagner Frank Schäfer 《Protein science : a publication of the Protein Society》2010,19(7):1312-1326
The genetic code is universal, but recombinant protein expression in heterologous systems is often hampered by divergent codon usage. Here, we demonstrate that reprogramming by standardized multi‐parameter gene optimization software and de novo gene synthesis is a suitable general strategy to improve heterologous protein expression. This study compares expression levels of 94 full‐length human wt and sequence‐optimized genes coding for pharmaceutically important proteins such as kinases and membrane proteins in E. coli. Fluorescence‐based quantification revealed increased protein yields for 70% of in vivo expressed optimized genes compared to the wt DNA sequences and also resulted in increased amounts of protein that can be purified. The improvement in transgene expression correlated with higher mRNA levels in our analyzed examples. In all cases tested, expression levels using wt genes in tRNA‐supplemented bacterial strains were outperformed by optimized genes expressed in non‐supplemented host cells. 相似文献
13.
Nadine Katzke Solmaz Arvani René Bergmann Franco Circolone Annette Markert Vera Svensson Karl-Erich Jaeger Achim Heck Thomas Drepper 《Protein expression and purification》2010,69(2):137-146
The functional expression of heterologous genes using standard bacterial expression hosts such as Escherichia coli is often limited, e.g. by incorrect folding, assembly or targeting of recombinant proteins. Consequently, alternative bacterial expression systems have to be developed to provide novel strategies for protein synthesis exceeding the repertoire of the standard expression host E. coli.Here, we report on the construction of a novel expression system that combines the high processivity of T7 RNA polymerase with the unique physiological properties of the facultative photosynthetic bacterium Rhodobacter capsulatus. This system basically consists of a recombinant R. capsulatus T7 expression strain (R. capsulatus B10S-T7) harboring the respective polymerase gene under control of a fructose inducible promoter. In addition, a set of different broad-host-range vectors (pRho) was constructed allowing T7 RNA polymerase dependent and independent target gene expression in R. capsulatus and other Gram-negative bacteria. The expression efficiency of the novel system was studied in R. capsulatus and E. coli using the yellow fluorescent protein (YFP) as model protein. Expression levels were comparable in both expression hosts and yielded up to 80 mg/l YFP in phototrophically grown R. capsulatus cultures. This result clearly indicates that the novel R. capsulatus-based expression system is well suited for the high-level expression of soluble proteins. 相似文献
14.
Mark Welch Sridhar Govindarajan Jon E. Ness Alan Villalobos Austin Gurney Jeremy Minshull Claes Gustafsson 《PloS one》2009,4(9)
Background
Production of proteins as therapeutic agents, research reagents and molecular tools frequently depends on expression in heterologous hosts. Synthetic genes are increasingly used for protein production because sequence information is easier to obtain than the corresponding physical DNA. Protein-coding sequences are commonly re-designed to enhance expression, but there are no experimentally supported design principles.Principal Findings
To identify sequence features that affect protein expression we synthesized and expressed in E. coli two sets of 40 genes encoding two commercially valuable proteins, a DNA polymerase and a single chain antibody. Genes differing only in synonymous codon usage expressed protein at levels ranging from undetectable to 30% of cellular protein. Using partial least squares regression we tested the correlation of protein production levels with parameters that have been reported to affect expression. We found that the amount of protein produced in E. coli was strongly dependent on the codons used to encode a subset of amino acids. Favorable codons were predominantly those read by tRNAs that are most highly charged during amino acid starvation, not codons that are most abundant in highly expressed E. coli proteins. Finally we confirmed the validity of our models by designing, synthesizing and testing new genes using codon biases predicted to perform well.Conclusion
The systematic analysis of gene design parameters shown in this study has allowed us to identify codon usage within a gene as a critical determinant of achievable protein expression levels in E. coli. We propose a biochemical basis for this, as well as design algorithms to ensure high protein production from synthetic genes. Replication of this methodology should allow similar design algorithms to be empirically derived for any expression system. 相似文献15.
David Hörnström Gen Larsson Antonius J.A. van Maris Martin Gustavsson 《生物化学与生物物理学报:生物膜》2019,1861(2):486-494
Display of recombinant enzymes on the cell surface of Gram-negative bacteria is a desirable feature with applications in whole-cell biocatalysis, affinity screening and degradation of environmental pollutants. One common technique for recombinant protein display on the Escherichia coli surface is autotransport. Successful autotransport of an enzyme largely depends on the following: (1) the size, sequence and structure of the displayed protein, (2) the cultivation conditions, and (3) the choice of the autotransporter expression system. Common problems with autotransporter-mediated surface display include low expression levels and truncated fusion proteins, which both limit the cell-specific activity. The present study investigated an autotransporter expression system for improved display of tyrosinase on the surface of E. coli by evaluating different variants of the autotransporter vector including: promoter region, signal peptide, the recombinant passenger, linker regions, and the autotransporter translocation unit itself. The impact of these changes on translocation to the cell surface was monitored by the cell-specific activity as well as antibody-based flow cytometric analysis of full-length and degraded passenger. Applying these strategies, the amount of displayed full-length tyrosinase on the cell surface was increased, resulting in an overall 5-fold increase of activity as compared to the initial autotransport expression system. Surprisingly, heterologous expression using 7 different translocation units all resulted in functional expression and only differed 1.6-fold in activity. This study provides a basis for broadening of the range of proteins that can be surface displayed and the development of new autotransporter-based processes in industrial-scale whole-cell biocatalysis. 相似文献
16.
Suppressing Posttranslational Gluconoylation of Heterologous Proteins by Metabolic Engineering of Escherichia coli
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Juan C. Aon Richard J. Caimi Alexander H. Taylor Quinn Lu Femi Oluboyede Jennifer Dally Michelle D. Kessler John J. Kerrigan Tia S. Lewis Lisa A. Wysocki Pramatesh S. Patel 《Applied microbiology》2008,74(4):950-958
Minimization of chemical modifications during the production of proteins for pharmaceutical and medical applications is of fundamental and practical importance. The gluconoylation of heterologously expressed protein which is observed in Escherichia coli BL21(DE3) constitutes one such undesired posttranslational modification. We postulated that formation of gluconoylated/phosphogluconoylated products of heterologous proteins is caused by the accumulation of 6-phosphogluconolactone due to the absence of phosphogluconolactonase (PGL) in the pentose phosphate pathway. The results obtained demonstrate that overexpression of a heterologous PGL in BL21(DE3) suppresses the formation of the gluconoylated adducts in the therapeutic proteins studied. When this E. coli strain was grown in high-cell-density fed-batch cultures with an extra copy of the pgl gene, we found that the biomass yield and specific productivity of a heterologous 18-kDa protein increased simultaneously by 50 and 60%, respectively. The higher level of PGL expression allowed E. coli strain BL21(DE3) to satisfy the extra demand for precursors, as well as the energy requirements, in order to replicate plasmid DNA and express heterologous genes, as metabolic flux analysis showed by the higher precursor and NADPH fluxes through the oxidative branch of the pentose phosphate shunt. This work shows that E. coli strain BL21(DE3) can be used as a host to produce three different proteins, a heterodimer of liver X receptors, elongin C, and an 18-kDa protein. This is the first report describing a novel and general strategy for suppressing this nonenzymatic modification by metabolic pathway engineering. 相似文献
17.
18.
Despite the fundamental importance of E. coli in the manufacture of a wide range of biotechnological and biomedical products, extensive process and/or target optimisation
is routinely required in order to achieve functional yields in excess of low mg/l levels. Molecular chaperones and folding
catalysts appear to present a panacea for problems of heterologous protein folding in the organism, due largely to their broad
substrate range compared with, e.g., protein-specific mutagenesis approaches. Painstaking investigation of chaperone overproduction has, however, met with mixed
– and largely unpredictable – results to date. The past 5 years have nevertheless seen an explosion in interest in exploiting
the native folding modulators of E. coli, and particularly cocktails thereof, driven largely by the availability of plasmid systems that facilitate simultaneous,
non-rational screening of multiple chaperones during recombinant protein expression. As interest in using E. coli to produce recombinant membrane proteins and even glycoproteins grows, approaches to reduce aggregation, delay host cell
lysis and optimise expression of difficult-to-express recombinant proteins will become even more critical over the coming
years. In this review, we critically evaluate the performance of molecular chaperones and folding catalysts native to E. coli in improving functional production of heterologous proteins in the bacterium and we discuss how they might best be exploited
to provide increased amounts of correctly-folded, active protein for biochemical and biophysical studies. 相似文献
19.
Jin Hu Jianquan Shi Yeshuang Yuan Bo Zhang Shengjie Li Haitao Dong 《Biochemistry and Biophysics Reports》2021
The widespread application of protein and peptide therapeutics is hampered by their poor stability, strong immunogenicity and short half-life. However, the existing protein modification technologies require the introduction of exogenous macromolecules, resulting in inevitable immunogenicity and decreased bioactivity. Herein, we reported an easy but universal protein modification approach, self-fused concatenation (SEC), to enhance the in vitro thermal stability and in vivo tumor retention of proteins. In this proof of concept study, we successfully obtained a set of green fluorescence protein (GFP) concatemers, monomer (GFP 1), dimer (GFP 2) and trimer (GFP 3) of GFP, and systematically studied the effects of SEC on the biological activity and stability of GFP. Notably, GFP concatemers displayed remarkable improvement in in vitro bioactivity and thermal stability over the monomeric GFP. In a murine tumor model, GFP 2 and GFP 3 exhibited significantly prolonged duration, with increases of 220- and 381-fold relative to GFP 1 in tumor retention 4 h after administration. Furthermore, the biological activity, thermal stability and tumor retention can be enhanced by the concatenated number of self-fused proteins. These findings demonstrate that SEC may be a promising alternative to design advanced protein and peptide therapeutics with enhanced pharmaceutic profiles. 相似文献
20.
Poblete-Castro Ignacio Binger Danielle Oehlert Rene Rohde Manfred 《BMC biotechnology》2014,14(1):1-11