共查询到20条相似文献,搜索用时 15 毫秒
1.
Patrick Sagmeister Clemens Schimek Andrea Meitz Christoph Herwig Oliver Spadiut 《Applied microbiology and biotechnology》2014,98(7):2937-2945
Controlling the recombinant protein production rate in Escherichia coli is of utmost importance to ensure product quality and quantity. Up to now, only the genetic construct, introduced into E. coli, and the specific growth rate of the culture were used to influence and stir the productivity. However, bioprocess technological means to control or even tune the productivity of E. coli are scarce. Here, we present a novel method for the process-technological control over the recombinant protein expression rate in E. coli. A mixed-feed fed-batch bioprocess based on the araBAD promoter expression system using both d-glucose and l-arabinose as assimilable C-sources was designed. Using the model product green fluorescent protein, we show that the specific product formation rate can be efficiently tuned even on the cellular level only via the uptake rate of l-arabinose. This novel approach introduces an additional degree of freedom for the design of recombinant bioprocesses with E. coli. We anticipate that the presented method will result in significant quality and robustness improvement as well as cost and process time reduction for recombinant bacterial bioprocesses in the future. 相似文献
2.
We constructed a recombinant expression plasmid encoding a protein A--neurotoxin fusion protein. The fused toxin is directly expressed in the periplasmic space of Escherichia coli and can be purified in the milligram range by a single immuno-affinity step. The LD50 values of the fused toxin and native toxin are 130 and 20 nmol/kg mouse respectively. The Kd values characterizing their binding to the nicotinic acetylcholine receptor (AcChoR) are respectively 4.8 +/- 0.8 and 0.07 +/- 0.03 nM. In contrast, the fused and native toxins are equally well recognized by a toxin-specific monoclonal antibody which recognizes the AcChoR binding site. The lower toxicity of the fused toxin might result, therefore, from a steric hindrance, due to the presence of the bulky protein A moiety (mol. wt = 31 kd) rather than to a direct alteration of the 'toxic' site. The fused toxin is more immunogenic than native toxin, since 1 nmol of hybrid toxin and 14 nmol of native toxin give rise to comparable titers of antitoxin antibodies which, furthermore, are equally potent at neutralizing neurotoxicity. The work described in this paper shows that the use of fused toxins may be of paramount importance for future development of serotherapy against envenomation by snake bites. 相似文献
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Jianying Luo Julie Choulet James C. Samuelson 《Protein science : a publication of the Protein Society》2009,18(8):1735-1744
We have designed a novel protein fusion partner (P8CBD) to utilize the co‐translational SRP pathway in order to target heterologous proteins to the E. coli inner membrane. SRP‐dependence was demonstrated by analyzing the membrane translocation of P8CBD‐PhoA fusion proteins in wt and SRP‐ffh77 mutant cells. We also demonstrate that the P8CBD N‐terminal fusion partner promotes over‐expression of a Thermotoga maritima polytopic membrane protein by replacement of the native signal anchor sequence. Furthermore, the yeast mitochondrial inner membrane protein Oxa1p was expressed as a P8CBD fusion and shown to function within the E. coli inner membrane. In this example, the mitochondrial targeting peptide was replaced by P8CBD. Several practical features were incorporated into the P8CBD expression system to aid in protein detection, purification, and optional in vitro processing by enterokinase. The basis of membrane protein over‐expression toxicity is discussed and solutions to this problem are presented. We anticipate that this optimized expression system will aid in the isolation and study of various recombinant forms of membrane‐associated protein. 相似文献
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《Enzyme and microbial technology》2008,42(6-7):859-866
E. coli proteome response to the stressor 2-HEDS was analyzed through two-dimensional gel electrophoresis (2-DE), and we identified DNA-directed RNA polymerase α-subunit (RpoA) as stress-responsive protein. Even under stress situation where the total number of soluble proteins decreased by 9.8%, the synthesis level of RpoA was increased 1.5-fold. As a fusion expression partner as well as solubility enhancer, RpoA facilitated the folding and increased significantly the solubility of many aggregation-prone heterologous proteins (human minipro-insulin, human epidermal growth factor, human prepro-ghrelin, human interleukin-2, human activation induced cytidine deaminase, human glutamate decarboxylase, Pseudomonas putida cutinase, human ferritin light chain, human granulocyte colony-stimulating factor, and cold inflammatory syndrome1 protein Nacht domain) in E. coli cytoplasm. Due probably to intrinsic high folding efficiency and/or chaperone-like activity, RpoA was very effective in shielding interactive surfaces of heterologous proteins that are associated with non-specific protein–protein interaction leading to the formation of inclusion bodies. RpoA was also well suited for the production of biologically active fusion mutant of Pseudomonas putida cutinase that is of much biotechnological and commercial interest. 相似文献
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Elizabeth Massey‐Gendel Anni Zhao Gabriella Boulting Hye‐Yeon Kim Michael A. Balamotis Len M. Seligman Robert K. Nakamoto James U. Bowie 《Protein science : a publication of the Protein Society》2009,18(2):372-383
A major barrier to the physical characterization and structure determination of membrane proteins is low yield in recombinant expression. To address this problem, we have designed a selection strategy to isolate mutant strains of Escherichia coli that improve the expression of a targeted membrane protein. In this method, the coding sequence of the membrane protein of interest is fused to a C‐terminal selectable marker, so that the production of the selectable marker and survival on selective media is linked to expression of the targeted membrane protein. Thus, mutant strains with improved expression properties can be directly selected. We also introduce a rapid method for curing isolated strains of the plasmids used during the selection process, in which the plasmids are removed by in vivo digestion with the homing endonuclease I‐CreI. We tested this selection system on a rhomboid family protein from Mycobacterium tuberculosis (Rv1337) and were able to isolate mutants, which we call EXP strains, with up to 75‐fold increased expression. The EXP strains also improve the expression of other membrane proteins that were not the target of selection, in one case roughly 90‐fold. 相似文献
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《Process Biochemistry》2010,45(8):1401-1405
Human insulin-like growth factor 1 (hIGF-1) is one kind of growth factor with clinical significance in medicine. The expression of TrxA-hIGF-1 fusion protein was rationally compared in three different Escherichia coli hosts (BL21 (DE3), Rosetta (DE3) and Rosetta-gami (DE3)) with the transformation of plasmid pET32-hIGF-1. The highest productivity of soluble hIGF-1 fusion protein was achieved in E. coli Rosetta-gami (DE3). Moreover, the effects of different expression conditions in this E. coli Rosetta-gami (DE3)/pET32-hIGF-1 host were systematically investigated to improve the expression level of the fusion protein. Under the optimized conditions, a high percent of the target fusion protein (96%) was expressed as soluble form with the volumetric production of soluble fusion protein reaching up to 2.06 g/L. After cell disruption, the soluble fusion protein was separated effectively by affinity chromatography and cleaved by enterokinase, with the concentration of mature hIGF-1 reaching up to 0.42 g/L in the mixture. The present work should be useful for the enhanced production of soluble protein with multiple disulfide bonds in E. coli. 相似文献
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While access to soluble recombinant proteins is essential for a number of proteome studies, preparation of purified functional proteins is often limited by the protein solubility. In this study, potent solubility-enhancing fusion partners were screened from the repertoire of endogenous E. coli proteins. Based on the presumed correlation between the intracellular abundance and folding efficiency of proteins, PCR-amplified ORFs of a series of highly abundant E. coli proteins were fused with aggregation-prone heterologous proteins and then directly expressed for quantitative estimation of the expression efficiency of soluble translation products. Through two-step screening procedures involving the expression of 552 fusion constructs targeted against a series of cytokine proteins, we were able to discover a number of endogenous E. coli proteins that dramatically enhanced the soluble expression of the target proteins. This strategy of cell-free expression screening can be extended to quantitative, global analysis of genomic resources for various purposes. 相似文献
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Chen R 《Biotechnology advances》2012,30(5):1102-1107
Escherichia coli expression system continues to dominate the bacterial expression systems and remain to be the preferred system for laboratory investigations and initial development in commercial activities or as a useful benchmark for comparison among various expression platforms. Some new developments in overcoming its shortcomings are reviewed in this paper, including antibiotics-free selection plasmids, extracellular production, and posttranslational modifications. The ability for E. coli to make mg glycosylated proteins promises even broader applications of the E. coli system in the future. Significant progresses have also been made over the past few years in alternative bacterial expression systems. Notably, the Lactoccocus lactis system has proven to be a viable choice for membrane proteins. Additionally, several Pseudomonas systems were developed and achieved product titers comparable to E. coli systems. Other bacterial systems such as Streptomyces, coryneform bacteria, and halophilic bacteria offer advantages in some niche areas, providing more choices of bacterial expression systems for recalcitrant proteins. 相似文献
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Protein expression in E. coli minicells by recombinant plasmids. 总被引:116,自引:0,他引:116
The polypeptides synthesized in E. coli minicells from recombinant plasmids containing DNA fragments from cauliflower mosaic virus, Drosophila melanogaster, and mouse mitochondria were examined. Molecularly cloned fragments of cauliflower mosaic virus DNA directed the synthesis of high levels of three polypeptides, which were synthesized entirely from within the cloned virus DNA fragments independent of their insertion into the plasmid vehicles. Several fragments of D. melanogaster DNA were capable of initiating polypeptide synthesis; however, termination of these polypeptides was dependent upon the insertion into the plasmid vehicle. The majority of D. melanogaster DNA fragments examined did not direct the detectable synthesis of any polypeptides. Insertion of DNA into the Eco RI site of ColE1 and pSC101 plasmids resulted in the altered expression of plasmid-encoded polypeptides. In the case of ColE1, this site of insertion lies within the colicin E1 structural gene, and insertion of foreign DNA into the site results in the synthesis of an inactive truncated colicin E1 molecule. It is probable that the Eco RI site in pSC101 lies within the structural gene for a polypeptide involved in tetracycline resistance, and insertion of DNA into this site may also result in the synthesis of a truncated or elongated polypeptide. 相似文献
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K Harper D E Jones M D Brennan R Lindahl 《Biochemical and biophysical research communications》1988,152(2):940-947
A cDNA encoding a rat liver inducible aldehyde dehydrogenase carried in a pUC8 plasmid is expressed in E. coli as a dimeric enzyme molecule functionally and physically identical to the authentic rat enzyme. The cDNA appears to be transcribed using the lac promoter, but is translated from an initiator codon 174 base pairs from the 5' end of the cDNA. The aldehyde dehydrogenase polypeptide is not produced as a fusion protein. This is the first example of the production by E. coli of a catalytically active, multimeric eukaryotic protein which is not a fusion protein. 相似文献
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《Peptides》2017
Scorpion venom could be a useful treatment for a variety of diseases, such as cancer, epilepsy and analgesia. BmKTX is a polypeptide extracts from scorpion venom (PESV), which have attracted much attention from researchers in recent years. mBmKTX is a mutant polypeptide according to the amino acid sequence of BmKTX. We expressed it with the vector pGEX-4T-1 in Escherichia coli, and Caenorhabditis elegans were used as the animal model and fed with the strains. In this study, the expression of pGEX-mBmKTX was analyzed by SDS-PAGE, and GST-mBmKTX purified from pGEX-mBmKTX as a glutathione S-transferase (GST)-tagged fusion protein is approximately 30 kDa. The secondary structure prediction shows that mBmKTX is mainly composed of approximately 13% β-sheet and 86% loop. A food clearance assay and brood size assay indicated that the worms fed pGEX-mBmKTX ate more and had greater fecundity than those fed the empty vector. A lifespan analysis demonstrated that mBmKTX could significantly prolong the lifespan of C. elegans, with an increase of 22.5% compared with the control. Behavioral assays confirmed that mBmKTX had no influence on the locomotion of C. elegans. In addition, microarray analysis and quantitative real-time PCR demonstrated that there are 320 differentially expressed genes, 182 of which are related to reproduction, growth and lifespan. In conclusion, the data suggested that mBmKTX has potential utility for increasing fecundity and animal survival. 相似文献
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Fusion ferritin, combined by heavy chain ferritin (21 kDa) and light chain ferritin (19 kDa), was expressed in recombinant E. coli. The fusion ferritin was easily purified by two-step sonications as well as gel filtration chromatography. SDS-gel electrophoresis showed a single band of 38 kDa with heavy and light chains. MALDI-TOF MS gave a molecular weight of fusion ferritin was 38 kDa. The specific activity and yield of purified fusion ferritin are 0.41 Fe3+ mg mg–1 of protein and 66%. Those values are larger than the previous ones of 0.2 Fe3+ mg mg–1 (Kim et al. 2001). 相似文献
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补体C3和杀菌通透性增加蛋白(BPI)对血液中的病原体均有黏附、促吞噬甚至杀灭作用,但两者的作用机制不同,制备两者活性区融合蛋白,可能具有更好的清除血液病原的作用。通过重叠延伸PCR融合人补体C3的补体受体Ⅰ、Ⅲ两个结合区,同时调取了杀菌通透性增加蛋白(BPI)活性区段rBPI,先后将补体C3活性区与BPI蛋白功能区基因克隆入原核表达载体pET28a中,获得融合蛋白(CB)表达载体pET28-CB,在大肠杆菌中进行了高表达产量、可溶性表达等条件的摸索,CB融合蛋白主要以包涵体形式表达,Western印迹证明CB具有C3的抗原活性,将包涵体蛋白变性与复性后,利用Ni2+固相化的螯合Sepharose Fast Flow亲和层析柱进行浓缩和纯化,最后得到了纯度较高的CB原核表达蛋白。CB融合蛋白的构建和高效表达、纯化为下步探讨其在促进血液病原清除上的功能鉴定和应用奠定了基础。 相似文献
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EGF-SEA融合蛋白在大肠杆菌中的表达和纯化 总被引:1,自引:0,他引:1
根据基因库中查到的金黄色葡萄球菌肠毒素A(SEA)基因序列和人体表皮生长因子(EGF)基因序列进行密码子优化,以适于大肠杆菌表达.人工合成SEA基因与EGF基因.将两目的基因克隆至原核表达栽体pFT22b中,经测序验证表明成功构建了重组表达质粒pET22b-EGF-SEA.将构建好的pET22b-EGF-SEA质粒转化大肠杆菌BL21(DE3),经IPTG诱导进行表达;SDS-PAGE分析表明融合基因EGF-SEA在大肠杆菌BL21(DE3)中以包涵体的形式得到了高效表达,产物相对分子质量约为44kDa,与理论值大小一致.包涵体经洗涤,变性、复性后用His Bind Kit进行分离纯化,所得蛋白纯度≥95%.高纯度EGF-SEA融合蛋白的获得为进一步研究其生物学活性及肿瘤治疗奠定了基础. 相似文献
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Romel Menacho-Melgar Zhixia Ye Eirik A. Moreb Tian Yang John P. Efromson John S. Decker Ruixin Wang Michael D. Lynch 《Biotechnology and bioengineering》2020,117(9):2715-2727
We report the scalable production of recombinant proteins in Escherichia coli, reliant on tightly controlled autoinduction, triggered by phosphate depletion in the stationary phase. The method, reliant on engineered strains and plasmids, enables improved protein expression across scales. Expression levels using this approach have reached as high as 55% of the total cellular protein. The initial use of the method in instrumented fed-batch fermentations enables cell densities of ∼30 gCDW/L and protein titers up to 8.1 ± 0.7 g/L (∼270 mg/gCDW). The process has also been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 μl (384-well plates), 100 μl (96-well plates), 20 ml, and 100 ml. In batch cultures, cell densities routinely reach ∼5–7 gCDW/L, offering protein titers above 2 g/L. The methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed-batch fermentation. 相似文献
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用基因重组技术将截短的HIV-1 p24基因和gp41基因连接成嵌合基因,插入质粒pGEX-4T3,构建成重组表达质粒pGEX-F。将pGEX-F转化大肠杆菌BL21。经IPTG诱导表达,pGEX-F在大肠杆菌BL21中获得了高效表达。融合蛋白P24-gp41经Glutathione-Sepharose4B亲和层析纯化后,用间接ELISA和免疫印迹检测HIV抗体阳性血清和正常人血清,P24-gp41只与HIV抗体阳性血清反应,证明获得的融合蛋白P24-gp41有很强的抗原特异性和免疫反应性,具有较高的应用价值。 相似文献