Development of clone with novel TrpE fusion tag in E. coli for overexpression of trypsin in a bench-scale bioreactor

Author(s):  
Santhosh Nagaraj Nanjundaiah ◽  
Jayasri MA ◽  
Sunilkumar Sukumaran ◽  
Ganesh Sambasivam
Keyword(s):  
E Coli ◽  
2009 ◽  
Vol 2009 ◽  
pp. 1-7 ◽  
Author(s):  
P. Azhahianambi ◽  
D. D. Ray ◽  
Pallab Chaudhuri ◽  
Rohita Gupta ◽  
Srikanta Ghosh

The use of tick vaccine in controlling ticks and tick borne diseases has been proved effective in integrated tick management format. For the control ofH. a. anatolicum, Bm86 ortholog ofH. a. anatolicumwas cloned and expressed as fusion protein inE. coliasE. coli-pETHaa86. The molecular weight of the rHaa86 was 97 kDa with a 19 kDa fusion tag of thioredoxin protein. The expressed protein was characterized immunologically and vaccine efficacy was evaluated. After 120 hours of challenge, only 26% tick could successfully fed on immunized animals. Besides significant reduction in feeding percentages, a significant reduction of 49.6 mg;P<.01in the weight of fed females in comparison to the females fed on control animals was recorded. Following oviposition, a significant reduction of 68.1 mg;P<.05in the egg masses of ticks fed on immunized animals in comparison to the ticks fed on control animals was noted. The reduction of number of females, mean weight of eggs, adult females and efficacy of immunogen were 73.8%, 31.3%, 15.8%, and 82.3%, respectively. The results indicated the possibility of development of rHaa86 based vaccine as a component of integrated control of tick species.


2021 ◽  
Author(s):  
Bayar Enkhtuya ◽  
Yuanyuan Ren ◽  
Yafang Hu ◽  
Yinghua Chen ◽  
Jiong Hu ◽  
...  

Abstract Tobacco etch virus protease (TEVp) is a powerful enzymatic reagent for removing fusion tag. In this work, we constructed nine TEVp variants with introducing one to three mutations of C19S, C110S and C130S into the soluble TEVp variant, TEVp5M. Using the C-terminal green fluorescent protein (GFP) variant reporter, all constructs showed different solubility levels among four E. coli strains. The TEVp5M containing the C110S and/or C130S mutations in the hyperoxic strain showed the enhanced the cleavage activity. Addition of dithiothreitol to the cultural medium increased the activity of certain constructs produced in the BL21(DE3), contrary to the added hydrogen peroxide, due to cytoplasmic redox change measured by the redox sensitive GFP construct. The more cysteine residues in the purified TEVp5M were modified specifically than those in the other variants. All purified constructs showed similar specific activities in the presence of 5 mM dithiothreitol. In the buffer containing the compounds to aid disulfide bond formation of the refolded protein, the double mutant TEVp5MC110S/C130S exhibited the highest cleavage efficiency. This variant was efficient for removing the fusion tag after refolding of cellulose-binding module tagged disulfide-rich proteins including bovine enteropeptidase and maize peroxidase absorbed on the regenerated amorphous cellulose.


2019 ◽  
Vol 16 (3) ◽  
pp. 162
Author(s):  
Le Duong Vuong ◽  
Le Thi Tuong Vy ◽  
Phan Thi Phuong Trang ◽  
Nguyen Duc Hoang

The Human rhinovirus 3C protease (HRV3C) is one of the most effective enzymes for removing fusion tag in purification process. This protease is often produced as fusion form GST-HRV3C but there is no study about the fusion form: GST-HRV3C-His. In this study, researchers conducted the purification GST-HRV3C-His expressed in E. coli, checked the activity and investigated its application. GST-HRV3C-His could be purified using His-tag column with 86.6% purity and GST column with 96.87%. The specific activity of GST-HRV3C-His was demonstrated to be about 4500 U/mg and its application in the purification of another proteins carrying HRV3C-specific recognition sequence, LEVLFQ¯GP based on His-tag or GST-tag was also proved in this study.


2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Hyunjun Ko ◽  
Minsik Kang ◽  
Mi-Jin Kim ◽  
Jiyeon Yi ◽  
Jin Kang ◽  
...  

Abstract Background Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli. Results A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. Conclusions The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli.


2008 ◽  
Vol 43 (1) ◽  
pp. 77-84 ◽  
Author(s):  
Lawrence H. Lee ◽  
Adam J. Arnold ◽  
Cutberto A. Santillan ◽  
Monica B. Emelko ◽  
Sarah E. Dickson ◽  
...  

Abstract Pulsed arc electrohydraulic discharge (PAED) offers concurrent treatment of chemical and microbial target compounds in water by several mechanisms. Here, Escherichia coli, Bacillus subtilis spore, and MS2 bacteriophage inactivation by PAED were investigated using two bench-scale reactors (0.7 and 3 L). A plasma channel was created between a pair of iron electrodes set 0.5 mm apart in these reactors. Pulsed applied voltage was supplied at approximately 0.3 kJ/pulse (~100 µsec). In the 0.7-L reactor, median E. coli, B. subtilis, and MS2 reductions of 2.4-, 4.6-, and 3.7-log, respectively, were observed after approximately 80 seconds of treatment in water with a conductivity of 14.7 mS; reductions of 2.8-, 4.0-, and 3.7-log, respectively, were observed in treated drinking water filter effluent (conductivity of 610 µS). In the 3-L reactor, at a conductivity of 500 to 600 µS, 1.9- and approximately 0.9-log median reductions of E. coli and B. subtilis were achieved after 500 pulses (~3,300 sec); and, at a conductivity of 4.3 mS, median E. coli reductions of 0.5-log were achieved after 50 pulses (400 sec). Further work is necessary to elucidate the primary mechanism(s) of disinfection acting in the PAED system and how they relate to the reactor design, applied power requirements, and disinfection of specific target microorganisms.


2015 ◽  
Vol 12 (9) ◽  
pp. 10276-10299 ◽  
Author(s):  
Mark Elliott ◽  
Christine Stauber ◽  
Francis DiGiano ◽  
Anna de Aceituno ◽  
Mark Sobsey

Author(s):  
D. E. Philpott ◽  
A. Takahashi

Two month, eight month and two year old rats were treated with 10 or 20 mg/kg of E. Coli endotoxin I. P. The eight month old rats proved most resistant to the endotoxin. During fixation the aorta, carotid artery, basil arartery of the brain, coronary vessels of the heart, inner surfaces of the heart chambers, heart and skeletal muscle, lung, liver, kidney, spleen, brain, retina, trachae, intestine, salivary gland, adrenal gland and gingiva were treated with ruthenium red or alcian blue to preserve the mucopolysaccharide (MPS) coating. Five, 8 and 24 hrs of endotoxin treatment produced increasingly marked capillary damage, disappearance of the MPS coating, edema, destruction of endothelial cells and damage to the basement membrane in the liver, kidney and lung.


Author(s):  
James A. Lake

The understanding of ribosome structure has advanced considerably in the last several years. Biochemists have characterized the constituent proteins and rRNA's of ribosomes. Complete sequences have been determined for some ribosomal proteins and specific antibodies have been prepared against all E. coli small subunit proteins. In addition, a number of naturally occuring systems of three dimensional ribosome crystals which are suitable for structural studies have been observed in eukaryotes. Although the crystals are, in general, too small for X-ray diffraction, their size is ideal for electron microscopy.


Author(s):  
Manfred E. Bayer

Bacterial viruses adsorb specifically to receptors on the host cell surface. Although the chemical composition of some of the cell wall receptors for bacteriophages of the T-series has been described and the number of receptor sites has been estimated to be 150 to 300 per E. coli cell, the localization of the sites on the bacterial wall has been unknown.When logarithmically growing cells of E. coli are transferred into a medium containing 20% sucrose, the cells plasmolize: the protoplast shrinks and becomes separated from the somewhat rigid cell wall. When these cells are fixed in 8% Formaldehyde, post-fixed in OsO4/uranyl acetate, embedded in Vestopal W, then cut in an ultramicrotome and observed with the electron microscope, the separation of protoplast and wall becomes clearly visible, (Fig. 1, 2). At a number of locations however, the protoplasmic membrane adheres to the wall even under the considerable pull of the shrinking protoplast. Thus numerous connecting bridges are maintained between protoplast and cell wall. Estimations of the total number of such wall/membrane associations yield a number of about 300 per cell.


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