scholarly journals Surface Immobilization of Human Arginase-1 with an Engineered Ice Nucleation Protein Display System in E. coli

PLoS ONE ◽  
2016 ◽  
Vol 11 (8) ◽  
pp. e0160367 ◽  
Author(s):  
Zhen Zhang ◽  
Rongxin Tang ◽  
Lu Bian ◽  
Meng Mei ◽  
Chunhua Li ◽  
...  
2009 ◽  
Vol 76 (2) ◽  
pp. 434-440 ◽  
Author(s):  
Chao Yang ◽  
Roland Freudl ◽  
Chuanling Qiao ◽  
Ashok Mulchandani

ABSTRACT A genetically engineered Escherichia coli strain coexpressing organophosphorus hydrolase (OPH) and methyl parathion hydrolase (MPH) was constructed for the first time by cotransforming two compatible plasmids. Since these two enzymes have different substrate specificities, the coexpression strain showed a broader substrate range than strains expressing either one of the hydrolases. To reduce the mass transport limitation of organophosphates (OPs) across the cell membrane, MPH and OPH were simultaneously translocated to the periplasm and cell surface of E. coli, respectively, by employing the twin-arginine translocation (Tat) pathway and ice nucleation protein (INP) display system. The resulting recombinant strain showed sixfold-higher whole-cell activity than the control strain expressing cytosolic OP hydrolases. The correct localization of MPH and OPH was demonstrated by cell fractionation, immunoblotting, and enzyme activity assays. No growth inhibition was observed for the recombinant E. coli strain, and suspended cultures retained almost 100% of the activity over a period of 2 weeks. Owing to its high level of activity and superior stability, the recombinant E. coli strain could be employed as a whole-cell biocatalyst for detoxification of OPs. This strategy of utilizing dual translocation pathways should open up new avenues for cotranslocating multiple functional moieties to different extracytosolic compartments of a bacterial cell.


2011 ◽  
Vol 63 (4) ◽  
pp. 943-948 ◽  
Author(s):  
Mohammed Sarhan

Surface display technology can be defined as that phenotype (protein or peptide) which is linked to a genotype (DNA or RNA) through an appropriate anchoring motif. A bacterial surface display system is based on expressing recombinant proteins fused to sorting signals (anchoring motifs) that direct their incorporation on the cell surface.


2021 ◽  
Author(s):  
Xinming Feng ◽  
Miaomiao Jin ◽  
Wei Huang ◽  
Wei Liu ◽  
Mo Xian

Abstract BackgroundFluorinases play a unique role in producing fluorinated organic molecules through a biological method. Whole-cell catalysis is a better choice in the large-scale fermentation processes, and over 60% of industrial biocatalysis uses this method. However, the in vivo catalytic efficiency of fluorinases is stuck with the mass transfer of the substrates.ResultsA gene sequence encoding a protein with fluorinase function was fused to the N-terminal of ice nucleation protein, and the fused protein was expressed in Escherichia coli BL21(DE3) cells. SDS-PAGE and Immunofluorescence microscopy were used to demonstrate the surface localization of the fusion protein. The fluorinase-containing surface display system with improved whole-cell catalytic efficiency and stability showed low growth pressure on the protein expressing host. The conversion rate of 5′-fluorodeoxyadenosine (5′-FDA) from S-adenosyl-L-methionine (SAM) achieved 55%.ConclusionsHere, we created the fluorinase-containing surface display system on E.coli cells for the first time. The fluorinase was successfully displayed on the surface of Escherichia coli and maintained its catalytic activity. The surface display offers a new solution for the industrial application of biological fluorination.


2019 ◽  
Author(s):  
Johannes Kassmannhuber ◽  
Sergio Mauri ◽  
Mascha Rauscher ◽  
Nadja Brait ◽  
Lea Schöner ◽  
...  

Abstract. An N-terminal truncated form of the ice nucleation protein (INP) of Pseudomonas syringae lacking the transport sequence for the localization of InaZ in the outer membrane was fused to N- and C- terminal inner membrane (IM) anchors and expressed in Escherichia coli C41. The ice nucleation (IN) activity of the corresponding living recombinant E. coli catalyzing heterogeneous ice formation of super-cooled water at high subzero temperatures was tested by droplet freezing assay. Median freezing temperature (T50) of the parental living E. coli C41 cells without INP was detected at −20.1 °C and with inner membrane anchored INPs at T50 value between −7 °C and −9 °C demonstrating that IM anchored INPs facing the luminal IM site are able to induce IN from the inside of the bacterium almost similar to bacterial INPs located at the outer membrane. Bacterial Ghosts (BGs) derived from the different constructs showed first droplet freezing values between −6 °C and −8 °C whereas C41 BGs alone without carrying IM anchored INPs exhibit a T50 of −18.9 °C. The more efficient IN of INP-BGs compared to their living parental strains can be explained by the free access of IM anchored INP constructs to ultrapure water filling the inner space of the BGs. The cell killing rate of -NINP carrying E. coli at subzero temperatures is higher when compared to survival rates of the parental C41 strain.


2021 ◽  
Author(s):  
Lilin Feng ◽  
Liang Gao ◽  
Daniel F. Sauer ◽  
Yu Ji ◽  
Haiyang Cui ◽  
...  

A facile and reversible method to immobilize His6-tagged proteins on the E. coli cell surface through the formation of an Fe(iii)-complex.


2012 ◽  
Vol 12 (22) ◽  
pp. 10667-10677 ◽  
Author(s):  
E. Attard ◽  
H. Yang ◽  
A.-M. Delort ◽  
P. Amato ◽  
U. Pöschl ◽  
...  

Abstract. Although ice nuclei from bacterial origin are known to be efficient at the highest temperatures known for ice catalysts, quantitative data are still needed to assess their role in cloud processes. Here we studied the effects of three typical cloud conditions (i) acidic pH (ii) NO2 and O3 exposure and (iii) UV-A exposure on the ice nucleation activity (INA) of four Pseudomonas strains. Three of the Pseudomonas syringae strains were isolated from cloud water and the phyllosphere and Pseudomonas fluorescens strain CGina-01 was isolated from Antarctic glacier ice melt. Among the three conditions tested, acidic pH caused the most significant effects on INA likely due to denaturation of the ice nucleation protein complex. Exposure to NO2 and O3 gases had no significant or only weak effects on the INA of two P. syringae strains whereas the INA of P. fluorescens CGina-01 was significantly affected. The INA of the third P. syringae strain showed variable responses to NO2 and O3 exposure. These differences in the INA of different Pseudomonas suggest that the response to atmospheric conditions could be strain-specific. After UV-A exposure, a substantial loss of viability of all four strains was observed whereas their INA decreased only slightly. This corroborates the notion that under certain conditions dead bacterial cells can maintain their INA. Overall, the negative effects of the three environmental factors on INA were more significant at the warmer temperatures. Our results suggest that in clouds where temperatures are near 0 °C, the importance of bacterial ice nucleation in precipitation processes could be reduced by some environmental factors.


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