efficient expression
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2022 ◽  
Vol 8 (1) ◽  
pp. 84
Author(s):  
Marilia M. Knychala ◽  
Angela A. dos Santos ◽  
Leonardo G. Kretzer ◽  
Fernanda Gelsleichter ◽  
Maria José Leandro ◽  
...  

In previous work, we developed a Saccharomyces cerevisiae strain (DLG-K1) lacking the main monosaccharide transporters (hxt-null) and displaying high xylose reductase, xylitol dehydrogenase and xylulokinase activities. This strain proved to be a useful chassis strain to study new glucose/xylose transporters, as SsXUT1 from Scheffersomyces stipitis. Proteins with high amino acid sequence similarity (78–80%) to SsXUT1 were identified from Spathaspora passalidarum and Spathaspora arborariae genomes. The characterization of these putative transporter genes (SpXUT1 and SaXUT1, respectively) was performed in the same chassis strain. Surprisingly, the cloned genes could not restore the ability to grow in several monosaccharides tested (including glucose and xylose), but after being grown in maltose, the uptake of 14C-glucose and 14C-xylose was detected. While SsXUT1 lacks lysine residues with high ubiquitinylation potential in its N-terminal domain and displays only one in its C-terminal domain, both SpXUT1 and SaXUT1 transporters have several such residues in their C-terminal domains. A truncated version of SpXUT1 gene, deprived of the respective 3′-end, was cloned in DLG-K1 and allowed growth and fermentation in glucose or xylose. In another approach, two arrestins known to be involved in the ubiquitinylation and endocytosis of sugar transporters (ROD1 and ROG3) were knocked out, but only the rog3 mutant allowed a significant improvement of growth and fermentation in glucose when either of the XUT permeases were expressed. Therefore, for the efficient heterologous expression of monosaccharide (e.g., glucose/xylose) transporters in S. cerevisiae, we propose either the removal of lysines involved in ubiquitinylation and endocytosis or the use of chassis strains hampered in the specific mechanism of membrane protein turnover.


2021 ◽  
Vol 17 ◽  
Author(s):  
Morena Miciaccia ◽  
Mariaclara Iaselli ◽  
Savina Ferorelli ◽  
Paola Loguercio Polosa ◽  
Maria Grazia Perrone ◽  
...  

Background: Recent findings enlightened the pivotal role of cyclooxygenases-1 and -2 (COX-1 and COX-2) in human diseases with inflammation as the committed earliest stage, such as cancer and neurodegenerative diseases. COXs are the main targets of nonsteroidal anti-inflammatory drugs and catalyze the bis-oxygenation of arachidonic acid into prostaglandin PGH2, then converted into prostaglandins, thromboxane, and prostacyclin by tissue-specific isomerases. A remarkable amount of pure COX-1 results is necessary to investigate COX-1 structure and function, as well as for in vitro disease biochemical pathway investigations. Methods: Spodoptera frugiperda cells were infected with Baculovirus that revealed to be an efficient expression system to obtain a high amount of ovine COX-1. Protein solubilization time in the presence of a non-ionic detergent was modified, and a second purification step was introduced. Results and Discussion: An improvement of a previously reported method for pure recombinant oCOX-1 production and isolation has been achieved, leading to a lower starting volume of infected cells for each purification, an increased cell density, and of the number of viral particles per cell, and a shortened infection period. The protocol for the recombinant oCOX-1 expression and purification has been in-depth elaborated to obtain 1 mg/L of protein. Conclusion: The optimized procedure could be suitable for producing other membrane proteins as well, for which an improvement in the solubilization step is necessary to have the availability of high concentration proteins.


2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Xianpu Wang ◽  
Lili Xu ◽  
Xiuxia Liu ◽  
Li Xin ◽  
Shujing Wu ◽  
...  

AbstractProtoplast transient expression is a powerful strategy for gene functional characterization, especially in biochemical mechanism studies. We herein developed a highly efficient transient expression system for apple protoplasts. The abilities of the Arabidopsis thaliana and Malus domestica ubiquitin-10 (AtUBQ10 and MdUBQ10) promoters to drive the expression of multiple genes were compared with that of the CaMV 35S promoter, and the results revealed that the AtUBQ10 and MdUBQ10 promoters were more efficient in apple protoplasts. With this system, we demonstrated that active AtMKK7ac could activate MAPK6/3/4 signaling cascades, which further regulated MdWRKY33 phosphorylation and stability in apple. Furthermore, the ligand-induced interaction between the immune receptor AtFLS2 and the coreceptor AtBAK1 was reconstituted in apple protoplasts. We also found that the stability of the bacterial effector AvrRpt2 was regulated by feedback involving auxin and the immune regulator RIN4. The system established herein will serve as a useful tool for the molecular and biochemical analyses of apple genes.


2021 ◽  
Vol 5 (1) ◽  
pp. 049-050
Author(s):  
Vittaladevaram Viswanath

One of the major challenges for vaccine producing companies is having favourable conditions for efficient expression system for living organisms in order to produce biologicals. Several companies across the globe looking for several alternative sources for better yield through efficient expression based system.


2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Hao You ◽  
Bin Sun ◽  
Na Li ◽  
Jun-Wei Xu

Abstract Background Ganoderma lucidum, a well-known medicinal mushroom, has received wide attention as a promising cell factory for producing bioactive compounds. However, efficient expression of heterologous genes remains a major challenge in Ganoderma, hindering metabolic regulation research and molecular breeding of this species. Results We show that the presence of glyceraldehyde-3-phosphate dehydrogenase gene (gpd) intron 1 at the 5′ end of, the 3′ end of, or within the heterologous phosphinothricin-resistant gene (bar) is efficient for its expression in G. lucidum. The enhanced expression of bar is exhibited by the higher accumulation of mRNA and increased amounts of protein. Moreover, the insertion of the gpd intron 1 in the β-glucuronidase gene (gus) elevates its mRNA accumulation and enzyme activity, which facilitates the use of this reporter gene in Ganoderma. Conclusions This study has demonstrated the importance of the introduction of gpd intron 1 for the efficient expression of bar and gus in G. lucidum. The presence of the gpd intron 1 in heterologous genes increases levels of mRNA accumulation and protein expression in basidiomycete Ganoderma. The developed method may be utilized in upregulating the expression of other heterologous genes in Ganoderma.


2021 ◽  
Author(s):  
Sven T. Stripp ◽  
Jonathan Oltmanns ◽  
Christina S. Müller ◽  
David Ehrenberg ◽  
Ramona Schlesinger ◽  
...  

The [4Fe-4S] cluster containing scaffold complex HypCD is the central construction site for the assembly of the [Fe](CN)2CO cofactor precursor of [NiFe]-hydrogenase. While the importance of the HypCD complex is well established, not much is known about the mechanism by which the CN– and CO ligands are transferred and attached to the iron ion. We report an efficient expression and purification system producing the HypCD complex from E. coli with complete metal content. This enabled in-depth spectroscopic characterizations. The results obtained by EPR and Mössbauer spectroscopy demonstrate that the [Fe](CN)2CO cofactor and the [4Fe-4S] cluster of the HypCD complex are redox active. The data indicate a potential-dependent interconversion of the [Fe]2+/3+ and [4Fe-4S]2+/+ couple, respectively. Moreover, ATR FTIR spectroscopy reveals potential-dependent disulfide formation, which hints at an electron confurcation step between the metal centers. MicroScale thermophoresis indicates preferable binding between the HypCD complex and its in vivo interaction partner HypE under reducing conditions. Together, these results provide comprehensive evidence for an electron inventory fit to drive multi-electron redox reactions required for the assembly of the CN– and CO ligands on the scaffold complex HypCD.


2021 ◽  
Vol 9 (8) ◽  
pp. 1551
Author(s):  
Zi-Xu Wang ◽  
Na Li ◽  
Jun-Wei Xu

A Vitreoscilla hemoglobin (VHb) gene was efficiently expressed by the optimization of codons and intron addition in G. lucidum. Expression of the VHb gene was confirmed by genome PCR, quantitative real-time PCR and carbon monoxide (CO)-difference spectrum analysis in the transformant. The effects of the efficient expression of VHb gene on production, monosaccharide compostion, and antioxidant activity of G. lucidum exopolysaccharides were studied. The maximum production of exopolysaccharides in the VHb gene-bearing transformant was 1.63 g/L, which was 1.5-fold higher than expression in the wild-type strain. Efficient expression of the VHb gene did not change the monosaccharide composition or distribution of molecular weight, but it increased the mole percentage ratio of galactose and mannose in G. lucidum exopolysaccharide. Exopolysaccharides from the transformant had higher scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl (OH) radical capacity and reducing power than those from the wild-type strain. These results may be helpful for increasing production and application of exopolysaccharides produced by G. lucidum fermentation.


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