Efficient production of active form of recombinant cassava hydroxynitrile lyase using Escherichia coli in low-temperature culture

Semba, Hisashi; Ichige, Eita; Imanaka, Tadayuki; Atomi, Haruyuki; Aoyagi, Haruhiko

doi:10.1007/s00253-008-1464-8

Cited by 20 publications

(9 citation statements)

References 16 publications

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“…The results assumed that PRV glycoproteins were not accumulated during the expression time and glycoproteins were degraded. It is known that recombinant protein synthesis in the pET system would be too rapid for protein expression (Semba et al, 2008). As expected, we observed that PRV glycoproteins expressed rapidly.…”

Section: Optimal Expression Conditionssupporting

confidence: 87%

Expression of Pseudorabies Virus (PRV) Glycoproteins gB, gC and gD using Bacterial Expression System

Yun¹,

Bae²,

Lee³

et al. 2011

International Journal of Industrial Entomology

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The Pseudorabies (PR), also called Aujeszky's disease (AD), is an infectious viral disease caused by an alpha herpes virus and has domestic and wild pigs, as well as a wide range of domestic and wild animals, as the natural host. Pseudorabies virus (PRV) virions contain several envelope glycoproteins. Among them, gB, gC and gD are regarded as the major immunogenic proteins. We expressed these glycoproteins using the bacterial expression system and analyzed recombinant proteins. Expression of glycoproteins gC and gD were observed on SDS-PAGE or Western blot analysis, but gB was not. Optimal concentration of IPTG and inducing time were determined as 1.0 mM and 4 h, respectively, for the expression of both gC and gD in E. coli. A sodium dodecyl sulfate (SDS) was the most efficient detergent in solubilizing insoluble recombinant protein.

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Section: Optimal Expression Conditionssupporting

confidence: 87%

Expression of Pseudorabies Virus (PRV) Glycoproteins gB, gC and gD using Bacterial Expression System

Yun¹,

Bae²,

Lee³

et al. 2011

International Journal of Industrial Entomology

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“…The whole fermentation process is divided into two stages; that is, the cell growth phage and the protein overexpression phage. The host strain is cultivated at the optimum growth temperature (the cell growth phase) and, subsequently, the cultivation temperature is reduced for the protein expression (the protein production phase) [ 35 ]. In the prophase study, we have investigated the effect of different post-induction temperatures on efficient expression of our target protein lipBJ10.…”

Section: Methodsmentioning

confidence: 99%

Development an effective system to expression recombinant protein in E. coli via comparison and optimization of signal peptides: Expression of Pseudomonas fluorescens BJ-10 thermostable lipase as case study

et al. 2018

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BackgroundThermostable lipases from microbial sources have been substantially overexpressed in E. coli, however, these enzymes are often produced with low-level enzymatic activity and mainly in the form of inclusion bodies. Several studies have reported that the secretory production of recombinant proteins fused their N-terminus to a signal peptide has been employed to resolve the problem. In general, the feasibility of this approach largely depends on the secretory pathway of signal peptide and the type of target protein to be secreted. This study was performed to compare and optimize signal peptides for efficient secretion of thermostable lipase lipBJ10 from Pseudomonas fluorescens BJ-10. Meanwhile, a comparative study between this method and cytoplasmic secretion was implemented in secreting soluble and active lipases.ResultsFusion expression using six signal peptides, i.e., PelB and five native E. coli signal peptides, as fusion partners produced more soluble and functional recombinant lipBJ10 than non-fusion expression. Recombinant lipBJ10, fused to these six diverse signal peptides, was secreted into the periplasm in E. coli. The total lipase activity in all cases of fusion expression was higher than those in non-fusion expression. The relative activity peaked when lipBJ10 was fused to DsbA, yielding a value 73.3 times greater than that of the non-fusion protein. When DsbA was used as the fusion partner, the highest activity (265.41 U/ml) was achieved with the least formation of inclusion bodies; the other four E. coli signal peptides, to some extent, led to low activity and insoluble inclusion bodies. Therefore, DsbA is the optimal signal peptide partner to fuse with lipBJ10 to efficiently produce soluble and functional protein.ConclusionWe found that fusing to these signal peptides, especially that of DsbA, can significantly decrease the formation of inclusion bodies and enhance the function and solubility of lipBJ10 compared to non-fusion lipBJ10. Our results reported here can provide a reference for the high-level expression of other lipases with respect to a possible industrial application.

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“…Various methods have been reported to increase the yield of solubilized recombinant proteins. The simplest and most widely applied method is the optimization of the culture conditions of the strain, e.g., lowering the cultivation temperature 6 and adding metal ions or precursors of cofactors to the cultures 7 . E. coli strains that express high levels of chaperones, such as DnaK or GroEL, are also effective for improving solubility 8 , 9 ; in this case, the chaperone assists with in vivo refolding of the proteins.…”

Section: Introductionmentioning

confidence: 99%

Rational identification of aggregation hotspots based on secondary structure and amino acid hydrophobicity

Matsui

Nakano

Dadashipour

et al. 2017

Sci Rep

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Insolubility of proteins expressed in the Escherichia coli expression system hinders the progress of both basic and applied research. Insoluble proteins contain residues that decrease their solubility (aggregation hotspots). Mutating these hotspots to optimal amino acids is expected to improve protein solubility. To date, however, the identification of these hotspots has proven difficult. In this study, using a combination of approaches involving directed evolution and primary sequence analysis, we found two rules to help inductively identify hotspots: the α-helix rule, which focuses on the hydrophobicity of amino acids in the α-helix structure, and the hydropathy contradiction rule, which focuses on the difference in hydrophobicity relative to the corresponding amino acid in the consensus protein. By properly applying these two rules, we succeeded in improving the probability that expressed proteins would be soluble. Our methods should facilitate research on various insoluble proteins that were previously difficult to study due to their low solubility.

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Efficient production of active form of recombinant cassava hydroxynitrile lyase using Escherichia coli in low-temperature culture

Cited by 20 publications

References 16 publications

Expression of Pseudorabies Virus (PRV) Glycoproteins gB, gC and gD using Bacterial Expression System

Expression of Pseudorabies Virus (PRV) Glycoproteins gB, gC and gD using Bacterial Expression System

Development an effective system to expression recombinant protein in E. coli via comparison and optimization of signal peptides: Expression of Pseudomonas fluorescens BJ-10 thermostable lipase as case study

Rational identification of aggregation hotspots based on secondary structure and amino acid hydrophobicity

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