Over-expression and characterization of the recombinant small heat shock protein from Pyrococcus furiosus

Chen, Huayou; Chu, Zema; Zhang, Yi; Yang, Shengli

doi:10.1007/s10529-006-9058-y

Cited by 14 publications

(3 citation statements)

References 10 publications

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“…It may because of the protection from HSPs is only short-acting [ 12 ] that once the duration of thermal stress is enhanced, the cells ultimately undergo cell death. Although the overexpression of HSP has been applied to improve the thermotolerance [ 49 , 50 ], the effects are not ideal. We assumed that preventing protein misfolding by OSR is more important than the repair by HSR after damage.…”

Section: Discussionmentioning

confidence: 99%

Augmented peroxisomal ROS buffering capacity renders oxidative and thermal stress cross-tolerance in yeast

Lin

et al. 2021

Microb Cell Fact

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Background Thermotolerant yeast has outstanding potential in industrial applications. Komagataella phaffii (Pichia pastoris) is a common cell factory for industrial production of heterologous proteins. Results Herein, we obtained a thermotolerant K. phaffii mutant G14 by mutagenesis and adaptive evolution. G14 exhibited oxidative and thermal stress cross-tolerance and high heterologous protein production efficiency. The reactive oxygen species (ROS) level and lipid peroxidation in G14 were reduced compared to the parent. Oxidative stress response (OSR) and heat shock response (HSR) are two major responses to thermal stress, but the activation of them was different in G14 and its parent. Compared with the parent, G14 acquired the better performance owing to its stronger OSR. Peroxisomes, as the main cellular site for cellular ROS generation and detoxification, had larger volume in G14 than the parent. And, the peroxisomal catalase activity and expression level in G14 was also higher than that of the parent. Excitingly, the gene knockdown of CAT encoding peroxisomal catalase by dCas9 severely reduced the oxidative and thermal stress cross-tolerance of G14. These results suggested that the augmented OSR was responsible for the oxidative and thermal stress cross-tolerance of G14. Nevertheless, OSR was not strong enough to protect the parent from thermal stress, even when HSR was initiated. Therefore, the parent cannot recover, thereby inducing the autophagy pathway and resulting in severe cell death. Conclusions Our findings indicate the importance of peroxisome and the significance of redox balance in thermotolerance of yeasts.

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Section: Discussionmentioning

confidence: 99%

Augmented peroxisomal ROS buffering capacity renders oxidative and thermal stress cross-tolerance in yeast

Lin

et al. 2021

Microb Cell Fact

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“…Genes encoding prefoldin, chaperonin (HSP60), sHSP, and extracellular α-amylase from P. furiosus (PFA) were PCRamplified from plasmids constructed in previous studies (Chen et al 2006(Chen et al , 2010. Primers used to amplify these g e n e s w e r e a s f o l l o w s : p r e f o l d i n : s e n s e , 5 ′ -GCCCATGGAAAACAATAAGGAAT-3′ (NcoI underlined); antisense, 5′-ACGCGTCGACGGAGCTCGAATTCTCATC-3′ (SalI underlined); HSP60: sense, 5′-ACTATCATATGGC CCAGTTAGCAGGC-3′ (NdeI underlined); antisense, 5′-CAGCTCGAGCTTGCTGGCAGCGATGAC-3′ (XhoI underlined); sHSP: sense, 5′-GCCCATGGTTCGTCGTATC-3′ (NcoI underlined); antisense, 5′ -AACG TC GAC TATTCAACTTT AACTTC-3′ (SalI underlined).…”

Section: Constructions Of Recombinant Plasmidsmentioning

confidence: 99%

Co-expression of chaperones from P. furiosus enhanced the soluble expression of the recombinant hyperthermophilic α-amylase in E. coli

Peng

Chu

et al. 2016

Cell Stress and Chaperones

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The extracellular α-amylase from the hyperthermophilic archaeum Pyrococcus furiosus (PFA) is extremely thermostable and of an industrial importance and interest. PFA aggregates and accumulates as insoluble inclusion bodies when expressed as a heterologous protein at a high level in Escherichia coli. In the present study, we investigated the roles of chaperones from P. furiosus in the soluble expression of recombinant PFA in E. coli. The results indicate that coexpression of PFA with the molecular chaperone prefoldin alone significantly increased the soluble expression of PFA. Although, co-expression of other main chaperone components from P. furiosus, such as the small heat shock protein (sHSP) or chaperonin (HSP60), was also able to improve the soluble expression of PFA to a certain extent. Co-expression of chaperonin or sHSP in addition to prefoldin did not further increase the soluble expression of PFA. This finding emphasizes the biotechnological potentials of the molecular chaperone prefoldin from P. furiosus, which may facilitate the production of recombinant PFA.

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“…As a typical hyperthermophilic archaeon, Pyrococcus furiosus has a simplified chaperone system (Chen et al 2006(Chen et al , 2010 and contains only one chaperonin gene per genome. Unlike yeast CCT complex which contains eight distinct but related subunits (Stoldt et al 1996), the chaperonin from P. furiosus (PfCPN) exists as homo-oligomer.…”

Section: Introductionmentioning

confidence: 99%

Gly-345 plays an essential role in Pyrococcus furiosus chaperonin function

et al. 2011

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Compared to the group I chaperonins, such as Escherichia coli GroEL, which facilitate protein folding, many aspects of the functional mechanism of archaeal group II chaperonins are unclear. Sequence homology between the chaperonin from Pyrococcus furiosus (PfCPN) and other group II chaperonins, together with the homo-oligomeric nature of PfCPN, suggest that PfCPN may serve as a model to clarify the role of the homologous position Gly-345 in the chaperonin-mediated protein folding. Here, we show that the purified chaperonin mutant in which the conserved residue Gly-345 is replaced by Asp (G345D) displays only about 25% ATP/ADP hydrolysis activities of the wild-type in the presence of Co(2+) and has a reduced capacity to promote folding of denatured malate dehydrogenase in vitro. This may be a reflection that Gly-345 plays an essential role in conformational change and protein refolding by archaeal group II chaperonins.

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Over-expression and characterization of the recombinant small heat shock protein from Pyrococcus furiosus

Cited by 14 publications

References 10 publications

Augmented peroxisomal ROS buffering capacity renders oxidative and thermal stress cross-tolerance in yeast

Augmented peroxisomal ROS buffering capacity renders oxidative and thermal stress cross-tolerance in yeast

Co-expression of chaperones from P. furiosus enhanced the soluble expression of the recombinant hyperthermophilic α-amylase in E. coli

Gly-345 plays an essential role in Pyrococcus furiosus chaperonin function

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