Enhanced Recombinant Protein Production Under Special Environmental Stress

Chen, Xinyi; Liang, Chun; Hu, Liu

doi:10.3389/fmicb.2021.630814

Cited by 23 publications

(21 citation statements)

References 102 publications

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“…T7-SHuffle cells thus enable the oxidative folding of multi-disulfide bonds, and DsbC isomerase can potentially reshuffle non-native SS bonds [ 26 ]. Further, lowering the expression temperature to 16 °C slows down protein expression and stabilizes the native structure, thereby aiding the folding process [ 27 ].…”

Section: Resultsmentioning

confidence: 99%

An Escherichia coli Expressed Multi-Disulfide Bonded SARS-CoV-2 RBD Shows Native-like Biophysical Properties and Elicits Neutralizing Antisera in a Mouse Model

Brindha

Yoshizue

Wongnak

et al. 2022

IJMS

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A large-scale Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 could yield a versatile and low-cost antigen for a subunit vaccine. Appropriately folded antigens can potentially elicit the production of neutralizing antisera providing immune protection against the virus. However, E. coli expression using a standard protocol produces RBDs with aberrant disulfide bonds among the RBD’s eight cysteines resulting in the expression of insoluble and non-native RBDs. Here, we evaluate whether E. coli expressing RBD can be used as an antigen candidate for a subunit vaccine. The expressed RBD exhibited native-like structural and biophysical properties as demonstrated by analytical RP-HPLC, circular dichroism, fluorescence, and light scattering. In addition, our E. coli expressed RBD binds to hACE2, the host cell’s receptor, with a binding constant of 7.9 × 10−9 M, as indicated by biolayer interferometry analysis. Our E. coli-produced RBD elicited a high IgG titer in Jcl:ICR mice, and the RBD antisera inhibited viral growth, as demonstrated by a pseudovirus-based neutralization assay. Moreover, the increased antibody level was sustained for over 15 weeks after immunization, and a high percentage of effector and central memory T cells were generated. Overall, these results show that E. coli-expressed RBDs can elicit the production of neutralizing antisera and could potentially serve as an antigen for developing an anti-SARS-CoV-2 subunit vaccine.

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Section: Resultsmentioning

confidence: 99%

An Escherichia coli Expressed Multi-Disulfide Bonded SARS-CoV-2 RBD Shows Native-like Biophysical Properties and Elicits Neutralizing Antisera in a Mouse Model

Brindha

Yoshizue

Wongnak

et al. 2022

IJMS

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show abstract

“…Concerning bioprocess optimization, the operational mode is a key factor in bioprocess design, batch or fedbatch being the most established modes for production at an industrial scale, while chemostat is more commonly used for strain characterization and bioprocess design (Looser et al, 2014;Kumar et al, 2020;Nieto-Taype et al, 2020a). Additionally, the application of different types of cell stress has been demonstrated to have a significant positive effect on RPP, so the established process strategies can be remarkably improved (García-Ortega et al, 2019;Chen et al, 2021). Some examples are the application of substrate starving periods (Garcia-Ortega et al, 2016;Çalık et al, 2018) and the implementation of oxygen limiting conditions (Baumann et al, 2008), which have shown a positive effect on RPP under P GAP regulation.…”

Section: Introductionmentioning

confidence: 99%

Innovative Bioprocess Strategies Combining Physiological Control and Strain Engineering of Pichia pastoris to Improve Recombinant Protein Production

Gasset

Garcia-Ortega

Garrigós-Martínez

et al. 2022

Front. Bioeng. Biotechnol.

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The combination of strain and bioprocess engineering strategies should be considered to obtain the highest levels of recombinant protein production (RPP) while assuring product quality and process reproducibility of heterologous products. In this work, two complementary approaches were investigated to improve bioprocess efficiency based on the yeast P. pastoris. Firstly, the performance of two Candida rugosa lipase 1 producer clones with different gene dosage under the regulation of the constitutive PGAP were compared in chemostat cultures with different oxygen-limiting conditions. Secondly, hypoxic conditions in carbon-limited fed-batch cultures were applied by means of a physiological control based on the respiratory quotient (RQ). Stirring rate was selected to maintain RQ between 1.4 and 1.6, since it was found to be the most favorable in chemostat. As the major outcome, between 2-fold and 4-fold higher specific production rate (qP) values were observed when comparing multicopy clone (MCC) and single-copy clone (SCC), both in chemostat and fed-batch. Additionally, when applying oxygen limitation, between 1.5-fold and 3-fold higher qP values were obtained compared with normoxic conditions. Thus, notable increases of up to 9-fold in the production rates were reached. Furthermore, transcriptional analysis of certain key genes related to RPP and central carbon metabolism were performed. Results seem to indicate the presence of a limitation in post-transcriptional protein processing steps and a possible transcription attenuation of the target gene in the strains with high gene dosage. The entire approach, including both strain and bioprocess engineering, represents a relevant novelty involving physiological control in Pichia cell factory and is of crucial interest in bioprocess optimization, boosting RPP, allowing bioproducts to be economically competitive in the market, and helping develop the bioeconomy.

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“… 2003 , Chen et al . 2021 ). On the other hand, the deletion of PTC1 reduces the cell wall β-1,6-glucan levels by activating Exg1p (Jiang et al .…”

Section: Resultsmentioning

confidence: 99%

CRISPR/Cas9-mediated point mutations improve α-amylase secretion in Saccharomyces cerevisiae

Wang

Chen

et al. 2022

FEMS Yeast Research

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The rapid expansion of the application of pharmaceutical proteins and industrial enzymes requires robust microbial workhorses for high protein production. The budding yeast Saccharomyces cerevisiae is an attractive cell factory due to its ability to perform eukaryotic post-translational modifications and to secrete proteins. Many strategies have been used to engineer yeast platform strains for higher protein secretion capacity. Herein, we investigated a line of strains that have previously been selected after UV random mutagenesis for improved α-amylase secretion. A total of 42 amino acid altering point mutations identified in this strain line were re-introduced into the parental strain AAC to study their individual effects on protein secretion. These point mutations included missense mutations (amino acid substitution), nonsense mutations (stop codon generation) and frameshift mutations. For comparison, single gene deletions for the corresponding target genes were also performed in this study. Eleven point mutations and seven gene deletions were found to effectively improve α-amylase secretion. These targets were involved in several bioprocesses, including cellular stresses, protein degradation, transportation, mRNA processing and export, DNA replication and repair, which indicates that the improved protein secretion capacity in the evolved strains is the result of the interaction of multiple intracellular processes. Our findings will contribute to the construction of novel cell factories for recombinant protein secretion.

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Enhanced Recombinant Protein Production Under Special Environmental Stress

Cited by 23 publications

References 102 publications

An Escherichia coli Expressed Multi-Disulfide Bonded SARS-CoV-2 RBD Shows Native-like Biophysical Properties and Elicits Neutralizing Antisera in a Mouse Model

An Escherichia coli Expressed Multi-Disulfide Bonded SARS-CoV-2 RBD Shows Native-like Biophysical Properties and Elicits Neutralizing Antisera in a Mouse Model

Innovative Bioprocess Strategies Combining Physiological Control and Strain Engineering of Pichia pastoris to Improve Recombinant Protein Production

CRISPR/Cas9-mediated point mutations improve α-amylase secretion in Saccharomyces cerevisiae

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