Optimization of recombinant protein expression level inEscherichia coli by flow cytometry and cell sorting

Abstract Background: Corynebacterium glutamicum is a traditional food-grade industrial microorganism， in which an efficient endotoxin-free recombinant protein expression factory is under developing in recent years. However, the intrinsic disadvantage of low recombinant protein expression level is still difficult to be solved. Here, according to the bacteria-specific polycistronic feature that multiple proteins can be translated in one mRNA, efforts have been made to insert a leading peptide gene upstream of target genes as an expression enhancer, and it is found that this can remarkably improve the expression level of proteins under the control of inducible tac promoter in C. glutamicum. Results: In this research, the Escherichia coli ( E. coli ) tac promoter combined with 24 different fore-cistron sequences were constructed in a bicistronic manner in C. glutamicum. Three strong bicistronic expression vectors were isolated and exhibited high efficiency under different culture conditions. The compatibility of these bicistronic vectors was further validated using six model proteins- aldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), RamA (regulator of acetate metabolism), Bovine interferon-a (BoIFN-a), glycoprotein D protein (gD) of infectious bovine rhinotracheitis virus (IBRV) and procollagen type I N-terminal peptide (PINP). All examined proteins were highly expressed compared with the original vector with tac promoter. Large-scale production of PINP was also performed in fed-batch cultivation, and the highest PINP production level was 1.2 g/L. Conclusion: In this study, the strength of the inducible tac promoter for C. glutamicum was improved by screening and inserting fore-cistron sequences in front of the target genes. Those vectors with bicistronic expression patterns have strong compatibility for expressing various heterogeneous proteins in high yield. This new strategy could be used to further improve the performance of inducible promoters, achieving double competence of inducible control and high yield.

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Recombinant protein expression and solubility screening inEscherichia coli: a comparative study

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444906031337 ◽

2006 ◽

Vol 62 (10) ◽

pp. 1218-1226 ◽

Cited By ~ 89

Author(s):

Nick S. Berrow ◽

K. Büssow ◽

B. Coutard ◽

J. Diprose ◽

M. Ekberg ◽

...

Keyword(s):

Recombinant Protein ◽

Protein Expression ◽

Comparative Study ◽

Recombinant Protein Expression ◽

Inescherichia Coli

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OR3 HLA-DP protein expression level and its impact on flow cytometry crossmatch (FXM)

Human Immunology ◽

10.1016/j.humimm.2019.07.005 ◽

2019 ◽

Vol 80 ◽

pp. 9-10

Author(s):

Lin Wang ◽

Ge Chen ◽

Marcelo Fernandez-Vina

Keyword(s):

Flow Cytometry ◽

Protein Expression ◽

Expression Level ◽

Protein Expression Level

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Comprehensive Analysis of ESRRA in Endometrial Cancer

Technology in Cancer Research & Treatment ◽

10.1177/1533033821992083 ◽

2021 ◽

Vol 20 ◽

pp. 153303382199208

Author(s):

Shufang Wang ◽

Xinlong Huo

Keyword(s):

Endometrial Cancer ◽

Protein Expression ◽

Immune Cell ◽

Enrichment Analysis ◽

Gene Set Enrichment Analysis ◽

The Cancer Genome Atlas ◽

Expression Level ◽

Operating Characteristics ◽

Protein Expression Level ◽

Normal Tissues

Background: Estrogen-related receptor alpha (ESRRA) was reported to play an important role in multiple biological processes of neoplastic diseases. The roles of ESRRA in endometrial cancer have not been fully investigated yet. Methods: Expression data and clinicopathological data of patients with uteri corpus endometrial carcinoma (UCEC) were obtained from The Cancer Genome Atlas (TCGA). Comprehensive bioinformatics analysis was performed, including receiver operating characteristics (ROC) curve analysis, Kaplan-Meier survival analysis, gene ontology (GO) enrichment analysis, and Gene Set Enrichment Analysis (GSEA). Immunohistochemistry was used to detect the protein expression level of ESRRA and CCK-8 assay was performed to evaluate the effect of ESRRA on the proliferation ability. Results: A total of 552 UCEC tissues and 35 normal tissues were obtained from the TCGA database. The mRNA and protein expression level of ESRRA was highly elevated in UCEC compared with normal tissues, and was closely associated with poor prognosis. ROC analysis indicated a very high diagnostic value of ESRRA for patients with UCEC. GO and GSEA functional analysis showed that ESRRA might be mainly involved in cellular metabolism processes, in turn, tumorigenesis and progression of UCEC. Knockdown of ESRRA inhibited the proliferation of UCEC cells in vitro. Further immune cell infiltration demonstrated that ESRRA enhanced the infiltration level of neutrophil cell and reduced that of T cell (CD4+ naïve), NK cell, and cancer associated fibroblast (CAF). The alteration of immune microenvironment will greatly help in developing immune checkpoint therapy for UCEC. Conclusions: Our study comprehensively analyzed the expression level, clinical value, and possible mechanisms of action of ESRRA in UCEC. These findings showed that ESRRA might be a potential diagnostic and therapeutic target.

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