scholarly journals Enhanced production of recombinant proteins in Corynebacterium glutamicum by construction a bicistronic gene expression system

2020 ◽  
Author(s):  
Manman Sun ◽  
Xiong Gao ◽  
Zihao Zhao ◽  
An Li ◽  
Yali Wang ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Corynebacterium Glutamicum ◽  
Target Genes ◽  
Recombinant Protein Expression ◽  
Expression Level ◽  
High Yield ◽  
Type I ◽  
Tac Promoter ◽  
Bicistronic Expression

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.

scholarly journals Enhanced production of recombinant proteins in Corynebacterium glutamicum by construction a bicistronic gene expression system

2020 ◽  
Author(s):  
Manman Sun ◽  
Xiong Gao ◽  
Zihao Zhao ◽  
An Li ◽  
Yali Wang ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Corynebacterium Glutamicum ◽  
Target Genes ◽  
Recombinant Protein Expression ◽  
Acetate Metabolism ◽  
Type I ◽  
Scale Production ◽  
Enhanced Production ◽  
Tac Promoter

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 disadvantages of low recombinant protein expression levels need to be solved. Here, according to the bacteria-specific polycistronic feature, trials have been made of inserting a leading peptide upstream of target genes as an expression enhancer, and we found it improving the expression level of proteins under the control of inducible tac promoter in C. glutamicum CGMCC1.15647 . 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 vectors were isolated and exhibited higher strength 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 of 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. Conclusions: In this study, we improved the strength of the inducible promoter tac promoter for C. glutamicum by screening and inserting fore-cistron in front of the target genes. Those vectors with bicistronic expression pattern have strong compatibility for expressing various heterogeneous proteins in high level. This new strategy could be used to further improve the performance of inducible promoters, achieving double competence of inducible control and high yield.

scholarly journals Transcriptome analysis of Corynebacterium glutamicum in the process of recombinant protein expression in bioreactors

PLoS ONE ◽  
2017 ◽  
Vol 12 (4) ◽  
pp. e0174824 ◽  
Author(s):  
Yang Sun ◽  
Wenwen Guo ◽  
Fen Wang ◽  
Chunjun Zhan ◽  
Yankun Yang ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Corynebacterium Glutamicum ◽  
Transcriptome Analysis ◽  
Recombinant Protein Expression

scholarly journals Choice of selectable marker affects recombinant protein expression in cells and exosomes

2021 ◽  
pp. 100838
Author(s):  
Chenxu Guo ◽  
Francis K. Fordjour ◽  
Shang Jui Tsai ◽  
James C. Morrell ◽  
Stephen J. Gould
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Selectable Marker ◽  
Recombinant Protein Expression ◽  
In Cells

BacMam System for Rapid Recombinant Protein Expression in Mammalian Cells

2019 ◽  
pp. 205-208 ◽  
Author(s):  
Deepak B. Thimiri Govinda Raj ◽  
Niamat Ali Khan ◽  
Srisaran Venkatachalam ◽  
Sivakumar Arumugam
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Mammalian Cells ◽  
Recombinant Protein Expression ◽  
Expression In Mammalian Cells

Evaluation of somatic embryos of alfalfa for recombinant protein expression

2014 ◽  
Vol 34 (2) ◽  
pp. 211-221 ◽  
Author(s):  
Guohua Fu ◽  
Vojislava Grbic ◽  
Shengwu Ma ◽  
Lining Tian
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Somatic Embryos ◽  
Recombinant Protein Expression

scholarly journals Mutation of Putative N-Glycosylation Sites on Dengue Virus NS4B Decreases RNA Replication

2015 ◽  
Vol 89 (13) ◽  
pp. 6746-6760 ◽  
Author(s):  
Nenavath Gopal Naik ◽  
Huey-Nan Wu
Keyword(s):  
Life Cycle ◽  
Recombinant Protein ◽  
Dengue Virus ◽  
Protein Expression ◽  
Nonstructural Protein ◽  
Recombinant Protein Expression ◽  
Rna Replication ◽  
Viral Production ◽  
Glycosylation Sites ◽  
Infected Cells

ABSTRACTDengue virus (DENV) nonstructural protein 4B (NS4B) is an endoplasmic reticulum (ER) membrane-associated protein, and mutagenesis studies have revealed its significance in viral genome replication. In this work, we demonstrated that NS4B is an N-glycosylated protein in virus-infected cells as well as in recombinant protein expression. NS4B is N glycosylated at residues 58 and 62 and exists in two forms, glycosylated and unglycosylated. We manipulated full-length infectious RNA clones and subgenomic replicons to generate N58Q, N62Q, and N58QN62Q mutants. Each of the single mutants had distinct effects, but the N58QN62Q mutation resulted in dramatic reduction of viral production efficiency without affecting secretion or infectivity of the virion in mammalian and mosquito C6/36 hosts. Real-time quantitative PCR (qPCR), subgenomic replicon, andtrans-complementation assays indicated that the N58QN62Q mutation affected RNA replication possibly by the loss of glycans. In addition, four intragenic mutations (S59Y, S59F, T66A, and A137T) were obtained from mammalian and/or mosquito C6/36 cell culture systems. All of these second-site mutations compensated for the replication defect of the N58QN62Q mutant without creating novel glycosylation sites.In vivoprotein stability analyses revealed that the N58QN62Q mutation alone or plus a compensatory mutation did not affect the stability of NS4B. Overall, our findings indicated that mutation of putative N-glycosylation sites affected the biological function of NS4B in the viral replication complex.IMPORTANCEThis is the first report to identify and reveal the biological significance of dengue virus (DENV) nonstructural protein 4B (NS4B) posttranslation N-glycosylation to the virus life cycle. The study demonstrated that NS4B is N glycosylated in virus-infected cells and in recombinant protein expression. NS4B is modified by glycans at Asn-58 and Asn-62. Functional characterization implied that DENV NS4B utilizes the glycosylation machinery in both mammalian and mosquito hosts. Four intragenic mutations were found to compensate for replication and subsequent viral production deficiencies without creating novel N-glycosylation sites or modulating the stabilities of the protein, suggesting that glycans may be involved in maintaining the NS4B protein conformation. NS4B glycans may be necessary elements of the viral life cycle, but compensatory mutations can circumvent their requirement. This novel finding may have broader implications in flaviviral biology as the most likely glycan at Asn-62 of NS4B is conserved in DENV serotypes and in some related flaviviruses.

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