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 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 Overview of Bacterial and Yeast Systems for Protein Expression

2021 ◽  
pp. 113-118
Author(s):  
Maheswara Reddy Mallu ◽  
Siva Reddy Golamari ◽  
Sree Rama Chandra Karthik Kotikalapudi ◽  
Renuka Vemparala
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Large Scale ◽  
Recombinant Protein Expression ◽  
Expression Vectors ◽  
Scale Production ◽  
Protein Coding ◽  
Recombinant Gene Expression ◽  
Vector Systems ◽  
Large Scale Production

Over the past decade the variety of hosts and vector systems for recombinant protein expression has increased dramatically. Researchers now select from among mammalian, insect, yeast, and prokaryotic hosts, and the number of vectors available for use in these organisms continues to grow. With the increased availability of cDNAs and protein coding sequencing information, it is certain that these and other, yet to be developed systems will be important in the future. Despite the development of eukaryotic systems, E. coli remains the most widely used host for recombinant protein expression. Optimization of recombinant protein expression in prokaryotic and eukaryotic host systems has been carried out by varying simple parameters such as expression vectors, host strains, media composition, and growth temperature. Recombinant gene expression in eukaryotic systems is often the only viable route to the large-scale production of authentic, post translationally modified proteins. It is becoming increasingly easy to find a suitable system to overexpress virtually any gene product, provided that it is properly engineered into an appropriate expression vector.

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 Reverse Engineering Targets for Recombinant Protein Production in Corynebacterium glutamicum Inspired by a Fast-Growing Evolved Descendant

2020 ◽  
Vol 8 ◽  
Author(s):  
Min Ju Lee ◽  
Jihoon Park ◽  
Kyunghoon Park ◽  
Jihyun F. Kim ◽  
Pil Kim
Keyword(s):  
Recombinant Protein ◽  
Reverse Engineering ◽  
Corynebacterium Glutamicum ◽  
Point Mutation ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Recombinant Protein Expression ◽  
Enhanced Production ◽  
Cell Reproduction ◽  
Ancestral Strain

We previously reported a Corynebacterium glutamicum JH41 strain with a 58% faster growth rate through application of adaptive laboratory evolution. To verify that the fast-reproducing strain was useful as a host for recombinant protein expression, we introduced a plasmid responsible for the secretory production of a recombinant protein. The JH41 strain harboring the plasmid indeed produced the secretory recombinant protein at a 2.7-fold greater rate than its ancestral strain. To provide the reverse engineering targets responsible for boosting recombinant protein production and cell reproduction, we compared the genome sequence of the JH41 strain with its ancestral strain. Among the 15 genomic variations, a point mutation was confirmed in the 14 bases upstream of NCgl1959 (encoding a presumed siderophore-binding protein). This mutation allowed derepression of NCgl1959, thereby increasing iron consumption and ATP generation. A point mutation in the structural gene ramA (A239G), a LuxR-type global transcription regulator involved in central metabolism, allowed an increase in glucose consumption. Therefore, mutations to increase the iron and carbon consumption were concluded as being responsible for the enhanced production of recombinant protein and cell reproduction in the evolved host.

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.

Reduction of adenovirus E1A mRNA by RNAi results in enhanced recombinant protein expression in transiently transfected HEK293 cells

Gene ◽  
2004 ◽  
Vol 341 ◽  
pp. 227-234 ◽  
Author(s):  
David L. Hacker ◽  
Martin Bertschinger ◽  
Lucia Baldi ◽  
Florian M. Wurm
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Protein Expression ◽  
Hek293 Cells ◽  
Adenovirus E1a
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