Cellular response to accumulation of recombinant proteins in the E. coli inner membrane: Implications for proteolysis and productivity of the secretory expression system

2008 ◽  
Vol 39 (1) ◽  
pp. 74-83 ◽  
Author(s):  
Balaji Balagurunathan ◽  
Guhan Jayaraman
Keyword(s):  
Recombinant Proteins ◽  
Cellular Response ◽  
Expression System ◽  
Secretory Expression ◽  
Inner Membrane ◽  
E Coli

Rapid E. coli-based cloning and expression system for production of recombinant proteins

2014 ◽  
Vol 185 ◽  
pp. S70
Author(s):  
Boguslaw Lupa ◽  
Krzysztof Stawujak ◽  
Igor Rozanski ◽  
Justyna Stec-Niemczyk
Keyword(s):  
Recombinant Proteins ◽  
Expression System ◽  
Cloning And Expression ◽  
E Coli

scholarly journals High efficient extracellular production of recombinant Streptomyces PMF phospholipase D in Escherichia coli

2020 ◽  
Author(s):  
Jing Wang ◽  
Sheng Xu ◽  
Yang Pang ◽  
Xin Wang ◽  
Kequan Chen ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Signal Peptide ◽  
Large Scale ◽  
Expression System ◽  
Cost Effective ◽  
Scale Production ◽  
Secretory Expression ◽  
E Coli ◽  
Cost Effective Method ◽  
High Efficient

Abstract Background Currently, Streptomyces is widely used in the preparation of phospholipase D (PLD) with high transphosphatidylation activity. However, the yield of PLD from Streptomyces was low and the culture period was long. Therefore, an efficient and cost-effective method is needed urgently.Results Firstly, PLDs from Streptomyces PMF and Streptomyces racemochromogenes were separately over-expressed in E. coli to compare their transphosphatidylation activity based on the synthesis of phosphatidylserine (PS), and PLDPMF was determined to have higher activity. To further improve PLDPMF synthesis, a secretory expression system suitable for PLDPMF was constructed and optimized with different signal peptides. The highest secretory efficiency was observed when the PLDPMF gene was expressed together with its native signal peptide (Nat) and the signal peptide PelB from E. coli. For the application of recombinant PLD to PS synthesis, the PLD properties were characterized and 30.2 g/L of PS was produced after 24 h of bioconversion when 50 g/L phosphatidylcholine (PC) was added.Conclusions We succeeded in over-expressing PLD from Streptomyces PMF in E. coli with high transphosphatidylation activity and enhanced the yield by secretory expression. The secreted PLD was successfully used in the production of PS. Our work makes the large-scale production of PLD and PS feasible.

scholarly journals Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Fei Du ◽  
Yun-Qi Liu ◽  
Ying-Shuang Xu ◽  
Zi-Jia Li ◽  
Yu-Zhou Wang ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Rna Polymerase ◽  
Recombinant Proteins ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
Expression Level ◽  
Foreign Protein ◽  
Atpase Subunit ◽  
E Coli ◽  
Prokaryotic Expression System

AbstractEscherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (PlacUV5), which is leakier and more active than wild-type lac promoter (PlacWT) under certain growth conditions. These characteristics are not advantageous for the production of those recombinant proteins with toxic or growth-burdened. On the one hand, leakage expression of T7 RNAP leads to rapid production of target proteins under non-inducing period, which sucks resources away from cellular growth. Moreover, in non-inducing or inducing period, high expression of T7 RNAP production leads to the high-production of hard-to-express proteins, which may all lead to loss of the expression plasmid or the occurrence of mutations in the expressed gene. Therefore, more BL21 (DE3)-derived variant strains with rigorous expression and different expression level of T7 RNAP should be developed. Hence, we replaced PlacUV5 with other inducible promoters respectively, including arabinose promoter (ParaBAD), rhamnose promoter (PrhaBAD), tetracycline promoter (Ptet), in order to optimize the production of recombinant protein by regulating the transcription level and the leakage level of T7 RNAP. Compared with BL21 (DE3), the constructed engineered strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the production of glucose dehydrogenase (GDH), a protein that causes host autolysis, the engineered strain BL21 (DE3::ara) exhibited higher biomass, cell survival rate and foreign protein expression level than that of BL21 (DE3). In addition, these engineered strains had been successfully applied to improve the production of membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and the E. coli F-ATPase subunit b (Ecb). The engineered strains constructed in this paper provided more host choices for the production of recombinant proteins.

scholarly journals Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21

2021 ◽  
Vol 12 ◽  
Author(s):  
Gema Lozano Terol ◽  
Julia Gallego-Jara ◽  
Rosa Alba Sola Martínez ◽  
Adrián Martínez Vivancos ◽  
Manuel Cánovas Díaz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Copy Number ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
E Coli

Recombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (PT7lac, Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB1′ and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.

scholarly journals Expression comparison between two genes encoding CSF3 recombinant proteins having different codon composition at N-terminal in Escherichia coli

2021 ◽  
Vol 948 (1) ◽  
pp. 012081
Author(s):  
K S Dewi ◽  
F D Wahyuni ◽  
S Salsabila ◽  
Aminah ◽  
N D Yanthi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Recombinant Plasmid ◽  
Synonymous Codon ◽  
Expression System ◽  
Negative Control ◽  
Translation Initiation Region ◽  
E Coli ◽  
Genes Encoding

Abstract Colony-stimulating factor 3 (CSF3) is a glycoprotein with many therapeutic applications. In the Escherichia coli expression system, mRNA folding and stability near the translation initiation region (TIR) are known to influence protein expression significantly. We have successfully constructed the recombinant plasmid carrying genes encoding CSF3.1 and CSF3.2, which have different synonymous codon usage at N-terminal. In this study, we compared both expressions of CSF3.1 and CSF3.2 recombinant proteins in E. coli host. Recombinant plasmid pJ414-CSF3.1 and pJ414-CSF3.2 were transformed individually into E. coli NiCo21(DE3) competent cells by a heat-shock method, then spread on solid Lysogeny Broth (LB) medium containing ampicillin. Eight transformant colonies were selected and then expressed in 2xYT medium with the addition of IPTG inducer. Expression analysis was carried out using 15% SDS-PAGE gel. No significantly different band was observed in CSF3.1 protein expression compared to the negative control. In contrast, CSF3.2 protein can be expressed with a good amount at its expected size of 18 kDa. This result was strengthened by bioinformatics analysis which demonstrated the more open TIR of CSF3.2 than that of CSF3.1 Our study highlighted that AU-rich mRNA at the N-terminal is essential for efficient recognition of the ribosome binding site.

scholarly journals Highly Efficient Extracellular Production of Recombinant Streptomyces PMF Phospholipase D in Escherichia coli

Catalysts ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1057
Author(s):  
Jing Wang ◽  
Sheng Xu ◽  
Yang Pang ◽  
Xin Wang ◽  
Kequan Chen ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Signal Peptide ◽  
Expression System ◽  
Signal Peptides ◽  
Extracellular Production ◽  
Secretory Expression ◽  
E Coli ◽  
Fusion Signal ◽  
Optimized Conditions ◽  
Native Signal Peptide

To achieve efficient bio-production of phospholipase D (PLD), PLDs from different organisms were expressed in E.coli. An efficient secretory expression system was thereby developed for PLD. First, PLDs from Streptomyces PMF and Streptomyces racemochromogenes were separately over-expressed in E.coli to compare their transphosphatidylation activity based on the synthesis of phosphatidylserine (PS), and PLDPMF was determined to have higher activity. To further improve PLDPMF synthesis, a secretory expression system suitable for PLDPMF was constructed and optimized with different signal peptides. The highest secretory efficiency was observed when the PLD * (PLDPMF with the native signal peptide Nat removed) was expressed fused with the fusion signal peptide PelB-Nat in E. coli. The fermentation conditions were also investigated to increase the production of recombinant PLD and 10.5 U/mL PLD was ultimately obtained under the optimized conditions. For the application of recombinant PLD to PS synthesis, the PLD properties were characterized and 30.2 g/L of PS was produced after 24 h of bioconversion when 50 g/L phosphatidylcholine (PC) was added.

scholarly journals Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters

2010 ◽  
Vol 9 (1) ◽  
pp. 18 ◽  
Author(s):  
Norma A Valdez-Cruz ◽  
Luis Caspeta ◽  
Néstor O Pérez ◽  
Octavio T Ramírez ◽  
Mauricio A Trujillo-Roldán
Keyword(s):  
Recombinant Proteins ◽  
Expression System ◽  
Inducible Expression ◽  
Phage Lambda ◽  
E Coli ◽  
Inducible Expression System

Allergen homologues, pathogenesis-related 1, polygalacturonase, and pectin methyl esterase from Japanese hop

2020 ◽  
Vol 27 ◽  
Author(s):  
Seok Woo Jang ◽  
Kyoung Yong Jeong ◽  
Ji Eun Yuk ◽  
Jongsun Lee ◽  
Kyung Hee Park ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Expression System ◽  
Allergic Diseases ◽  
Nitrilotriacetic Acid ◽  
Cross Reactivity ◽  
Molecular Characteristics ◽  
Pectin Methyl Esterase ◽  
E Coli ◽  
Ige Reactivity ◽  
Pathogenesis Related

Background: Japanese hop is an important cause of weed pollinosis in East Asia. Its pollen is abundant in autumn. This pollen is known to be the cause of many allergic diseases. However, molecular characteristics of its allergens have not been elucidated. Objective: In this study, we produced recombinant proteins of allergen homologues from Japanese hop by the analysis of expressed sequence tags (EST), and evaluated its allergenicity. Methods: cDNA library was constructed using as little as 50 ng of total RNA from Japanese hop pollen. Allergen homologues were identified by the initial screening of 963 EST clones. Recombinant proteins were overexpressed in the E. coli expression system and purified using Ni-nitrilotriacetic acid-agarose. Purified proteins were analyzed by ELISA. Results: Japanese hop pathogenesis-related 1 protein (PR-1) shares 37.0 to 44.4% of amino acid sequence identity with Art v 2, Cuc m 3, and Cyn d 24. Pectin methyl esterase (PME) shows 23.2 to 50.2% of identities to Act d 7, Ole e 11, and Sal k 1. Polygalacturonase (PGs) shows 16.7 to 19.3% of identities to Phl p 13, Cry j 2, Cha o 2, Jun a 2, Pla a 2, and Pla or 2. IgE antibodies from Japanese hop allergy patients’ sera recognized PR-1 (3.4%), PME (13.8%), PGs (3.7%), and profilin (13.8%), respectively. Conclusion: Novel allergenic components were identified, even though low IgE reactivity was displayed reflecting the low degree of cross-reactivity with other pollen allergens. We believe that these molecules have worth further studies.

scholarly journals Recent Developments of Recombinant Protein Expression for Therapeutic Purposes

2021 ◽  
Vol 3 (4) ◽  
Author(s):  
Anam Amir
Keyword(s):  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Expression System ◽  
Cell Penetrating Peptides ◽  
Ovary Cell ◽  
Expression Systems ◽  
E Coli ◽  
Mammalian Expression ◽  
Mammalian Expression System ◽  
High Level

In the most recent seven to eight years, the therapeutic recombinant proteins have rapidly expanded in the biotechnology domain due to its wide variety of needs. There has been significant development in the mammalian expression system for fine purification and increased level of expressed recombinant proteins [1,2]. Many drugs like tetracycline have been demonstrated on the Chinese Hamster Ovary cell line for promising multi control strategies and effective cytotoxicity. Mammalian expression system improves the proper glycosylation of recombinant proteins which are very helpful to increase solubility of product [3-6].             Meanwhile on the prokaryotic expression system, E. coli has proven to be an easier to handle, friendly and economical strain [2]. Recently these expression systems are using to work on antibody fragment productions and their proper folding with co-expression of chaperones [7]. Moreover E. coli has been used for the production of cancer cell penetrating peptides which promises the targeted delivery of drugs to specific effector cells only.  Yeast systems are also being used for the antibody fragments production and the high level production of insulin. Interestingly cell free expression systems are also participating in this game and that would be very fascinating to see in the coming years about cell extract medium for production of high level recombinant protein [8, 9]. Purification and optimization of recombinant protein has always been a challenging situation for scientists and they paid more attention to increase the overall yield of the product. Many affinity chromatography techniques has been introduced for efficient purification of protein of interest [10]. Despite these research and developments in methodologies to produce and purify the recombinant therapeutic protein, scientists still face the hurdles and challenges with all expression systems. Rationally E. coli produces inclusion bodies and many mammalian cell types do not show the same results with the same recombinant protein. [11]. So there is a requirement for adding the appropriate features to the expression systems focused to better improvising recovery, production and purification of recombinant protein. Copyright(c) The Author

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