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

Fasciola gigantica cathepsin (cysteine) proteases are potential diagnostic antigens for animal and human fasciolosis. These include cathepsin-L proteases that have been exploited in the diagnosis of animal fasciolosis. However, no scientific data on the diagnostic potential of F. gigantica cathepsin B proteases is available. Therefore, three recombinant antigens of F. gigantica viz. cathepsin (cat) B-1, cat B-2 and cat B-3 were expressed in prokaryotic expression system. The recombinant antigens were purified under denaturing conditions by Nickel affinity chromatography and an optimal level of the recombinant proteins was obtained. These recombinant proteins will further be evaluated for their potential in the early prepatent diagnosis of F. gigantica infection in domestic ruminants.

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Expression in Escherichia coli of human ARHGAP6 gene and purification of His-tagged recombinant protein.

Acta Biochimica Polonica ◽

10.18388/abp.2003_3732 ◽

2003 ◽

Vol 50 (1) ◽

pp. 239-247 ◽

Cited By ~ 3

Author(s):

Anna-Maria Ochocka ◽

Marzena Czyzewska ◽

Tadeusz Pawełczyk

Keyword(s):

Escherichia Coli ◽

Fusion Protein ◽

Rho Gtpase ◽

Expression System ◽

Cloning And Expression ◽

Soluble Form ◽

E Coli ◽

Escherichia Coli Cells ◽

Step Procedure ◽

Shock Proteins

In this report we describe cloning and expression of human Rho GTPase activating protein (ARHGAP6) isoform 4 in Escherichia coli cells as a fusion protein with 6xHis. We cloned the ARHGAP6 cDNA into the bacterial expression vector pPROEX-1. Induction of the 6xHis-ARHGAP6 protein in BL21(DE3) and DH5alpha cells caused lysis of the cells irrespective of the kind of culture medium used. Successful expression of the fusion protein was obtained in the MC4100Deltaibp mutant strain lacking the small heat-shock proteins IbpA and IbpB. Reasonable yield was obtained when the cells were cultured in Terrific Broth + 1% glucose medium at 22 degrees C for 16 h. The optimal cell density for expression of soluble 6xHis-ARHGAP6 protein was at A(600) about 0.5. Under these conditions over 90% of the fusion protein was present in a soluble form. The 6xHis-ARHGAP6 protein was purified to near homogeneity by a two step procedure comprising chromatography on Ni-nitrilotriacetate and cation exchange columns. The expression system and purification procedure employed made it possible to obtain 1-2 mg of pure 6xHis-ARHGAP6 protein from 300 ml (1.5 g of cells) of E. coli culture.

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

Microbial Cell Factories ◽

10.1186/s12934-021-01680-6 ◽

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.

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Cloning and expression of LTB in Escherichia coli and Bacillus subtilis

Science and Technology Development Journal ◽

10.32508/stdj.v16i1.1392 ◽

2013 ◽

Vol 16 (1) ◽

pp. 13-22 ◽

Cited By ~ 1

Author(s):

Trang Thi Phuong Phan ◽

Anh Le Tuan Nguyen ◽

Hoang Duc Nguyen

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Fusion Protein ◽

Expression System ◽

Pharmaceutical Products ◽

Cloning And Expression ◽

Gene Encoding ◽

E Coli ◽

B Subunit ◽

The Common

LTB is the B subunit of heat labile toxins (LT) in Escherichia coli ETEC. This subunit is non-toxic but has a high immune response. Therefore, LTB is considered a suitable antigen for partial vaccine against the diarrhea caused by E. coli ETEC. The most important component of partial vaccine is antigen protein. Nowadays, with the advancement of recombinant protein technology, these antigens are mainly produced by the common bacterial expression system as E. coli. However, the recombinant proteins produced by E. coli are often miscellaneous with enterotoxins, which should be removed from pharmaceutical products. Thus, the production of antigen proteins in other expression system without endotoxins like Bacillus subtilis is in attention. We conducted the experiments of cloning and expressing LTB using a novel pHT plasmid that allow the protein to be expressed in both of E. coli and B. subtilis. We were successful to generate plasmid pHT326 and express the gene encoding for the fusion protein of LTB and LysSN-6xHis-TEV in B. subtilis and E. coli. The binding of fusion protein on the columns that have affinity with His-tag was confirmed. This result is about to be applied for the development of partial vaccine aganst the diarrhea as well as the development of some diagnostic kits for ETEC in food or medical waste and kits to detect antibodies against LTB in animals.

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

Frontiers in Microbiology ◽

10.3389/fmicb.2021.682001 ◽

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.

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Cloning and Expression of the Benzoate Dioxygenase Genes from Rhodococcus sp. Strain 19070

Applied and Environmental Microbiology ◽

10.1128/aem.67.6.2507-2514.2001 ◽

2001 ◽

Vol 67 (6) ◽

pp. 2507-2514 ◽

Cited By ~ 29

Author(s):

Sandra Haddad ◽

D. Matthew Eby ◽

Ellen L. Neidle

Keyword(s):

Reductase Activity ◽

Expression System ◽

Open Reading Frames ◽

Cloning And Expression ◽

Positive Bacterium ◽

E Coli ◽

C Terminus ◽

Genes Encoding ◽

Benzoate Dioxygenase ◽

Reading Frames

ABSTRACT The bopXYZ genes from the gram-positive bacteriumRhodococcus sp. strain 19070 encode a broad-substrate-specific benzoate dioxygenase. Expression of the BopXY terminal oxygenase enabled Escherichia coli to convert benzoate or anthranilate (2-aminobenzoate) to a nonaromaticcis-diol or catechol, respectively. This expression system also rapidly transformed m-toluate (3-methylbenzoate) to an unidentified product. In contrast, 2-chlorobenzoate was not a good substrate. The BopXYZ dioxygenase was homologous to the chromosomally encoded benzoate dioxygenase (BenABC) and the plasmid-encoded toluate dioxygenase (XylXYZ) of gram-negative acinetobacters and pseudomonads. Pulsed-field gel electrophoresis failed to identify any plasmid inRhodococcus sp. strain 19070. Catechol 1,2- and 2,3-dioxygenase activity indicated that strain 19070 possesses bothmeta- and ortho-cleavage degradative pathways, which are associated in pseudomonads with the xyl andben genes, respectively. Open reading frames downstream ofbopXYZ, designated bopL and bopK, resembled genes encoding cis-diol dehydrogenases and benzoate transporters, respectively. The bop genes were in the same order as the chromosomal ben genes of P. putida PRS2000. The deduced sequences of BopXY were 50 to 60% identical to the corresponding proteins of benzoate and toluate dioxygenases. The reductase components of these latter dioxygenases, BenC and XylZ, are 201 residues shorter than the deduced BopZ sequence. As predicted from the sequence, expression of BopZ in E. coli yielded an approximately 60-kDa protein whose presence corresponded to increased cytochrome c reductase activity. While the N-terminal region of BopZ was approximately 50% identical in sequence to the entire BenC or XylZ reductases, the C terminus was unlike other known protein sequences.

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

IOP Conference Series Earth and Environmental Science ◽

10.1088/1755-1315/948/1/012081 ◽

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.

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

Microbial Cell Factories ◽

10.1186/1475-2859-9-18 ◽

2010 ◽

Vol 9 (1) ◽

pp. 18 ◽

Cited By ~ 83

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

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

Protein and Peptide Letters ◽

10.2174/0929866527666200813201924 ◽

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.

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