Protein fusion tags for efficient expression and purification of recombinant proteins in the periplasmic space of E. coli

Bm-TFF2 was isolated from Bombina maxima. The B.maxima Trefoil factors 2 Variant (vBm-TFF2) could be developed as a novel administered wound healing agent. The challenge is its preparation from refolding insoluble protein expressed in Escherichia coli. A recombinant vBm-TFF2 was overexpressed in E. coli cells as a fusion protein with bacterial N-utilizing substance A (NusA). The fusion protein NusA-Bm-TFF2 can promote the wound healing.

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Expression and purification of four truncated 56 kDa antigens from Orientia tsutsugamushi in Escherichia coli

Vietnam Journal of Biotechnology ◽

10.15625/1811-4989/16/3/12929 ◽

2018 ◽

Vol 16 (3) ◽

pp. 533-541

Author(s):

Le Thi Lan Anh ◽

Trinh Van Toan ◽

Pham Thi Ha Giang ◽

Bui Thi Thanh Nga ◽

Vo Viet Cuong ◽

...

Keyword(s):

Recombinant Proteins ◽

Inclusion Bodies ◽

Scrub Typhus ◽

Febrile Illness ◽

Affinity Column ◽

Orientia Tsutsugamushi ◽

Expression And Purification ◽

E Coli ◽

Clinical Presentations ◽

Insoluble Form

Scrub typhus is an acute febrile illness caused by Orientia tsutsugamushi, transmitted to humans by the bite of the larva of trombiculid mites. Diagnosis of scrub typhus is normally based on the clinical presentations. However, it is difficult to differentiate scrub typhus from other acute febrile illnesses, such as dengue fever, malaria and leptospirosis due to similar symptoms. For differential diagnosis of scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. In order to produce an ELISA kit for detection of antibodies against O. tsutsugamushi in Vietnam, four truncated 56 kDa antigenic genes of O. tsutsugamushi including Karp (HT-09), Gilliam (HT-11), TA763 (HT-49), and Kato (YB-50) íolated from the most prevalent cases in Vietnam were cloned and expressed in E. coli Rosetta 1 cells. The recombinant proteins formed inclusion bodies when expressed in E. coli. The recombinant 56 kDa proteins in insoluble form were solubilized in 6M urea and were successfully purified by Ni2+affinity column. The purity of four recombinant proteins,HT-09, HT-11, HT-49 and YB-50,reached more than 95% and their concentrations are 12,57 mg/ml; 11,6 mg/ml; 8,98 mg/ml và 8,02 mg/ml, respectively.

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Expression, Purification, and Functional Analysis of Novel RelE Operon fromX. nematophila

The Scientific World JOURNAL ◽

10.1155/2014/428159 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 2

Author(s):

Jitendra Singh Rathore ◽

Lalit Kumar Gautam

Keyword(s):

Escherichia Coli ◽

Functional Analysis ◽

Recombinant Proteins ◽

Bacterial Toxin ◽

Type Ii ◽

Xenorhabdus Nematophila ◽

Expression And Purification ◽

Toxicity Assay ◽

Neutralization Activity ◽

E Coli

Bacterial toxin-antitoxin (TA) complexes induce programmed cell death and also function to relieve cell from stress by various response mechanisms.Escherichia coliRelB-RelE TA complex consists of a RelE toxin functionally counteracted by RelB antitoxin. In the present study, a novel homolog of RelE toxin designated as Xn-relE toxin fromXenorhabdus nematophilapossessing its own antitoxin designated as Xn-relEAT has been identified. Expression and purification of recombinant proteins under native conditions with GST and Ni-NTA chromatography prove the existence of novel TA module. The expression of recombinant Xn-relE under tightly regulated ara promoter inE. coliTop 10 cells confirms its toxic nature in endogenous toxicity assay. The neutralization activity in endogenous toxicity assay by Xn-relEAT antitoxin confirms its antidote nature when studying the whole TA operon under ara regulated promoter. This study promotes newly discovered TA module to be regarded as important as other proteins of type II toxin-antitoxin system.

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Efficient Expression and Purification of Cryptochrome1 from Columbia livia in E. coli

Protein and Peptide Letters ◽

10.2174/0929866525666181004112308 ◽

2018 ◽

Vol 25 (11) ◽

pp. 986-995

Author(s):

Xiaoxia Yuan ◽

Wenhao Wang ◽

Wenjian Wu ◽

Jing Wang

Keyword(s):

Expression And Purification ◽

E Coli ◽

Efficient Expression ◽

Columbia Livia

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Cytoplasmic glycoengineering enables biosynthesis of nanoscale glycoprotein assemblies

Nature Communications ◽

10.1038/s41467-019-13283-2 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 12

Author(s):

Hanne L. P. Tytgat ◽

Chia-wei Lin ◽

Mikail D. Levasseur ◽

Markus B. Tomek ◽

Christoph Rutschmann ◽

...

Keyword(s):

Recombinant Proteins ◽

Biomedical Applications ◽

Biological Properties ◽

Cytoplasmic Localization ◽

Periplasmic Space ◽

Site Specific ◽

E Coli ◽

Self Assembling ◽

Mammalian Origin ◽

Bacterial Cytoplasm

AbstractGlycosylation of proteins profoundly impacts their physical and biological properties. Yet our ability to engineer novel glycoprotein structures remains limited. Established bacterial glycoengineering platforms require secretion of the acceptor protein to the periplasmic space and preassembly of the oligosaccharide substrate as a lipid-linked precursor, limiting access to protein and glycan substrates respectively. Here, we circumvent these bottlenecks by developing a facile glycoengineering platform that operates in the bacterial cytoplasm. The Glycoli platform leverages a recently discovered site-specific polypeptide glycosyltransferase together with variable glycosyltransferase modules to synthesize defined glycans, of bacterial or mammalian origin, directly onto recombinant proteins in the E. coli cytoplasm. We exploit the cytoplasmic localization of this glycoengineering platform to generate a variety of multivalent glycostructures, including self-assembling nanomaterials bearing hundreds of copies of the glycan epitope. This work establishes cytoplasmic glycoengineering as a powerful platform for producing glycoprotein structures with diverse future biomedical applications.

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A generic protocol for the expression and purification of recombinant proteins in Escherichia coli using a combinatorial His6-maltose binding protein fusion tag

Nature Protocols ◽

10.1038/nprot.2007.50 ◽

2007 ◽

Vol 2 (2) ◽

pp. 383-391 ◽

Cited By ~ 56

Author(s):

Sreedevi Nallamsetty ◽

David S Waugh

Keyword(s):

Escherichia Coli ◽

Recombinant Proteins ◽

Binding Protein ◽

Maltose Binding Protein ◽

Protein Fusion ◽

Expression And Purification ◽

Fusion Tag ◽

Maltose Binding Protein Fusion

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CONSTRUCTION, EXPRESSION AND PURIFICATION OF BRUCELLA SPP. RECOMBINANT PROTEINS L7/L12 AND SODC IN E. COLI

SERIES OF BIOLOGICAL AND MEDICAL ◽

10.32014/10.32014/2020.2519-1629.9 ◽

2020 ◽

Vol 2 (338) ◽

pp. 20-30

Author(s):

E. T. Tailakova ◽

S. О. Sadikaliyeva ◽

G. O. Shynybekova ◽

A. K. Abubakirova ◽

K. T. Sultankulova ◽

...

Keyword(s):

Recombinant Proteins ◽

Target Genes ◽

Guanidine Hydrochloride ◽

Public Health Problem ◽

Sodium Deoxycholate ◽

Expression And Purification ◽

Important Public Health Problem ◽

E Coli ◽

Wash Buffer ◽

Cellular Immune

Brucellosis is still an important public health problem as long as natural reservoirs of infection exist. Currently, live attenuated vaccines based on strains S19, RB51 and Rev1 are used for the prevention of brucellosis in animals, the main disadvantage of which is virulence for humans. However, animal immunization programs should be implemented to reduce the incidence of humans. The development of safe and effective new generation vaccines using “omix” technology is a promising direction of vaccinology. A number of immunogenic Brucella proteins that elicit both a humoral and cellular immune response has been identified. The aim of these research was to optimize the expression and purification conditions of the Brucella spp. recombinant proteins L7/L12 and SodC. As a result, expressing plasmids pET/Br-L7/L12 and pET/Br-SodC were obtained. The parameters of target genes expression in E. coli were established and the method for purification of recombinant proteins was optimized. Purification of the L7/L12 protein was performed under hybrid conditions on HisPur agarose using a binding buffer containing 6 M guanidine hydrochloride, a wash buffer with 20 mM imidazole and an elution buffer with 300 mM imidazole. Protein SodC was purified under denaturing conditions with the addition of 1 % Triton X-100 and 1 % sodium deoxycholate to the lysis buffer. Inclusions were solubilized with a buffer containing 8 M urea and 5 mM imidazole. The target protein was eluted from HisPur agarose with buffer containing 8 M urea and 100 mM imidazole. The use of modified purification protocols made it possible to obtain purified recombinant proteins with a yield of 13 mg/L for the L7/L12 protein and 10 mg/L for the protein SodC, respectively. The specificity of the proteins was confirmed by a Western blot. Immunization of mice with recombinant proteins led to the production of specific antibodies, the titer of which in ELISA was 1:20480 and 1:20480, respectively.

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Electron Inventory of the Iron-Sulfur Scaffold Complex HypCD Essential in [NiFe]-Hydrogenase Cofactor Assembly

Biochemical Journal ◽

10.1042/bcj20210224 ◽

2021 ◽

Author(s):

Sven T. Stripp ◽

Jonathan Oltmanns ◽

Christina S. Müller ◽

David Ehrenberg ◽

Ramona Schlesinger ◽

...

Keyword(s):

Redox Reactions ◽

Expression And Purification ◽

Metal Centers ◽

E Coli ◽

Iron Sulfur ◽

Reducing Conditions ◽

Redox Active ◽

Purification System ◽

Efficient Expression

The [4Fe-4S] cluster containing scaffold complex HypCD is the central construction site for the assembly of the [Fe](CN)2CO cofactor precursor of [NiFe]-hydrogenase. While the importance of the HypCD complex is well established, not much is known about the mechanism by which the CN– and CO ligands are transferred and attached to the iron ion. We report an efficient expression and purification system producing the HypCD complex from E. coli with complete metal content. This enabled in-depth spectroscopic characterizations. The results obtained by EPR and Mössbauer spectroscopy demonstrate that the [Fe](CN)2CO cofactor and the [4Fe-4S] cluster of the HypCD complex are redox active. The data indicate a potential-dependent interconversion of the [Fe]2+/3+ and [4Fe-4S]2+/+ couple, respectively. Moreover, ATR FTIR spectroscopy reveals potential-dependent disulfide formation, which hints at an electron confurcation step between the metal centers. MicroScale thermophoresis indicates preferable binding between the HypCD complex and its in vivo interaction partner HypE under reducing conditions. Together, these results provide comprehensive evidence for an electron inventory fit to drive multi-electron redox reactions required for the assembly of the CN– and CO ligands on the scaffold complex HypCD.

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