Recombinant protein expression in Escherichia coli: advances and challenges

Rosano, Germán L.; Ceccarelli, Eduardo A.

doi:10.3389/fmicb.2014.00172

Cited by 1,831 publications

(1,471 citation statements)

References 222 publications

Supporting

Mentioning

1,333

Contrasting

Unclassified

Order By: Relevance

“…Human IL-2 is folded by three Cys residues including Cys-58, Cys-105, and Cys-125, which needs oxidation environment to formation (22). Therefore, nascent proteins should be translocated to periplasm of the bacteria as oxidative compartment (23). E. coli uses Sec, TAT, and SPR pathways to translocate nascent proteins to periplasm based on signal peptides at N terminal of the protein.…”

Section: Discussionmentioning

confidence: 99%

Engineering, Cloning and Expression of DNA Sequence Coding of OMP31 Epitope of Brucella melitensis linked to IL-2 in Escherichia coli

et al. 2018

View full text Add to dashboard Cite

Brucellosis is a zoonetic disease that is caused by Brucella melitensis, which affects human health and animal husbandry industry. Omp31 is the most exposed outer membrane proteins (OMPs) and is a dominant antigen in smooth strains of B. melitensis, thus it is considered a desirable protein in subunit vaccine against brucellosis disease. Besides, IL-2 acts as a growth and differentiation stimulator of T-cells in cellular immune responses, therefore, it can serve as an adjuvant in subunit vaccines against intracellular pathogens. It seems that chimera scaffold, consisting of some copies of OMP31 experimental epitope region and IL-2, can provide a good immunization. In this regard, these tow fragments are linked together through using rigid linker and splicing overlap extension (SOE) PCR technique. Successful PCR products were TA cloned and then sub-cloned into pET-22b (+) vector as an expression vector. Recombinant protein was expressed in Escherichia coli BL21 (DE3) as an expressing lineage bacterium and then confirmed by SDS-PAGE and Western-blotting analysis. Secondary and tertiary structure prediction of recombinant protein and its immunity characterization were evaluated using in silico process. Further studies on the immunogenicity properties of this protein are expected to be carried out in the future to confirm the results in vivo.

show abstract

Section: Discussionmentioning

confidence: 99%

Engineering, Cloning and Expression of DNA Sequence Coding of OMP31 Epitope of Brucella melitensis linked to IL-2 in Escherichia coli

et al. 2018

View full text Add to dashboard Cite

show abstract

“…17, 18, 19, 20 However, the development of effective recombinant chimeras is often laborious;17 bioactivities can be reduced;21 and there are limited opportunities through biosynthesis to further improve pharmacokinetic parameters and therapeutic efficacy such as the attachment of stabilizing polymers or fusion of multiple copies of biomolecules, particularly cyclic peptides 22, 23, 24, 25, 26. Even though synthetic approaches have managed to overcome some of these technological hurdles, the application of chemical strategies alone may be insufficient to prepare multidomain protein conjugates with structural precision and bioactivity 27, 28.…”

Section: Introductionmentioning

confidence: 99%

Boosting Antitumor Drug Efficacy with Chemically Engineered Multidomain Proteins

et al. 2018

View full text Add to dashboard Cite

A facile chemical approach integrating supramolecular chemistry, site‐selective protein chemistry, and molecular biology is described to engineer synthetic multidomain protein therapeutics that sensitize cancer cells selectively to significantly enhance antitumor efficacy of existing chemotherapeutics. The desired bioactive entities are assembled via supramolecular interactions at the nanoscale into structurally ordered multiprotein complexes comprising a) multiple copies of the chemically modified cyclic peptide hormone somatostatin for selective targeting and internalization into human A549 lung cancer cells expressing SST‐2 receptors and b) a new cysteine mutant of the C3bot1 (C3) enzyme from Clostridium botulinum, a Rho protein inhibitor that affects and influences intracellular Rho‐mediated processes like endothelial cell migration and blood vessel formation. The multidomain protein complex, SST3‐Avi‐C3, retargets C3 enzyme into non‐small cell lung A549 cancer cells and exhibits exceptional tumor inhibition at a concentration ≈100‐fold lower than the clinically approved antibody bevacizumab (Avastin) in vivo. Notably, SST3‐Avi‐C3 increases tumor sensitivity to a conventional chemotherapeutic (doxorubicin) in vivo. These findings show that the integrated approach holds vast promise to expand the current repertoire of multidomain protein complexes and can pave the way to important new developments in the area of targeted and combination cancer therapy.

show abstract

“…rapid transformation process, high yield of expression, and the whole expression is less expensive in comparison with other hosts (Rosano and Ceccarelli, 2014). However, the higher expression rates of recombinant proteins in E. coli are often accompanied with the formation of insoluble aggregates of the target protein called inclusion bodies (IBs) (Singh et al, 2015).…”

Section: Primermentioning

confidence: 99%