scholarly journals Active Protein Aggregates Produced in Escherichia coli

2011 ◽  
Vol 12 (11) ◽  
pp. 8275-8287 ◽  
Author(s):  
Špela Peternel ◽  
Radovan Komel
Keyword(s):  
Escherichia Coli ◽  
Protein Aggregates ◽  
Active Protein

Recombinant expression of antimicrobial peptides using a novel self-cleaving aggregation tag in Escherichia coli

2014 ◽  
Vol 60 (3) ◽  
pp. 113-120 ◽  
Author(s):  
Chao Luan ◽  
Yong Gang Xie ◽  
Yu Tian Pu ◽  
Hai Wen Zhang ◽  
Fei Fei Han ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Peptides ◽  
Recombinant Expression ◽  
Expression System ◽  
Effective Means ◽  
Protein Aggregates ◽  
The Self ◽  
Active Protein ◽  
Cleavage Reaction ◽  
Amphipathic Peptide

Antimicrobial peptides (AMPs) are part of the innate immune system of complex multicellular organisms. Despite the fact that AMPs show great potential as a novel class of antibiotics, the lack of a cost-effective means for their mass production limits both basic research and clinical use. In this work, we describe a novel expression system for the production of antimicrobial peptides in Escherichia coli by combining ΔI-CM mini-intein with the self-assembling amphipathic peptide 18A to drive the formation of active aggregates. Two AMPs, human β-defensin 2 and LL-37, were fused to the self-cleaving tag and expressed as active protein aggregates. The active aggregates were recovered by centrifugation and the intact antimicrobial peptides were released into solution by an intein-mediated cleavage reaction in cleaving buffer (phosphate-buffered saline supplemented with 40 mmol/L Bis–Tris, 2 mmol/L EDTA, pH 6.2). The peptides were further purified by cation-exchange chromatography. Peptides yields of 0.82 ± 0.24 and 0.59 ± 0.11 mg/L were achieved for human β-defensin 2 and LL-37, respectively, with demonstrated antimicrobial activity. Using our expression system, intact antimicrobial peptides were recovered by simple centrifugation from active protein aggregates after the intein-mediated cleavage reaction. Thus, we provide an economical and efficient way to produce intact antimicrobial peptides in E. coli.

scholarly journals The Chaperone Activities of DsbG and Spy Restore Peptidoglycan Biosynthesis in the elyC Mutant by Preventing Envelope Protein Aggregation

2018 ◽  
Vol 200 (19) ◽  
Author(s):  
Imène Kouidmi ◽  
Laura Alvarez ◽  
Jean François Collet ◽  
Felipe Cava ◽  
Catherine Paradis-Bleau
Keyword(s):  
Escherichia Coli ◽  
Protein Folding ◽  
Low Temperatures ◽  
Protein Aggregates ◽  
Cell Lysis ◽  
Content Type ◽  
Regulatory Pathways ◽  
Peptidoglycan Biosynthesis ◽  
Folding Defect ◽  
Content Factor

ABSTRACT Peptidoglycan (PG) is the main structural component of bacterial envelopes. It protects bacterial cells against variations in osmotic pressure and cell lysis. The newly discovered Escherichia coli factor ElyC has been shown to be important for peptidoglycan biosynthesis at low temperatures. PG production in ΔelyC mutant cells is totally blocked after a few hours of growth at 21°C, triggering cell lysis. In this study, we took a candidate approach to identify genetic suppressors of the ΔelyC mutant cell lysis phenotype. We identified the periplasmic proteins DsbG and Spy as multicopy suppressors and showed that their overproduction restores PG biosynthesis in the ΔelyC mutant. Interestingly, we found that DsbG acts by a novel mechanism, which is independent of its known reductase activity and substrates. DsbG, like Spy, acts as a chaperone to reduce the amounts of protein aggregates in the envelopes of ΔelyC cells. In fact, we found that the amount of protein aggregates was greater in the ΔelyC mutant than in the wild type. Taken together, our results show a protein-folding defect in the envelope compartments of ΔelyC cells that blocks PG production, and they reveal a new physiological activity of DsbG. IMPORTANCE Peptidoglycan biosynthesis is a dynamic and well-controlled pathway. The molecular assembly of PG and the regulatory pathways ensuring its maintenance are still not well understood. Here we studied the newly discovered Escherichia coli factor ElyC, which is important for PG biosynthesis at low temperatures. We revealed an important protein-folding defect in the ΔelyC mutant and showed that overproduction of the periplasmic chaperone DsbG or Spy was sufficient to correct the protein-folding defect and restore PG biosynthesis. These results show that the PG defect in the absence of ElyC is caused, at least in part, by a protein-folding problem in the cell envelope. Furthermore, we showed, for the first time, that the periplasmic protein DsbG has chaperone activity in vivo.

Expression and characterization of the 42 kDa chitinase of the biocontrol fungusMetarhizium anisopliaeinEscherichia coli

2003 ◽  
Vol 49 (11) ◽  
pp. 723-726 ◽  
Author(s):  
César Milton Baratto ◽  
Marcia Vanusa da Silva ◽  
Lucélia Santi ◽  
Luciane Passaglia ◽  
Irene Silveira Schrank ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Metarhizium Anisopliae ◽  
Entomopathogenic Fungus ◽  
Expression Vector ◽  
Hydrolytic Enzymes ◽  
Active Protein ◽  
Chitinase Genes

Albeit Metarhizium anisopliae is the best-characterized entomopathogenic fungus, the role of some hydrolytic enzymes during host cuticle penetration has not yet been established. Three chitinase genes (chit1, chi2, chi3) from Metarhizium have already been isolated. To characterize the chitinase coded by the chit1 gene, we expressed the active protein (CHIT42) in Escherichia coli using a T7-based promoter expression vector. The recombinant protein, CHIT42, is active against glycol chitin and synthetic N-acetylglucosamine (GlcNAc) dimer and tetramer substrates. These activities suggest that the recombinant CHIT42 acts as an endochitinase.Key words: Metarhizium anisopliae, chitinases, chit genes, recombinant protein, enthomopathogenic fungi.

Self-assembling amphipathic alpha-helical peptides induce the formation of active protein aggregates in vivo

2013 ◽  
Vol 166 ◽  
pp. 243 ◽  
Author(s):  
Zhanglin Lin ◽  
Bihong Zhou ◽  
Wei Wu ◽  
Lei Xing ◽  
Qing Zhao
Keyword(s):  
Protein Aggregates ◽  
Active Protein ◽  
Helical Peptides ◽  
Self Assembling ◽  
Alpha Helical Peptides

scholarly journals Two-State Allosteric Modeling Suggests Protein Equilibrium as an Integral Component for Cyclic AMP (cAMP) Specificity in the cAMP Receptor Protein of Escherichia coli

2008 ◽  
Vol 190 (13) ◽  
pp. 4532-4540 ◽  
Author(s):  
Hwan Youn ◽  
Junseock Koh ◽  
Gary P. Roberts
Keyword(s):  
Escherichia Coli ◽  
Binding Pocket ◽  
Receptor Protein ◽  
Ligand Specificity ◽  
Wild Type ◽  
Camp Receptor Protein ◽  
Active Protein ◽  
Common Basis ◽  
Affinity Ligand ◽  
Camp Receptor

ABSTRACT Activation of the cAMP receptor protein (CRP) from Escherichia coli is highly specific to its allosteric ligand, cAMP. Ligands such as adenosine and cGMP, which are structurally similar to cAMP, fail to activate wild-type CRP. However, several cAMP-independent CRP variants (termed CRP*) exist that can be further activated by both adenosine and cGMP, as well as by cAMP. This has remained a puzzle because the substitutions in many of these CRP* variants lie far from the cAMP-binding pocket (>10 Å) and therefore should not directly affect that pocket. Here we show a surprising similarity in the altered ligand specificity of four CRP* variants with a single substitution in D53S, G141K, A144T, or L148K, and we propose a common basis for this phenomenon. The increased active protein population caused by an equilibrium shift in these variants is hypothesized to preferentially stabilize ligand binding. This explanation is completely consistent with the cAMP specificity in the activation of wild-type CRP. The model also predicts that wild-type CRP should be activated even by the lower-affinity ligand, adenosine, which we experimentally confirmed. The study demonstrates that protein equilibrium is an integral factor for ligand specificity in an allosteric protein, in addition to the direct effects of ligand pocket residues.

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